Developing a Paper-Based Antigen Assay to Differentiate between Coronaviruses and SARS-CoV-2 Spike Variants.

COVID-19 first appeared in December of 2019 in Wuhan, China. Since then, it has become a global pandemic. A robust and scalable diagnostics strategy is crucial for containing and monitoring the pandemic. RT-PCR is a known, reliable method for COVID-19 diagnostics, which can differentiate between SARS-CoV-2 and other viruses. However, PCR is location-dependent, time-consuming, and relatively expensive. Thus, there is a need for a more flexible method, which may be produced in an off-the-shelf format and distributed more widely. Paper-based immunoassays can fulfill this function. Here, we present the first steps toward a paper-based test, which can differentiate between different spike proteins of various coronaviruses, SARS-CoV-1, SARS-CoV-2, and CoV-HKU1, with negligible cross-reactivity for HCoV-OC43 and HCoV-229E in a single assay, which takes less than 30 min. Furthermore, our test can distinguish between fractions of the same spike protein. This is done by an altered assay design with four test line locations where each antigen builds a unique, identifiable binding pattern. The effect of several factors, such as running media, immunoprobe concentration, and antigen interference, is considered. We find that running media has a significant effect on the final binding pattern where human saliva provides results while human serum leads to the lowest signal quality.

Designing Paper-Based Immunoassays for Biomedical Applications.

Paper-based sensors and assays have been highly attractive for numerous biological applications, including rapid diagnostics and assays for disease detection, food safety, and clinical care. In particular, the paper immunoassay has helped drive many applications in global health due to its low cost and simplicity of operation. This review is aimed at examining the fundamentals of the technology, as well as different implementations of paper-based assays and discuss novel strategies for improving their sensitivity, performance, or enabling new capabilities. These innovations can be categorized into using unique nanoparticle materials and structures for detection via different techniques, novel biological species for recognizing biomarkers, or innovative device design and/or architecture.

Effect of the Protein Corona on Antibody-Antigen Binding in Nanoparticle Sandwich Immunoassays.

We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, KDeff. Results show that corona formation mediates the specificity and sensitivity of the antibody-antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.

A comparison of nanoparticle-antibody conjugation strategies in sandwich immunoassays.

Point-of-care (POC) diagnostics such as lateral flow and dipstick immunoassays use gold nanoparticle (NP)-antibody conjugates for visual readout. We investigated the effects of NP conjugation, surface chemistries, and antibody immobilization methods on dipstick performance. We compared orientational, covalent conjugation, electrostatic adsorption, and a commercial conjugation kit for dipstick assays to detect dengue virus NS1 protein. Assay performance depended significantly on their conjugate properties. We also tested arrangements of multiple test lines within strips. Results show that orientational, covalent conjugation with PEG shield could improve NS1 detection. These approaches can be used to optimize immunochromatographic detection for a range of biomarkers.

Applications of Gold Nanoparticles in Plasmonic and Nanophotonic Biosensing.

The unique properties of plasmonic nanoparticles and nanostructures have enabled a broad range of applications in a diverse set of fields, ranging from biological sensing, cancer therapy, to catalysis. They have been some of the most studied nanomaterials due in part to their chemical stability and biocompatibility as well as supporting theoretical efforts. The synthesis and fabrication of plasmonic nanoparticles and nanostructures have now reached high precision and sophistication. We review here their fundamental optical properties, discuss their tailoring for biological environments, and then detail examples on how they have been used to innovate in the biological and biomedical fields.

Surface composition tuning of Au-Pt bimetallic nanoparticles for enhanced carbon monoxide and methanol electro-oxidation.

The ability to direct bimetallic nanoparticles to express desirable surface composition is a crucial step toward effective heterogeneous catalysis, sensing, and bionanotechnology applications. Here we report surface composition tuning of bimetallic Au-Pt electrocatalysts for carbon monoxide and methanol oxidation reactions. We establish a direct correlation between the surface composition of Au-Pt nanoparticles and their catalytic activities. We find that the intrinsic activities of Au-Pt nanoparticles with the same bulk composition of Au0.5Pt0.5 can be enhanced by orders of magnitude by simply controlling the surface composition. We attribute this enhancement to the weakened CO binding on Pt in discrete Pt or Pt-rich clusters surrounded by surface Au atoms. Our finding demonstrates the importance of surface composition control at the nanoscale in harnessing the true electrocatalytic potential of bimetallic nanoparticles and opens up strategies for the development of highly active bimetallic nanoparticles for electrochemical energy conversion.

