Antibody-nanoparticle Conjugates Research Articles

A review on immunoglobulin Y (IgY) conjugated with metal nanoparticles and biomedical uses.

Today, the use of nanoparticles has attracted considerable attention in biomedical investigations and applications. Antibody-nanoparticle conjugates have proven to be useful tools for raising accuracy and sensitivity in in vitro diagnostics. IgY antibodies have benefits over different antibodies in terms of minimizing animal harm, reducing reactivity with mammalian factors, and cost-effective extraction. Metal nanoparticles are widely used for various medical and biological applications and are potential candidates for identifying pathogens and treating them, which can be mostly related to their special properties, including their shape and size. Avian IgY antibodies conjugated with nanoparticles have been widely used for the detection of parasitic, viral, and bacterial infections as well as allergens and toxicological and pharmaceutical molecules. This review aimed to investigate avian antibodies conjugated with metal nanoparticles and their biological applications.

Site-Specific Antibody Assembly on Nanoparticles via a Versatile Coating Method for Improved Cell Targeting.

Antibody-nanoparticle conjugates are promising candidates for precision medicine. However, developing a controllable method for conjugating antibodies to nanoparticles without compromising the antibody activity represents a critical challenge. Here, a facile and generalizable film-coating method is presented using zeolitic imidazole framework-8 (ZIF-8) to immobilize antibodies on various nanoparticles in a favorable orientation for enhanced cell targeting. Different model and therapeutic antibodies (e.g., Herceptin) are assembled on nanoparticles via a biomineralized film-coating method and exhibited high antibody loading and targeting efficiencies. Importantly, the antibodies selectively bind to ZIF-8 via their Fc regions, which favorably exposes the functional Fab regions to the biological target, thus improving the cell targeting ability of antibody-coated nanoparticles. In combination, molecular dynamics simulations and experimental studies on antibody immobilization, orientation efficiency, and biofunctionality collectively demonstrate that this versatile site-specific antibody conjugation method provides effective control over antibody orientation and leads to improved cell targeting for a variety of nanoparticles.

Smartphone-based paper microfluidic competitive immunoassay for the detection of α-amanitin from mushrooms.

α-Amanitin is often considered the most poisonous mushroom toxin produced by various mushroom species, which are hard to identify from edible, non-toxic mushrooms. Conventional detection methods require expensive and bulky equipment or fail to meet high analytical sensitivity. We developed a smartphone-based fluorescence microscope platform to detect α-amanitin from dry mushroom tissues. Antibody-nanoparticle conjugates were captured by immobilized antigen-hapten conjugates while competing with the free analytes in the sample. Captured fluorescent nanoparticles were excited at 460nm and imaged at 500nm. The pixel numbers of such nanoparticles in the test zone were counted, showing a decreasing trend with increasing analyte concentration. The detection method exhibited a low detection limit (1pg/mL), high specificity, and selectivity, allowing us to utilize a simple rinsing for toxin extraction and avoiding the need for high-speed centrifugation. In addition, this assay's short response time and portable features enable field detection of α-amanitin from amanitin-producing mushrooms.

Evaluation of Cellular Targeting by Fab' vs Full-Length Antibodies in Antibody-Nanoparticle Conjugates (ANCs) Using CD4 T-cells.

Targeted delivery of chemotherapeutic drugs can improve their therapeutic efficiency by localizing their toxic effects at the diseased site. This is often achieved either by direct conjugation of drugs to antibodies targeting overexpressed receptors on cancer cells (antibody-drug conjugates/ADCs) or by conjugating antibodies to nanoparticles bearing drugs (antibody-nanoparticle conjugates/ANCs). Here, we report a platform for utilizing hinge cysteines on antigen-binding fragment (Fab') of an anti-CD4 antibody for site-specific conjugation to nanoparticles giving rise to anti-CD4 Fab'-nanoparticle conjugates (Fab'-NCs). We demonstrate a convenient route for obtaining functional anti-CD4 Fab' from full-length antibody and examine the targeted delivery efficiencies of anti-CD4 Fab'-NCs vs ANCs for selective delivery to CD4high mT-ALL cells. Our results indicate that higher avidity of full-length anti-CD4 antibody, i.e., protein alone translated to higher binding ability to CD4high mT-ALL cells in comparison with anti-CD4 Fab' alone. However, the targeted delivery efficiency of anti-CD4 Fab'-NCs was comparable to ANCs indicating that the avidity of Fab' is restored in a nanoparticle-conjugate format. Fab'-NCs are equally capable of achieving targeted drug delivery to CD4high T-cells as ANCs and are a versatile alternative to ANCs by offering site-selective modification strategy while retaining their advantages.

