Amplified visual immunosensor integrated with nanozyme for ultrasensitive detection of avian influenza virus

Abstract

Nanomaterial-based artificial enzymes or nanozymes exhibit superior properties such as stability, cost effectiveness and ease of preparation in comparison to conventional enzymes. However, the lower catalytic activity of nanozymes limits their sensitivity and thereby practical applications in the bioanalytical field. To overcome this drawback, herein we propose a very simple but highly sensitive, specific and low-cost dual enhanced colorimetric immunoassay for avian influenza A (H5N1) virus. 3,3´,5,5´- Tetramethylbenzidine (TMBZ) was used as a reducing agent to produce gold nanoparticles (Au NPs) with blue colored solution from a viral target-specific antibody-gold ion mixture at first step. The developed blue color from the sensing design was further amplified through catalytic activity of Au NPs in presence of TMBZ-hydrogen peroxide (H2O2) solution in second step. Hence, the developed dual enhanced colorimetric immunosensor enables the detection of avian influenza virus A (H5N1) with a limit of detection (LOD) of 1.11 pg/mL. Our results confirmed that the developed assay has superior sensitivity than the conventional ELISA method, plasmonic-based bioassay and commercial flu diagnostic kits. Proposed sensing method further showed its capability to detect viruses, avian influenza A (H4N6) and A (H9N2) virus, in blood samples with limit of detection of 0.0269 HAU and 0.0331 HAU respectively.

Direct immunosensing of avian influenza A virus in whole blood using hybrid nanocomposites

“…3(B)). These results are agreeable with previously developed ELISA and nanoenzyme techniques (Ahmed et al, 2017). It is quite possible that the biosensor can detect concentrations greater than 1 µg/mL; however, higher concentrations have yet to be tested.…”

Recent Progress of Nanozymes in the Detection of Pathogenic Microorganisms

“…With great advantages of tunability in activities, high stability as well as easy storage, noble-metal nanozymes show great potential in biosensing. [48][49][50][51][52][53][54][55][56][57][58][59][60] Numerous noble metal nanomaterials such as AuNPs, [48][49][50][51] Pt NPs, [52,53] PdPt, [54] PtAu, [55,56] PdCu, [57] FeCu NPs, [58] Pd@Pt NPs [59] and Au@Pt core/shell nanorods, [46,60] etc. have been widely studied for the detection of microbial pathogens.…”

“…AuNPs are the most widely studied noble metal-based nanozymes due to their excellent enzymatic activities, good biocompatibility and the availability of versatile bioconjugation means. [22,[48][49][50][51] Recently, Ahmed et al proposed a dual enhanced visual immunosensor integrated with AuNPs for the detection of avian influenza A virus (IAV) (H5N1). 3,3',5,5'-tetramethylbenzidine (TMB) first catalyzed the reduction of an antibodygold ion mixture to blue colored AuNPs.…”

“…The developed colorimetric immunoassay could also detect avian IAV (H4N6) and (H9N2) in blood samples through the replacement of corresponding virus-specific antibodies. [48] In addition to the combination of AuNPs and specific antibodies, researchers also prepared aptamer labeled AuNPs to detect viruses or bacteria. For example, Weerathunge et al [49] combined the peroxidase-like AuNPs with the MNV-specific AG3 aptamers for murine norovirus (MNV) detection.…”

Inorganic Complexes and Metal-Based Nanomaterials for Infectious Disease Diagnostics

“…Beyond magnetite, several other types of nanoparticles, including gold, 426 platinum, 427 hybrid particles, 428−432 and MOFs, 433 have been utilized as enzyme mimics in bioassays for targets such as respiratory syncytial virus, 432 avian influenza A, 426 Salmonella , and E. coli , 431 hepatitis C and HIV, 428 and Staphylococcus aureus . 433 Many of the assays that utilize nanozymes as catalytic turnover reagents for detection are formatted similarly to immunoassays on the surface of well plates.…”