Biophysical and biochemical insights in the design of immunoassays.

BACKGROUND: Rapid antigen assays have been attractive for decentralized, point of care diagnostics because of their low cost, robustness, and ease of use. The development of a diagnostic assay for a newly emerging infectious disease needs to take into account the progression of a disease, whether there is human to human transmission, and patient biomarker levels with time, and these all impact the choice of antigen targets and affinity agents. SCOPE OF REVIEW: The factors involved in the biophysical design of rapid antigen immunoassays are discussed, focusing on antigen selection and designing for cross-reactivity. State of the art in the biophysical characterization of protein-ligand or antigen-antibody interactions, the different types of affinity agents used in immunoassays, and biochemical conjugation strategies are described. MAJOR CONCLUSIONS: Antigen choice is a critical factor in immunoassay diagnostic development, and should account for the properties of the virion, virus, and disease progression. Biophysical and biochemical aspects of immunoassays are critical for performance. GENERAL SIGNIFICANCE: This review can serve as an instructive guide to aid in diagnostic development for future emerging diseases.

Gold Nanoparticle Paper Immunoassays for Sensing the Presence of Vibrio parahaemolyticus in Oyster Hemolymph.

Seafood contamination with Vibrio bacteria is a problem for aquaculture, especially with oysters, which are often consumed raw. Current methods for diagnosing bacterial pathogens in seafood involve lab-based assays such as polymerase chain reaction or culturing, which are time consuming and must occur in a centralized location. Detection of Vibrio in a point-of-care assay would be a significant tool for food safety control measures. We report here a paper immunoassay that can detect the presence of Vibrio parahaemolyticus (Vp) in buffer and oyster hemolymph. The test uses gold nanoparticles conjugated to polyclonal anti-Vibrio antibodies in a paper-based sandwich immunoassay. A sample is added to the strip and wicked through by capillary action. If Vp is present, it results in a visible color at the test area that can be read out by eyes or a standard mobile phone camera. The assay has a limit of detection of 6.05 × 105 cfu/mL and a cost estimate of $5 per test. Receiver operating characteristic curves with validated environmental samples showed a test sensitivity of 0.96 and a specificity of 1.00. Because the assay is inexpensive and can be used on Vp directly without the requirement for culturing, or sophisticated equipment, it has the potential to be used in fieldable settings.

Engineering the interface between glucose oxidase and nanoparticles.

The behavior of glucose oxidase (GOx) on gold nanoparticles (NPs) was investigated as a function of (1) NP surface chemistry, (2) stabilizing protein additives, and (3) protein microenvironment. GOx secondary structure and unfolding was probed by circular dichroism (CD) spectroscopy and fluorescence, and GOx enzymatic activity was measured by a colorimetric assay. We also examined the activity and structure of GOx after displacement from the NP surface. Generally, GOx behavior was negatively impacted by conjugation to the NP, and conjugation conditions could vary the influence of the NP. Surface chemistry and protein microenvironment could improve behavior, but addition of stabilizing proteins negatively influenced activity. After displacement from the NPs, GOx tended to remain unfolded, indicating that the interactions with the NP were irreversible.

Stability of gold nanorods passivated with amphiphilic ligands.

The stability of gold nanorods (NRs) coated with amphiphilic ligands (ALs) was investigated. NRs coated with cetyltrimethylammonium bromide (CTAB) were ligand exchanged with polyoxyethylene [10] cetyl ether (Brij56), Oligofectamine (OF), and phosphatidylserine (PS). An aggregation index based on the longitudinal surface plasmon resonance peak broadening was used to measure stability of the NR-ALs under different conditions including the number of washes, pH, ionic concentration, and temperature. The aggregation index was also used to measure the stability of the NR-ALs under ultrafast laser irradiation and in the presence of proteins commonly used in cell culture. Differences in NR-AL stability were found, which were due to differences in the physical and chemical properties of the ALs. Apart from the charge on the AL headgroup, we suggest the Gibbs free energy of passivation (ΔG(p)) and enthalpy of passivation (ΔH(p)) of the AL could potentially aid in the selection of amphiphiles that can effectively passivate NRs for stability and optimize their properties and desired biological impact.

Site-directed nanoparticle labeling of cytochrome c.