Retention of Activity by Antibodies Immobilized on Gold Nanoparticles of Different Sizes: Fluorometric Method of Determination and Comparative Evaluation.

Antibody–nanoparticle conjugates are widely used analytical reagents. An informative parameter reflecting the conjugates’ properties is the number of antibodies per nanoparticle that retain their antigen-binding ability. Estimation of this parameter is characterized by a lack of simple, reproducible methods. The proposed method is based on the registration of fluorescence of tryptophan residues contained in proteins and combines sequential measurements of first the immobilized antibody number and then the bound protein antigen number. Requirements for the measurement procedure have been determined to ensure reliable and accurate results. Using the developed technique, preparations of spherical gold nanoparticles obtained by the most common method of citrate reduction of gold salts (the Turkevich–Frens method) and varying in average diameter from 15 to 55 nm have been characterized. It was shown that the number of antibodies (immunoglobulins G) bound by one nanoparticle ranged from 30 to 194 during adsorptive unoriented monolayer immobilization. C-reactive protein was considered as the model antigen. The percentage of antibody valences that retained their antigen-binding properties in the conjugate increased from 17 to 34% with an increase in the diameter of gold nanoparticles. The proposed method and the results of the study provide tools to assess the capabilities of the preparations of gold nanoparticles and their conjugates as well as the expediency of seeking the best techniques for various practical purposes.

Targeted sonodynamic destruction of glioblastoma cells using antibody-titanium dioxide nanoparticle conjugates.

Aim: We present data on sonodynamic therapy (SDT) against glioblastoma cells utilizing titanium dioxide (TiO2) nanoparticlesconjugated to anti-EGFRantibody. Materials & methods: TiO2 nanoparticles were bound to anti-EGFR antibody to form antibody-nanoparticle conjugates (ANCs), then characterized by x-ray photoelectron spectroscopy and transmission electron microscopy. Cells underwent ultrasound and assessment on viability, reactive oxygen speciesand apoptosis were performed. Results: X-ray photoelectron spectroscopy analysis revealed the formation of an ANC. Transmission electron microscopy showed internalization of the ANCs by glioblastoma cells. With SDT, cell viabilities were reduced in the presence of ANCs, reactive oxygen species production was formed, but minimal effect on apoptosis was seen. Conclusion: For the first time, an ANC can be used with SDT to kill glioblastoma cells.

Abstract A096: Oncogenic TRIM37 is a genetic determinant of racial disparity in TNBC patients

Abstract Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that disproportionately affects younger African American (AA) women. A 5-year survival rate for AA TNBC patients is only 14% compared to 36% of Caucasian patients, which is attributed to early relapse, and metastasis to lung, liver and brain. Although disparities in treatment, co-morbid disease, and access to health care contribute to poor prognosis in AA women, a race-specific genetic component to TNBC disparity cannot be ruled out. To this end, our comprehensive preliminary studies have discovered genetic variations in tripartite motif-containing protein 37 (TRIM37) protein that strongly correlates with TNBC disparity and aggressive metastatic phenotype. Originally, we discovered and characterized TRIM37 as a new breast cancer oncogene that catalyzes histone H2A ubiquitination, a repressive epigenetic modification (Bhatnagar et. al., Nature 2014). Our recent follow-up studies combined with analysis of ethnically diverse genotype-tissue expression, quantitative trait loci, and 1000 genome datasets have discovered single nucleotide polymorphism in TRIM37 that segregate racially. Critically, we have identified TRIM37 polymorphic variants that are predominant in AA women and strongly associate with high TRIM37 in breast tissue. Furthermore, our genetic and biochemical studies establish a strong correlation between TRIM37 expression, metastasis and poor overall-survival using murine TNBC models and TNBC patient tissue analysis. Our comparative mechanistic investigations provide compelling evidence that TRIM37 accelerates TNBC metastasis by enhancing resistance to chemotherapy and inhibiting anoikis pathway, preferentially in AA TNBC relative to non-African American (non-AA) TNBC cells. Therefore, we hypothesize that TRIM37 is a genetic determinant of a high predisposition to metastatic TNBC in AA women, and a strategic target for clinical intervention. Clinical data indicate that AA women have a higher rate of metastatic recurrences compared to non-AA patients. An aggressive clinical course and our preliminary findings strongly suggest that TRIM37 preferentially influences clinical outcome of TNBC in AA women and represents a novel therapeutic target. In this proposal, we will test a highly innovative TRIM37 targeting approach that makes use of cutting-edge antibody-nanoparticle conjugates and antisense oligonucleotide strategy. The overall objectives of this proposal are to demonstrate that TRIM37 is a genetic variant associated with a high risk of metastatic TNBC in AA women and generate proof-of-concept data to treat metastatic TNBC by inhibiting TRIM37 in vivo. Our high precision-targeted approach offers a promising path for selective and effective TNBC treatment. If successful, the described strategy also fits into a broader goal of effective and superior TNBC treatments, and therefore, the potential to generate and replicate additional therapies using other TNBC regulators is very high. Citation Format: Rachisan Gabriel Djiake Tihagam. Oncogenic TRIM37 is a genetic determinant of racial disparity in TNBC patients [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A096.

