Peptide nucleic acid and antifouling peptide based biosensor for the non-fouling detection of COVID-19 nucleic acid in saliva.

The electrochemical biosensor with outstanding sensitivity and low cost is regarded as a viable alternative to current clinical diagnostic techniques for various disease biomarkers. However, their actual analytical use in complex biological samples is severely hampered due to the biofouling, as they are also highly sensitive to nonspecific adsorption on the sensing interfaces. Herein, we have constructed a non-fouling electrochemical biosensor based on antifouling peptides and the electroneutral peptide nucleic acid (PNA), which was used as the recognizing probe for the specific binding of the viral RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Different from the negatively charged DNA probes that will normally weaken the biosensors' antifouling capabilities owing to the charge attraction of positively charged biomolecules, the neutral PNA probe will generate no side-effects on the biosensor. The biosensor demonstrated remarkable sensitivity in detecting SARS-CoV-2 viral RNA, possessing a broad linear range (1.0 fM - 1.0 nM) and a detection limit down to 0.38 fM. Furthermore, the sensing performance of the constructed electrochemical biosensor in human saliva was nearly similar to that in pure buffer, indicating satisfying antifouling capability. The combination of PNA probes with antifouling peptides offered a new strategy for the development of non-fouling sensing systems capable of assaying trace disease biomarkers in complicated biological media.

Electrochemical Biosensor with Enhanced Antifouling Capability for COVID-19 Nucleic Acid Detection in Complex Biological Media.

Biofouling caused by the accumulation of biomolecules on sensing surfaces is one of the major problems and challenges to realize the practical application of electrochemical biosensors, and an effective way to counter this problem is the construction of antifouling biosensors. Herein, an antifouling electrochemical biosensor was constructed based on electropolymerized polyaniline (PANI) nanowires and newly designed peptides for the detection of the COVID-19 N-gene. The inverted Y-shaped peptides were designed with excellent antifouling properties and two anchoring branches, and their antifouling performances against proteins and complex biological media were investigated using different approaches. Based on the biotin-streptavidin affinity system, biotin-labeled probes specific to the N-gene (nucleocapsid phosphoprotein) of COVID-19 were immobilized onto the peptide-coated PANI nanowires, forming a highly sensitive and antifouling electrochemical sensing interface for the detection of COVID-19 nucleic acid. The antifouling genosensor demonstrated a wide linear range (10-14 to 10-9 M) and an exceptional low detection limit (3.5 fM). The remarkable performance of the genosensor derives from the high peak current of PANI, which is chosen as the sensing signal, and the extraordinary antifouling properties of designed peptides, which guarantee accurate detection in complex systems. These crucial features represent essential elements for future rapid and decentralized clinical testing.