Although nanoparticle-protein conjugates have been synthesized for numerous applications, bioconjugation remains a challenge, often resulting in denaturation or loss of protein function. This is partly because the protein-nanoparticle interface is poorly understood, which impedes the use of nanoparticles in nanomedicine. Although the effects of nanoparticle ligand and material on protein structure have been explored, the choice of the labeling site on the protein has not yet been systematically studied. To address this issue, we label cytochrome c site-specifically with a negatively charged Au nanoparticle via a covalent thiol-Au bond. The attachment site is controlled by cysteine mutations of surface residues. The effect of labeling on protein structure is probed by circular dichroism. Protein unfolding is the most severe when the nanoparticle is attached to the N- and C-terminal foldon, the core motif of cytochrome c. Also, when the nanoparticle is attached in the vicinity of charged residues, the amount of structural damage is greater because of salt-dependent electrostatic interactions with charged ligand bis(p-sulfonatophenyl) phenylphosphine on the nanoparticle. Molecular dynamics simulations also elucidate local to global structural perturbation depending on labeling site. These results suggest that the labeling site must be considered as one of the main design criteria for nanoparticle-protein conjugates.

Nanomaterial and interface advances in immunoassay biosensors.

Biosensors have been used for a remarkable array of applications, including infectious diseases, environmental monitoring, cancer diagnosis, food safety, and numerous others. In particular, the global COVID-19 pandemic has exposed a need for rapid tests, so the type of biosensor that has gained considerable interest recently are immunoassays, which are used for rapid diagnostics. The performance of paper-based lateral flow and dipstick immunoassays is influenced by the physical properties of the nanoparticles (NPs), NP-antibody conjugates, and paper substrate. Many materials innovations have enhanced diagnostics by increasing sensitivity or enabling unique readouts. However, negative side effects can arise at the interface between the biological sample and biomolecules and the NP or paper substrate, such as non-specific adsorption and protein denaturation. In this Perspective, we discuss the immunoassay components and highlight chemistry and materials innovations that can improve sensitivity. We also explore the range of bio-interface issues that can present challenges for immunoassays.

Correction: SARS-CoV-2 and approaches for a testing and diagnostic strategy.

Correction for 'SARS-CoV-2 and approaches for a testing and diagnostic strategy' by Delyan R. Hristov et al., J. Mater. Chem. B, 2021, 9, 8157-8173, DOI: https://doi.org/10.1039/D1TB00674F.

The Influence of Preforming Protein Coronas on the Performance of Dengue NS1 Immunoassays.

The effect of preformed protein coronas on immunoassays for Dengue nonstructural protein 1 (NS1) immunoassays was investigated. The composition of the protein corona that forms around nanoparticle-antibody conjugates in human serum was characterized, and selected proteins from the corona were used for preformed coronas (human serum albumin and apolipoprotein A1). Coronas were formed and characterized by dynamic light scattering (DLS), and the nanoparticle-conjugate was probed by optical absorption spectroscopy. Immunoassays were run, and performance was quantified by analyzing the strip intensity as a function of NS1 concentration. The preformed coronas influenced the limit of detection (LOD) of the assay and the affinity for the NS1 target (KD). The resulting KD and LODs for the NP-Ab-ApoA1 immunoprobes were 0.83 nM and 1.24 nM, respectively. For the NP-Ab -HSA coronas, the test line intensity was lower by 33% at a given NS1 concentration than for the NP-Ab immunoprobes, and KD was 0.14 nM, a slightly higher affinity. Due to the relatively large error of the negative control, a meaningful LOD for the NP-Ab with HSA coronas could not be determined.

Multiplexed rapid antigen tests developed using multicolored nanoparticles and cross-reactive antibody pairs: Implications for pandemic preparedness.

Global public health infrastructure is unprepared for emerging pathogen epidemics, in part because diagnostic tests are not developed in advance. The recent Zika, Ebola, and SARS-CoV-2 virus epidemics are cases in point. We demonstrate here that multicolored gold nanoparticles, when coupled to cross-reactive monoclonal antibody pairs generated from a single immunization regimen, can be used to create multiple diagnostics that specifically detect and distinguish related viruses. The multiplex approach for specific detection centers on immunochromatography with pairs of antibody-conjugated red and blue gold nanoparticles, coupled with clustering algorithms to detect and distinguish related pathogens. Cross-reactive antibodies were used to develop rapid tests for i) Dengue virus serotypes 1-4, ii) Zika virus, iii) Ebola and Marburg viruses, and iv) SARS-CoV and SARS-CoV-2 viruses. Multiplexed rapid antigen tests based on multicolored nanoparticles and cross-reactive antibodies and can be developed prospectively at low cost to improve preparedness for epidemic outbreaks.

Quantifying the nanomachinery of the nanoparticle-biomolecule interface.