Rituximab conjugated iron oxide nanoparticles for targeted imaging and enhanced treatment against CD20-positive lymphoma.

Since its launch in 1997, rituximab (RTX) has extensively improved the treatment of CD20-positive follicular and diffuse large B cell non-Hodgkin lymphoma (NHL). The application of RTX is limited usually by the failed therapy because of resistance. Iron oxide nanomaterials have been explored for cancer detection and treatment in recent years. In this study, a multivalent nanoprobe comprising one Fe3O4 nanoparticle and several RTX antibodies was constructed for the targeted imaging and enhanced treatment of NHL. Poly(ethylene glycol) (PEG)-coated Fe3O4 nanoparticles were fabricated via a thermal decomposition method and ligand exchange. RTX was conjugated onto the surface of the Fe3O4-PEG nanoparticles to form Fe3O4-PEG-nAb (n = 2, 5 or 8) multivalent nanoprobes. These multivalent nanoprobes, with a core size of approximately 11 nm and a hydrodynamic diameter of about 22 nm, showed colloidal stability in buffer solution. The r2 relaxation rate of Fe3O4-PEG-nAb was similar to that of Fe3O4-PEG (309 ± 3.08 mM-1 s-1). The specificity of nanoprobes for CD20-positive Raji cells was assessed on a clinical magnetic resonance imaging scanner. The receptor binding site of one multivalent nanoprobe was more than that of one RTX, exhibiting valence-dependent induction of Raji cell apoptosis, and this effect could be enhanced by complement activation from blood serum added. A similar activity was observed in vivo in a NHL xenograft model. The multivalent nanoprobe treatment significantly reduced tumor burden and enhanced survival in comparison to the RTX group. Our studies demonstrate that the appropriate design and preparation of anticancer antibody-nanoparticle conjugates enable the generation of improved anticancer nanomedicines and could thus provide an efficient cancer theranostic strategy.

Single-Domain Antibody-Functionalized pH-Responsive Amphiphilic Block Copolymer Nanoparticles for Epidermal Growth Factor Receptor Targeted Cancer Therapy.

Biocompatible antibody-nanoparticle conjugates have attracted interest as anticancer agents due to their potential to selectively target therapeutic agents at disease sites. However, new formulation and conjugation approaches are urgently needed to improve their uniformity for clinical applications. Here, a pH-responsive benzaldehyde-functionalized poly[oligo(ethylene glycol) methacrylate-st-para-formyl phenyl methacrylate]-b-poly[2-(diisopropyl)aminoethyl methacrylate] [P(OEGMA-st-pFPMA)-b-PDPA] block copolymer, prepared by reversible addition-fragmentation chain transfer polymerization, produced PEGylated nanoparticles (pH ∼ 7.4) by a single emulsion-solvent evaporation formulation approach. Efficient site-specific attachment of an aminooxy-functionalized anti-EGFR single-domain antibody (sdAb) on these benzaldehyde-decorated nanoparticles is achieved by oxime bond formation. These nanoconjugates can specifically bind EGFR (modified ELISA) and have enhanced uptake over nonfunctionalized controls in EGFR-positive HeLa cells. Encapsulation of rhodamine 6G dye and its dispersion upon cellular uptake, consistent with nanoparticle stability loss at pH < 5.7, prove their ability to facilitate triggered release in endosomal compartments and highlight their potential for use as next-generation antibody-drug nanoconjugates for therapeutic drug delivery.

Updated Regulatory Considerations for Nanomedicines.

