Antibody-Driven Assembly of Plasmonic Core–Satellites to Increase the Sensitivity of a SERS Vertical Flow Immunoassay

Here, we describe a SERS-based vertical flow assay as a platform technology suitable for point-of-care (POC) diagnostic testing. A capture substrate is constructed from filter paper embedded with spherical gold nanoparticles (AuNPs) and functionalized with an appropriate capture antibody. The capture substrate is loaded into a filtration device and connected to a syringe to rapidly and repeatedly pass the sample through the sensor for efficient antigen binding. The antigen is then labeled with a SERS-active detection probe. We show that only a few Raman reporter molecules, exclusively located adjacent to the plasmonic capture substrate, generate detectible signals. To maximize the signal from underutilized Raman reporter molecules, we employ a secondary signal enhancing probe that undergoes antibody-directed assembly to form plasmonic core–satellites. This facile enhancement step provides a 3.5-fold increase in the signal and a detection limit of 0.23 ng/mL (1.6 pM) for human IgG. This work highlights the potential to rationally design plasmonic architectures using widely available and reproducible spherical AuNPs to achieve large SERS enhancements for highly sensitive POC diagnostics

Immobilization of Thiol-Modified Horseradish Peroxidase on Gold Nanoparticles Enhances Enzyme Stability and Prevents Proteolytic Digestion

The specificity and efficiency of enzyme-mediated reactions have the potential to positively impact many biotechnologies; however, many enzymes are easily degraded. Immobilization on a solid support has recently been explored to improve enzyme stability. This study aims to gain insights and facilitate enzyme adsorption onto gold nanoparticles (AuNPs) to form a stable bioconjugate through the installation of thiol functional groups that alter the protein chemistry. In specific, the model enzyme, horseradish peroxidase (HRP), is thiolated via Traut’s reagent to increase the robustness and enzymatic activity of the bioconjugate. This study compares HRP and its thiolated analog (THRP) to deduce the impact of thiolation and AuNP-immobilization on the enzyme activity and stability. HRP, THRP, and their corresponding bioconjugates, HRP-AuNP and THRP-AuNP, were analyzed via UV–vis spectrophotometry, circular dichroism, zeta potential, and enzyme–substrate kinetics assays. Our data show a 5-fold greater adsorption for THRP on the AuNP, in comparison to HRP, that translated to a 5-fold increase in the THRP-AuNP bioconjugate activity. The thiolated and immobilized HRP exhibited a substantial improvement in stability at elevated temperatures (50 °C) and storage times (1 month) relative to the native enzyme in solution. Moreover, HRP, THRP, and their bioconjugates were incubated with trypsin to assess the susceptibility to proteolytic digestion. Our results demonstrate that THRP-AuNP bioconjugates maintain full enzymatic activity after 18 h of incubation with trypsin, whereas free HRP, free THRP, and HRP-AuNP conjugates are rendered inactive by trypsin treatment. These results highlight the potential for protein modification and immobilization to substantially extend enzyme shelf life, resist protease digestion, and enhance biological function to realize enzyme-enabled biotechnologies

Rapid and Sensitive Detection of Rotavirus Molecular Signatures Using Surface Enhanced Raman Spectroscopy

Human enteric virus infections range from gastroenteritis to life threatening diseases such as myocarditis and aseptic meningitis. Rotavirus is one of the most common enteric agents and mortality associated with infection can be very significant in developing countries. Most enteric viruses produce diseases that are not distinct from other pathogens, and current diagnostics is limited in breadth and sensitivity required to advance virus detection schemes for disease intervention strategies. A spectroscopic assay based on surface enhanced Raman scattering (SERS) has been developed for rapid and sensitive detection of rotavirus. The SERS method relies on the fabrication of silver nanorod array substrates that are extremely SERS-active allowing for direct structural characterization of viruses. SERS spectra for eight rotavirus strains were analyzed to qualitatively identify rotaviruses and to classify each according to G and P genotype and strain with >96% accuracy, and a quantitative model based on partial least squares regression analysis was evaluated. This novel SERS-based virus detection method shows that SERS can be used to identify spectral fingerprints of human rotaviruses, and suggests that this detection method can be used for pathogen detection central to human health care

Detection of Mycoplasma pneumoniae in Simulated and True Clinical Throat Swab Specimens by Nanorod Array-Surface-Enhanced Raman Spectroscopy

