RESUMO
Rapid design, screening, and characterization of biorecognition elements (BREs) is essential for the development of diagnostic tests and antiviral therapeutics needed to combat the spread of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address this need, we developed a high-throughput pipeline combining in silico design of a peptide library specific for SARS-CoV-2 spike (S) protein and microarray screening to identify binding sequences. Our optimized microarray platform allowed the simultaneous screening of ~ 2.5 k peptides and rapid identification of binding sequences resulting in selection of four peptides with nanomolar affinity to the SARS-CoV-2 S protein. Finally, we demonstrated the successful integration of one of the top peptides into an electrochemical sensor with a clinically relevant limit of detection for S protein in spiked saliva. Our results demonstrate the utility of this novel pipeline for the selection of peptide BREs in response to the SARS-CoV-2 pandemic, and the broader application of such a platform in response to future viral threats.
Assuntos
COVID-19/imunologia , Técnicas de Química Combinatória , Peptídeos/química , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , COVID-19/virologia , Biologia Computacional , Eletroquímica/métodos , Ensaio de Imunoadsorção Enzimática , Humanos , Interferometria , Cinética , Biblioteca de Peptídeos , Análise Serial de Proteínas , Engenharia de Proteínas , Saliva/imunologiaRESUMO
Because of the sharp distance dependence of surface-enhanced Raman spectroscopy (SERS), analyte molecules that do not exhibit strong affinity for Au/Ag often elude detection. New methods of integrating such analytes with SERS substrates are required to circumvent this limitation and expand the sensitivity of SERS to new molecules and applications. We communicate here a solution-phase, capture agent-free method of aggregating Au nanospheres in the presence of five neurotransmitters (dopamine, epinephrine, norepinephrine, serotonin, and histamine) and preventing sedimentation by encapsulating the aggregated nanospheres with polyvinylpyrrolidone, thereby trapping the neurotransmitters in close proximity to the Au nanospheres and enabling SER detection. The primary advantages of this physicochemical trapping method, which is generalizable to analytes beyond the scope of this work, are the high signal-to-noise ratio and spectral consistency down to nM levels. Normal Raman spectra and density functional theory calculations corroborate the accuracy of the spectra. Spectra collected over a wide range of concentrations were used to construct adsorption isotherms for all five neurotransmitters, from which adsorption dissociation constants were calculated, spanning from 5.7 × 10-4 M to 1.7 × 10-10 M. We expect this method to produce high quality SER spectra of any molecule with an Au affinity known or expected (based on functional groups) to be within that range. Our results have implications for plasmonic detection of these neurotransmitters, particularly for mixtures of those that exhibited disparate Au affinity in our study. We also present evidence that this method produces spectra of sufficient resolution to explore hypotheses related to surface adsorption behavior.