Nanomedicine is a branch which deals with medicinal products, devices, nonbiological complex drugs and antibody-nanoparticle conjugates and general health products that are manufactured using nanotechnology. Nano-medicine provides the same efficacies as traditional medicines owing to their improved solubility and bioavailability with reduced dosages. However, there are currently safety concerns due to the difficulties related to nanomaterial characterization; this might be the reason for unawareness of such medicines among the patients. The absence of clear regulatory guidelines further complicates matters, as it makes the path to registering them with regulatory bodies difficult. However, some products have overcome these obstacles and have been registered. While there are many international initiatives to harmonize the regulatory requirements and helps the industry to determine the most important characteristics that influence in vivo product performance. This review focuses on the various types of nanopharmaceuticals, and developments process with strategies tailored to upcoming regulations may satisfy the patients' needs.

Frizzled7 Antibody-Functionalized Nanoshells Enable Multivalent Binding for Wnt Signaling Inhibition in Triple Negative Breast Cancer Cells.

Antibodies that antagonize cell signaling pathways specific to their targeted receptor are invaluable tools to study and treat malignancies, but their utility is limited by high production costs and treatment dosages. Researchers have shown that antibodies conjugated to nanoparticles display increased affinity for their target relative to freely delivered antibodies due to multivalency, and this study investigates how this multivalency can enable antibody-nanoparticle conjugates to inhibit oncogenic cell signaling more effectively than freely delivered antibodies. This effect was evaluated using triple negative breast cancer (TNBC) cells that are characterized by hyperactive Wnt signaling mediated through overexpressed Frizzled7 (FZD7) transmembrane receptors. Through analysis of the expression of β-catenin and Axin2, two downstream targets in the Wnt pathway, the results demonstrate that FZD7 antibody-nanoshell conjugates (FZD7-NS) are drastically more effective at inhibiting Wnt signaling in TNBC cells than freely delivered FZD7 antibodies. Additionally, cells treated with FZD7-NS, but not cells treated with freely delivered FZD7 antibodies, have decreased viability, indicating the therapeutic potential of this technology. The results demonstrate that antibody-functionalized nanoparticles can exploit multivalency for improved signal cascade interference over free antibodies, and this may ultimately permit lower antibody dosages to be administered to study signaling pathways or to manage diseases.

Antibody-nanoparticle conjugates to enhance the sensitivity of ELISA-based detection methods.

Accurate antigen detection is imperative for clinicians to diagnose disease, assess treatment success, and predict patient prognosis. The most common technique used for the detection of disease-associated biomarkers is the enzyme linked immunosorbent assay (ELISA). In an ELISA, primary antibodies are incubated with biological samples containing the biomarker of interest. Then, detectible secondary antibodies conjugated with horseradish peroxidase (HRP) bind the primary antibodies. Upon addition of a color-changing substrate, the samples provide a colorimetric signal that directly correlates to the targeted biomarker concentration. While ELISAs are effective for analyzing samples with high biomarker content, they lack the sensitivity required to analyze samples with low antigen levels. We hypothesized that the sensitivity of ELISAs could be enhanced by replacing freely delivered primary antibodies with antibody-nanoparticle conjugates that provide excess binding sites for detectible secondary antibodies, ultimately leading to increased signal. Here, we investigated the use of nanoshells (NS) decorated with antibodies specific to epidermal growth factor receptor (EGFR) as a model system (EGFR-NS). We incubated one healthy and two breast cancer cell lines, each expressing different levels of EGFR, with EGFR-NS, untargeted NS, or unconjugated EGFR antibodies, as well as detectable secondary antibodies. We found that EGFR-NS consistently increased signal intensity relative to unconjugated EGFR antibodies, with a substantial 13-fold enhancement from cells expressing high levels of EGFR. Additionally, 40x more unconjugated antibodies were required to detect EGFR compared to those conjugated to NS. Our results demonstrate that antibody-nanoparticle conjugates lower the detection limit of traditional ELISAs and support further investigation of this strategy with other antibodies and nanoparticles. Owing to their enhanced sensitivity, we anticipate that nanoparticle-modified ELISAs can be used to detect low levels of biomarkers found in various diseases, such as cancers, tuberculosis, and rheumatoid arthritis, and may ultimately enable earlier diagnosis, better prognostication, and improved treatment monitoring.

Marked enhancement of lysosomal targeting and efficacy of ErbB2-targeted drug delivery by HSP90 inhibition.