The prokaryote Mycoplasma pneumoniae is a major cause of respiratory disease in humans, accounting for 20% of all community-acquired pneumonia and the leading cause of pneumonia in older children and young adults. The limitations of existing options for mycoplasma diagnosis highlight a critical need for a new detection platform with high sensitivity, specificity, and expediency. Here we evaluated silver nanorod arrays (NA) as a biosensing platform for detection and differentiation of M. pneumoniae in culture and in spiked and true clinical throat swab samples by surface-enhanced Raman spectroscopy (SERS). Three M. pneumoniae strains were reproducibly differentiated by NA-SERS with 95%–100% specificity and 94–100% sensitivity, and with a lower detection limit exceeding standard PCR. Analysis of throat swab samples spiked with M. pneumoniae yielded detection in a complex, clinically relevant background with >90% accuracy and high sensitivity. In addition, NA-SERS correctly classified with >97% accuracy, ten true clinical throat swab samples previously established by real-time PCR and culture to be positive or negative for M. pneumoniae. Our findings suggest that the unique biochemical specificity of Raman spectroscopy, combined with reproducible spectral enhancement by silver NA, holds great promise as a superior platform for rapid and sensitive detection and identification of M. pneumoniae, with potential for point-of-care application

Low-Pathogenic Avian Influenza Viruses in Wild House Mice

Background: Avian influenza viruses are known to productively infect a number of mammal species, several of which are commonly found on or near poultry and gamebird farms. While control of rodent species is often used to limit avian influenza virus transmission within and among outbreak sites, few studies have investigated the potential role of these species in outbreak dynamics. Methodology/Principal Findings: We trapped and sampled synanthropic mammals on a gamebird farm in Idaho, USA that had recently experienced a low pathogenic avian influenza outbreak. Six of six house mice (Mus musculus) caught on the outbreak farm were presumptively positive for antibodies to type A influenza. Consequently, we experimentally infected groups of naïve wild-caught house mice with five different low pathogenic avian influenza viruses that included three viruses derived from wild birds and two viruses derived from chickens. Virus replication was efficient in house mice inoculated with viruses derived from wild birds and more moderate for chicken-derived viruses. Mean titers (EID50 equivalents/mL) across all lung samples from seven days of sampling (three mice/day) ranged from 103.89 (H3N6) to 105.06 (H4N6) for the wild bird viruses and 102.08 (H6N2) to 102.85 (H4N8) for the chicken-derived viruses. Interestingly, multiple regression models indicated differential replication between sexes, with significantly (p\u3c0.05) higher concentrations of avian influenza RNA found in females compared with males. Conclusions/Significance: Avian influenza viruses replicated efficiently in wild-caught house mice without adaptation, indicating mice may be a risk pathway for movement of avian influenza viruses on poultry and gamebird farms. Differential virus replication between males and females warrants further investigation to determine the generality of this result in avian influenza disease dynamics

Asymmetrically Functionalized Antibody–Gold Nanoparticle Conjugates to Form Stable Antigen-Assembled Dimers

Biomolecular assays based on the aggregation of modified gold nanoparticles (AuNPs) have been developed to provide low detection limits and rapid results with a simple one-step, wash-free procedure. However, a relatively narrow dynamic range, low sensitivity, and poor precision due to time-sensitive readout limit the application of these assay platforms. In this work we synthesized asymmetrically functionalized antibody–AuNP conjugates that are rationally designed to overcome the limitations of aggregation-based immunoassays. Solid-phase synthesis was used to chemically passivate the majority of the AuNP surface and restrict antibody immobilization into a small area of the AuNP surface. These asymmetric conjugates assembled into dimers with the addition of antigen and were stable for over 24 h. In contrast, conventional antibody–AuNP conjugates which are symmetrically modified with antibody assembled into large aggregates that continuously increased in size with the addition of target antigen. These results suggest that asymmetric antibody–AuNP conjugates have the potential to significantly improve the analytical performance of aggregation-based immunoassays

Accelerated Surface-Enhanced Raman Spectroscopy (SERS)-Based Immunoassay on a Gold-Plated Membrane

A rapid and simple SERS-based immunoassay has been developed to overcome diffusion-limited binding kinetics that often impedes rapid analysis in conventional heterogeneous immunoassays. This paper describes the development of an antibody-modified membrane as a flow-through capture substrate for a nanoparticle-enabled SERS immunoassay to enhance antibody–antigen binding kinetics. A thin layer of gold is plated onto polycarbonate track-etched nanoporous membranes via electroless deposition. Capture antibody is immobilized onto the surface of a gold-plated membrane via thiolate coupling chemistry to serve as a capture substrate. A syringe is then used to actively transport the analyte and extrinsic Raman labels to the capture substrate. The fabrication of the gold-plated membrane is thoroughly investigated and established as a viable capture substrate for a SERS-based immunoassay in the absence of sample/SERS label flow. A syringe pump is used to systematically investigate the effect of flow rate on antibody–antigen binding kinetics and demonstrate that active transport to the capture membrane surface expedites antibody–antigen binding. Mouse IgG and goat anti-mouse IgG are selected as a model antigen–antibody system to establish proof of principle. It is demonstrated that the assay for mouse IgG is reduced from 24 h to 10 min and a 10-fold improvement in detection limit is achieved with the flow assay developed herein relative to the passive, i.e., no flow, assay. Moreover, mouse serum is directly analyzed and IgG level is determined using the flow assay