Switch-on Fluorescence Analysis of Protease Activity with the Assistance of a Nickel Ion-Nitrilotriacetic Acid-Conjugated Magnetic Nanoparticle.

Heterogeneous protease biosensors show high sensitivity and selectivity but usually require the immobilization of peptide substrates on a solid interface. Such methods exhibit the disadvantages of complex immobilization steps and low enzymatic efficiency induced by steric hindrance. In this work, we proposed an immobilization-free strategy for protease detection with high simplicity, sensitivity and selectivity. Specifically, a single-labeled peptide with oligohistidine-tag (His-tag) was designed as the protease substrate, which can be captured by a nickel ion-nitrilotriacetic acid (Ni-NTA)-conjugated magnetic nanoparticle (MNP) through the coordination interaction between His-tag and Ni-NTA. When the peptide was digested by protease in a homogeneous solution, the signal-labeled segment was released from the substrate. The unreacted peptide substrates could be removed by Ni-NTA-MNP, and the released segments remained in solution to emit strong fluorescence. The method was used to determine protease of caspase-3 with a low detection limit (4 pg/mL). By changing the peptide sequence and signal reporters, the proposal could be used to develop novel homogeneous biosensors for the detection of other proteases.

Development of a Modular NTA:His Tag Viral Vaccine for Co-delivery of Antigen and Adjuvant.

The SARS-CoV-2 pandemic has highlighted the need for vaccines that are effective, but quickly produced. Of note, vaccines with plug-and-play capabilities that co-deliver antigen and adjuvant to the same cell have shown remarkable success. Our approach of utilizing a nitrilotriacetic acid (NTA) histidine (His)-tag chemistry with viral adjuvants incorporates both of these characteristics: plug-and-play and co-delivery. We specifically utilize the cowpea mosaic virus (CPMV) and the virus-like particles from bacteriophage Qß as adjuvants and bind the model antigen ovalbumin (OVA). Successful binding of the antigen to the adjuvant/carrier was verified by SDS-PAGE, western blot, and ELISA. Immunization in C57BL/6J mice demonstrates that with Qß - but not CPMV - there is an improved antibody response against the target antigen using the Qß-NiNTA:His-OVA versus a simple admixture of antigen and adjuvant. Antibody isotyping also shows that formulation of the vaccines can alter T helper biases; while the Qß-NiNTA:His-OVA particle produces a balanced Th1/Th2 bias the admixture was strongly Th2. In a mouse model of B16F10-OVA, we further demonstrate improved survival and slower tumor growth in the vaccine groups compared to controls. The NiNTA:His chemistry demonstrates potential for rapid development of future generation vaccines enabling plug-and-play capabilities with effectiveness boosted by co-delivery to the same cell.

Effect of intra-nasal nitrilotriacetic acid trisodium salt in lowering elevated calcium cations and improving olfactory dysfunction in COVID-19 patients.

PURPOSE: An association between COVID-19 and olfactory dysfunction has been noted in many patients worldwide. The olfactory adaptation process leads to an increase in intracellular calcium cation levels. Nitrilotriacetic acid trisodium salt has high selective chelation for calcium cations from olfactory mucus. The aim of this work is to test the effect of an intranasal nitrilotriacetic acid trisodium salt to lower the elevated calcium cations in COVID-19 patients with relevant symptoms of olfactory dysfunction. METHODS: Fifty-eight COVID-19 adult patients with relevant symptoms of olfactory dysfunction were enrolled in a prospective randomized controlled trial. They received a nasal spray containing either 0.9% sodium chloride or 2% nitrilotriacetic acid trisodium salt. Olfactory function was assessed before and after treatment using the Sniffin' Sticks test. Quantitative analysis of calcium cation concentration in nasal secretions was performed using a carbon paste ion-selective electrode. RESULTS: After the application of nitrilotriacetic acid trisodium salt compared to sodium chloride, a significant improvement from functional anosmia to healthy normosmia with significant decrease in calcium cation concentration was observed. CONCLUSIONS: Further collaborative research is needed to fully investigate the effect of an intranasal nitrilotriacetic acid trisodium salt in the treatment of olfactory disorders.