Targeted delivery of anticancer drugs to tumor cells using monoclonal antibodies against oncogenic cell surface receptors is an emerging therapeutic strategy. These strategies include drugs directly conjugated to monoclonal antibodies through chemical linkers (Antibody-Drug Conjugates, ADCs) or those encapsulated within nanoparticles that in turn are conjugated to targeting antibodies (Antibody-Nanoparticle Conjugates, ANPs). The recent FDA approval of the ADC Trastuzumab-TDM1 (Kadcyla®; Genentech; San Francisco) for the treatment of ErbB2-overexpressing metastatic breast cancer patients has validated the strong potential of these strategies. Even though the activity of ANPs and ADCs is dependent on lysosomal traffic, the roles of the endocytic route traversed by the targeted receptor and of cancer cell-specific alterations in receptor dynamics on the efficiency of drug delivery have not been considered in these new targeted therapies. For example, constitutive association with the molecular chaperone HSP90 is thought to either retard ErbB2 endocytosis or to promote its recycling, traits undesirable for targeted therapy with ANPs and ADCs. HSP90 inhibitors are known to promote ErbB2 ubiquitination, targeting to lysosome and degradation. We therefore hypothesized that ErbB2-targeted drug delivery using Trastuzumab-conjugated nanoparticles could be significantly improved by HSP90 inhibitor-promoted lysosomal traffic of ErbB2. Studies reported here validate this hypothesis and demonstrate, both in vitro and in vivo, that HSP90 inhibition facilitates the intracellular delivery of Trastuzumab-conjugated ANPs carrying a model chemotherapeutic agent, Doxorubicin, specifically into ErbB2-overexpressing breast cancer cells, resulting in improved antitumor activity. These novel findings highlight the need to consider oncogene-specific alterations in receptor traffic in the design of targeted drug delivery strategies. We suggest that combination of agents that enhance receptor endocytosis and lysosomal routing can provide a novel strategy to significantly improve therapy with ANPs and ADCs.

Anti Human Fibronectin–Gold Nanoparticle Complex, a Potential Nanobiosensor Tool for Detection of Fibronectin in ECM of Cultured Cells

Specific protein detection by means of antibody-nanoparticle conjugates is a new field in medical nanobiotechnology. Among many nanoparticles used, gold nanoparticles show strong light-absorption properties which have been exploited in designing nanobiosensors. Fibronectin (FN) plays an important role in extracellular matrix (ECM) structure and function of normal cells; however, in conditions like lung carcinoma, its expression increases, especially in non small cell lung carcinoma (NSCLC). In this study, we conjugated gold nanoparticles to human fibronectin antibody (anti-hFN) to design a colorimetric nanobiosensor for detection of FN present in ECM of cultured cells. Three different cell lines, namely A549 (target cells), AGO-1522 (control cells), and Nalm-6 (negative control cells), were used to compare changes in color resulting from aggregation of gold nanoparticles due to higher amount of FN. Our construct was able to detect increased level of FN which was distinguishable visually by change in color and could be confirmed by spectrophotometer as well.

Development of Gold Nanoparticle Based Colorimetric Biosensor for Detection of Fibronectin in Lung Cancer Cell Line

Specific protein detection by means of antibody-nanoparticle conjugates is a new field in medical nanobiotechnology. Among many nanoparticles used, gold nanoparticles show strong light-absorption properties which have been exploited in designing nanobiosensors. Fibronectin (FN) plays an important role in extracellular matrix (ECM) structure and function of normal cells; however, in conditions like lung carcinoma its expression increases, especially in non-small cell lung carcinoma (NSCLC). In this study, we conjugated gold nanoparticles to human fibronectin antibody (anti-hFN) to design a colorimetric nanobiosensor for detection of FN present in ECM of cultured cells. Three different cell lines namely A549 (target cells), AGO-1522 (control cells) and Nalm-6 (negative control cells) were used to compare changes in color resulting from aggregation of gold nanoparticles due to higher amount of FN. Our construct was able to detect increased level of FN which was distinguishable visually by change in color and could be confirmed by spectrophotometer as well.

Antibody-Targeted Nanoparticles for Cancer Therapy

In recent years, nanoparticulate-mediated drug delivery research has examined a full spectrum of nanoparticles that can be used in diagnostic and therapeutic cancer applications. A key aspect of this technology is in the potential to specifically target the nanoparticles to diseased cells using a range of molecules, in particular antibodies. Antibody-nanoparticle conjugates have the potential to elicit effective targeting and release of therapeutic targets at the disease site, while minimizing off-target side effects caused by dosing of normal tissues. This article provides an overview of various antibody-conjugated nanoparticle strategies, focusing on the rationale of cell-surface receptors targeted and their potential clinical application.