A study is presented of the nanomechanical phenomena experienced by nanoparticle-conjugated biomolecules. A thermodynamic framework is developed to describe the binding of thrombin-binding aptamer (TBA) to thrombin when the TBA is conjugated to nanorods. Binding results in nanorod aggregation (viz. directed self-assembly), which is detectable by absorption spectroscopy. The analysis introduces the energy of aggregation, separating it into TBA-thrombin recognition and surface-work contributions. Consequently, it is demonstrated that self-assembly is driven by the interplay of surface work and thrombin-TBA recognition. It is shown that the work at the surface is about -10 kJ mol(-1) and results from the accumulation of in-plane molecular forces of pN magnitude and with a lifetime of <1 s, which arises from TBA nanoscale rearrangements fuelled by thrombin-directed nanorod aggregation. The obtained surface work can map aggregation regimes as a function of different nanoparticle surface conditions. Also, the thermodynamic treatment can be used to obtain quantitative information on surface effects impacting biomolecules on nanoparticle surfaces.

SARS-CoV-2 and approaches for a testing and diagnostic strategy.

The COVID-19 pandemic has led to an unprecedented global health challenge, creating sudden, massive demands for diagnostic testing, treatment, therapies, and vaccines. In particular, the development of diagnostic assays for SARS-CoV-2 has been pursued as they are needed for quarantine, disease surveillance, and patient treatment. One of the major lessons the pandemic highlighted was the need for fast, cheap, scalable and reliable diagnostic methods, such as paper-based assays. Furthermore, it has previously been suggested that paper-based tests may be more suitable for settings with lower resource availability and may help alleviate some supply chain challenges which arose during the COVID-19 pandemic. Therefore, we explore how such devices may fit in a comprehensive diagnostic strategy and how some of the challenges to the technology, e.g. low sensitivity, may be addressed. We discuss the properties of the SARS-CoV-2 virus itself, the COVID-19 disease pathway, and the immune response. We then describe the different diagnostic strategies that have been pursued, focusing on molecular strategies for viral genetic material, antigen tests, and serological assays, and innovations for improving the diagnostic sensitivity and capabilities. Finally, we discuss pressing issues for the future, and what needs to be addressed for the ongoing pandemic and future outbreaks.

Platinum-gold nanoparticles: a highly active bifunctional electrocatalyst for rechargeable lithium-air batteries.

PtAu nanoparticles (NPs) were shown to strongly enhance the kinetics of the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) in rechargeable Li-O(2) cells. Li-O(2) cells with PtAu/C were found to exhibit the highest round-trip efficiency reported to date. During ORR via xLi(+) + O(2) + xe(-) --> Li(x)O(2), the discharge voltage with PtAu/C was considerably higher than that of pure carbon and comparable to that of Au/C. During OER via Li(x)O(2) --> xLi(+) + O(2) + xe(-), the charge voltages with PtAu/C fell in the range from 3.4 to 3.8 V(Li), which is slightly lower than obtained with Pt. It is hypothesized that PtAu NPs exhibit bifunctional catalytic activity, having surface Au and Pt atoms primarily responsible for ORR and OER kinetics in Li-O(2) cells, respectively.

Effect of ligands on thermal dissipation from gold nanorods.

Thermal interface conductance was measured for soluble gold nanorods (NRs) coated with mercaptocarboxylic acids (HS-(CH(2))(n)COOH, n = 5, 10, 15), thiolated polyethylene glycols (MW = 356, 1000, 5000), and HS-(CH(2))(15)-COOH-coated NRs further coated with alternating layers of poly(diallyldimethylammonium chloride) and poly(sodium styrenesulfonate). Ferguson analysis determined ligand thickness. The thermal-diffusion-dominated regime of transient absorption spectra was fit to a continuum heat diffusion finite element model to obtain the thermal interface conductance, G, which varied with ligand chemistry but not molecule length. The results suggest that the ability to exclude water from the NR surface governs ligand G values.

Effective size and zeta potential of nanorods by Ferguson analysis.

The effective hydrodynamic size and free mobility of particles of varying aspect ratio were evaluated by Ferguson analysis of gel electrophoresis. The ligand layer thickness was estimated from the difference between the effective size and the size of the metal core from TEM imaging. The zeta potential of the particles was calculated from the Ferguson analysis result by applying conventional electrophoresis theories for spheres and cylinders. The results show that Henry's solution for spherical particles can be used to obtain the zeta potential of cylindrical particles without requiring the use of TEM for size analysis.