ABSTRACT
SARS-CoV-2 rapid spread required urgent, accurate, and prompt diagnosis to control the virus dissemination and pandemic management. Several sensors were developed using different biorecognition elements to obtain high specificity and sensitivity. However, the task to achieve these parameters in combination with fast detection, simplicity, and portability to identify the biorecognition element even in low concentration remains a challenge. Therefore, we developed an electrochemical biosensor based on polypyrrole nanotubes coupled via Ni(OH)2 ligation to an engineered antigen-binding fragment of heavy chain-only antibodies (VHH) termed Sb#15. Herein we report Sb#15-His6 expression, purification, and characterization of its interaction with the receptor-binding domain (RBD) of SARS-CoV-2 in addition to the construction and validation of a biosensor. The recombinant Sb#15 is correctly folded and interacts with the RBD with a dissociation constant (KD) of 27.1 ± 6.4 nmol/L. The biosensing platform was developed using polypyrrole nanotubes and Ni(OH)2, which can properly orientate the immobilization of Sb#15-His6 at the electrode surface through His-tag interaction for the sensitive SARS-CoV-2 antigen detection. The quantification limit was determined as 0.01 pg/mL using recombinant RBD, which was expressively lower than commercial monoclonal antibodies. In pre-characterized saliva, both Omicron and Delta SARS-CoV-2 were accurately detected only in positive samples, meeting all the requirements recommended by the World Health Organization for in vitro diagnostics. A low sample volume of saliva is needed to perform the detection, providing results within 15 min without further sample preparations. In summary, a new perspective allying recombinant VHHs with biosensor development and real sample detection was explored, addressing the need for accurate, rapid, and sensitive biosensors.
ABSTRACT
LTA zeolite is used both in industry as well as in laboratories, because of its spatial-specific structure which is useful in gas adsorption and in ion exchange separation. At-line reaction monitoring and multivariate analysis of data, such as Principal Component Analysis (PCA), are fundamentals of the Process Analytical Technology (PAT), which consists of the use of analyzers with rapid detection and low sample preparation for analysis during the process stream. In this work, an optimization of zeolite LTA synthesis was performed aiming to obtain nanocrystals and the synthesis was monitored using Raman spectroscopy and PCA. A reaction mixture of 6.2Na2O : Al2O3 : 2SiO2 : 128H2O was used and it was possible to obtain a small particle size and high crystallinity after 72 h of synthesis at 25 °C. The synthesis was monitored at-line, using Raman spectroscopy in both liquid and solid phases. The extension of the reaction could be clearly observed by the PCA scores. As expected, the liquid phase presented changes over time, but the solid phase presented three specific stationary conditions at 0-24 h, 32-56 h and 72-80, related to the beginning of the reaction, the nucleation process, and the crystal formation, respectively. In addition, it was possible to identify the intermediates of the reaction and with the aid of PCA to monitor the reaction close to the real time.
