Ultrasensitive Colorimetric Detection of Murine Norovirus Using NanoZyme Aptasensor

Abstract

Human norovirus (NoV) remains the most common cause of viral gastroenteritis and the leading cause of viral foodborne outbreaks globally. NoV is highly pathogenic with an estimated median viral infective dose (ID 50 ) ranging from 18 to 1015 genome copies. For NoV detection, the only reliable and sensitive method available for detection and quantification is reverse transcription quantitative polymerase chain reaction (RTqPCR). NoV detection in food is particularly challenging, requiring matrix specific concentration of the virus and removal of inhibitory compounds to detection assays. Hence, the RTqPCR method poses some challenges for rapid in-field or point-of-care diagnostic applications. We propose a new colorimetric NanoZyme aptasensor strategy for rapid (10 min) and ultrasensitive (calculated Limit of Detection (LoD) of 3 viruses per assay equivalent to 30 viruses/mL of sample and experimentally demonstrated LoD of 20 viruses per assay equivalent to 200 viruses/mL) detection of the infective murine norovirus (MNV), a readily cultivable surrogate for NoV. Our approach combines the enzyme-mimic catalytic activity of gold nanoparticles with high target specificity of an MNV aptamer to create sensor probes that produce a blue color in the presence of this norovirus, such that the color intensity provides the virus concentrations. Overall, our strategy offers the most sensitive detection of norovirus or a norovirus surrogate achieved to date using a biosensor approach, enabling for the first time, the detection of MNV virion corresponding to the lower end of the ID 50 for NoV. We further demonstrate the robustness of the norovirus NanoZyme aptasensor by testing its performance in the presence of other nontarget microorganisms, human serum and shellfish homogenate, supporting the potential of detecting norovirus in complex matrices. This new assay format can, therefore, be of significant importance as it allows ultrasensitive norovirus detection rapidly within minutes, while also offering the simplicity of use and need for nonspecialized laboratory infrastructure.

Virus detection using nanosensors

“…They integrated the gold nanoparticles that have enzymeÀmimic catalytic activity with a murine norovirus aptamer and noticed that this nanosensor created a blue color in the being of norovirus. They also found the detection limit as three viruses per assay, which is equal to 30 viruses/mL of the sample [111].…”

A Survey of Analytical Techniques for Noroviruses

“…Similarly, a study conducted by Kitajima et al [68] utilized an AG3 aptamer to develop a miniaturized and potentially portable electrochemical biosensor for detection of MNV. More recently, by using the AG3 aptamer, Weerathunge et al [69] developed a rapid (10 min) and ultrasensitive colorimetric NanoZyme aptasensor for the detection of MNV with an experimental LOD of 200 viruses/mL and calculated LOD of 30 viruses/mL. Specifically, gold nanoparticles (AuNPs) that have enzyme-mimic catalytic activity (i.e., NanoZyme activity) were immobilized with the AG3 aptamer (Kd of 18.5 nM) for MNV detection.…”

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.…”

“…[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. MNV is considered a satisfying alternative to human norovirus in scientific research.…”