Dramatically Enhanced Immunochromatographic Assay Using Cascade Signal Amplification for Ultrasensitive Detection of Escherichia coli O157:H7 in Milk

Abstract

The conventional colloidal gold immunochromatographic assay (AuNP-ICA) cannot meet the requirements for the rapid and sensitive detection of Escherichia coli (E. coli) O157:H7 because of its poor sensitivity. Herein, a novel two-step cascade signal amplification strategy that integrates in situ gold growth and nanozyme-mediated catalytic deposition was proposed to enhance the detection sensitivity of conventional AuNP-ICA dramatically. The enhanced strip displayed ultrahigh sensitivity in E. coli O157:H7 detection and had a detection limit of 1.25 × 101 CFU/mL, which is approximately 400-fold lower than that of traditional AuNP-ICA (5 × 103 CFU/mL). The amplified strip had no background signal in the T-line zone in the absence of E. coli O157:H7 even after one round of cascade signal amplification. The enhanced strip demonstrated excellent selectivity against E. coli O157:H7 with a negligible cross-reaction to nine other common pathogens. Intra-assays and interassays showed that the improved strip has acceptable accuracy and precision for determining E. coli O157:H7. The average recoveries in a real milk sample ranged from 87.33 to 112.15%, and the coefficients of variation were less than 10%. The enhanced strip also showed great potential in detecting a single E. coli O157:H7 cell in a 75 μL solution.

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.…”

“…Due to the electrochemical activity of TMB, the nanozyme-based colorimetric method could be transformed into an electrochemical assay, achieving the dual mode detection of P. aeruginosa. Fu and colleagues [51] proposed a novel two-step cascade signal amplification strategy to improve the detection sensitivity of conventional colloidal gold immunochromatographic assay (AuNPÀ ICA). As shown in Figure 1, in-situ gold growth was used to amplify the detection signals of the T and C lines on the strip.…”

“…This strategy has been applied for rapid and ultrasensitive detection of Escherichia coli with an LOD of 1.25 × 10 1 CFU mL À 1 , which is about 400 times lower than that of the conventional AuNP-ICA strip. [51] Besides AuNPs, PtNPs were also widely studied as highly sensitive and stable catalysts. [52,53] For instance, Yang's group [52] reported that PtNPs generated more than 400 times more O 2 per second than common catalase, resulting in much higher detection sensitivity.…”

“…Schematic of the cascade signal-amplification strategy based on the AuNP-ICA platform for E. coli detection. Reprinted with permission from ref [51]…”