Single-Domain Antibody-Nanoparticles: Promising Architectures for Increased Staphylococcus aureus Detection Specificity and Sensitivity

Because antibodies are highly target-specific and nanoparticles possess diverse, material-dependent properties that can be exploited in order to label and potentially identify biomolecules, the development of antibody-nanoparticle conjugates (nanoconjugates) has huge potential in biodiagnostics. Here, we describe a novel superparamagnetic nanoconjugate, one whose recognition component is a single-domain antibody. It is highly active toward its target Staphylococcus aureus, displays long shelf life, lacks cross-reactivity inherent to traditional homologue whole antibodies, and captures a few dozen S. aureus cells in a mixed cell population with ~100% efficiency and specificity. We ascribe the excellent performance of our nanoconjugate to its single-domain antibody component and recommend it as a general purpose recognition element.

Comparative Analysis of Nanoparticle-Antibody Conjugations: Carbodiimide Versus Click Chemistry

The ability to modify the physical, chemical, and biologic properties of nanoparticles has led to their use as multifunctional platforms for drug delivery and diagnostic imaging applications. Typically, these applications involve functionalizing the nanoparticles with targeting agents. Antibodies remain an attractive choice as targeting agents because of their large epitope space and high affinity; however, implementation of antibody-nanoparticle conjugates is plagued by low coupling efficiencies and the high cost of reagents. Click chemistry may provide a solution to this problem, with reported coupling efficiencies nearing 100%. Although click chemistries have been used to functionalize nanoparticles with small molecules, they have not previously been used to functionalize nanoparticles with antibodies. Concerns associated with extending this procedure to antibodies are that reaction catalysts or the ligands required for cross-linking may result in loss of functionality. We evaluated the efficiency of conjugations between antibodies and superparamagnetic iron oxide nanoparticles using click chemistry as well as the functionality of the product. The results were compared with conjugates formed through carbodiimide cross-linking. The click reaction allowed for a higher extent and efficiency of labeling compared with carbodiimide, thus requiring less antibody. Further, conjugates prepared via the click reaction exhibited improved binding to target receptors.

Detection and Identification of Virus Particles on a Microfluidic Platform

We have developed a nanoparticle-based assay to capture and detect targeted virus particles on a microfluidic platform. Antibody-decorated magnetic nanoparticles were used to capture virions and detection was afforded by measuring the change in magnetic birefringence relaxation rates. Vesicular Stomatitis Virus (VSV), a bullet-shaped, negative-strand RNA virus, is used as a model for air, water or blood-borne pathogenic viruses. Antibodies against the single envelope glycoprotein of either the Indiana or New Jersey strains of VSV were conjugated to 30nm superparamagnetic nanoparticles to generate the capturing reagent. The size distribution of the antibody-nanoparticle conjugates was determined using a CONTIN analysis of dynamic light scattering (DLS) data. Our results were confirmed by TEM analysis. Antibody-decorated nanoparticles were combined with one of the VSV strains and subjected to Ismagilov mixing, utilizing two immiscible phases in micro-channels to increase the speed of capture. Virus binding was determined by changes in hydrodynamic volume of the virus-nanoparticle complexes as measured by their birefringence relaxation rate under stopped flow conditions. In addition, the dynamics of particle complex formation and aggregation was studied by DLS at different scattering angles. We compared the results of these two methods for measuring time-dependent increases in the size of the nanoparticle/virus complexes. The birefringence relaxation method is more adaptable to field and other applications than DLS since it is relatively insensitive to particle concentration or to the presence of dust and sample impurities. Our approach is superior to existing techniques such as qPCR or ELISA assays due to the speed of detection and insensitivity to environmental contaminants. We are looking into the suitability of this assay for point-of-care applications.This work was supported in part by NIH grant R44 ES012515.

Protein-Directed Self-Assembly of Gold Nanoparticles

AbstractWe have employed protein-protein interactions for controlled assembly of gold nanoparticles. Stoichiometric 1:1 protein:nanoparticle conjugates were prepared for proteins known to strongly interact with each other and these interactions were used to self-assemble nanoparticles. Mixing equivalent amounts of the antigen-nanoparticle and antibody-nanoparticle conjugates resulted in the formation of nanoparticle dimers with a yield of about 60%. Trimers (yield ∼30%) can be obtained by mixing 2:1 antigen-nanoparticle with 1:1 antibody-nanoparticle conjugates in a molar ratio of 1:2. The structures are destroyed at low pH when the antibody-antigen complex dissociates.