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.

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.

SERS-based immunoassay on a plasmonic syringe filter for improved sampling and labeling efficiency of biomarkers.

Rapid, sensitive, and quantitative detection of biomarkers is needed for early diagnosis of disease and surveillance of infectious outbreaks. Here, we exploit a plasmonic syringe filter and surface-enhanced Raman spectroscopy (SERS) in the development of a rapid detection system, using human IgG as a model diagnostic biomarker. The novel assay design facilitates multiple passages of the sample and labeling solution through the detection zone enabling us to investigate and maximize sampling efficiency to the capture substrate. The vertical flow immunoassay process in this study involves the utilization of filter paper embedded with gold nanoparticles (AuNPs) to form a plasmonic substrate. Capture antibody (anti-human IgG) is then immobilized onto the prepared plasmonic paper and inserted into a vertical flow device (syringe filter holder). Sample solution is passed through the filter paper and the target antigen (human IgG) is selectively captured by the immobilized antibody to form an antibody-antigen complex. Next, functionalized AuNPs as extrinsic Raman labels (ERLs) are passed through the filter paper to label the captured biomarker molecules forming a layered structure. This sandwiched geometry enhances plasmonic coupling and SERS signal to provide highly sensitive detection of biomolecules. Systematic studies to investigate the impact of multiple infuse/withdraw cycles of the sample and labeling solutions reveal that antigen and ERL binding are maximized with 10 and 20 cycles, respectively. The optimized assay achieves a detection limit of ∼0.2 ng mL-1 for human IgG with a total assay time of less than 5 minutes, meeting the demands for rapid point of care diagnostics. Additionally, the optimized platform was implemented in the quantitative analysis of the SARS-CoV-2 nucleocapsid protein, the typical target in commercial, FDA-approved rapid antigen tests for COVID-19.

Controlled Temporal Release of Serum Albumin Immobilized on Gold Nanoparticles.

Proteins adsorbed to gold nanoparticles (AuNPs) form bioconjugates and are critical to many emerging technologies for drug delivery, diagnostics, therapies, and other biomedical applications. A thorough understanding of the interaction between the immobilized protein and AuNP is essential for the bioconjugate to perform as designed. Here, we explore a correlation between the number of solvent-accessible thiol groups on a protein and the protein desorption rate from the AuNP surface in the presence of a competing protein. The chemical modification of human serum albumin (HSA) was carried out to install additional free thiols using Traut's reagent and create a library of HSA analogues by tailoring the molar excess of the Traut's reagent. We pre-adsorbed HSA variants onto the AuNP surface, and the resulting bioconjugates were then exposed to IgG antibody, and protein exchange was monitored as a function of time. We found that the rate of HSA displacement from the AuNP correlated with the experimentally measured number of accessible free thiol groups. Additionally, bioconjugates were synthesized using thiolated analogues of bovine serum albumin (BSA) and suspended in serum as a model for a complex sample matrix. Similarly, desorption rates with serum proteins were modulated with solvent-accessible thiols on the immobilized protein. These results further highlight the key role of Au-S bonds in the formation of protein-AuNP conjugates and provide a pathway to systematically control the number of free thiols on a protein, enabling the controlled release of protein from the surface of AuNP.

Structure and activity of native and thiolated α-chymotrypsin adsorbed onto gold nanoparticles.

A detailed understanding of protein-nanoparticle interactions is critical to realize the full potential of bioconjugate-enabled technologies. Parameters that lead to conformational changes in protein structure upon adsorption must be identified and controlled to mitigate loss of biological function. We hypothesized that the installation of thiol functional groups on a protein will facilitate robust adsorption to gold nanoparticles (AuNPs) and prevent protein unfolding to achieve thermodynamic stability. Here we investigated the adsorption behavior of α-chymotrypsin (ChT) and a thiolated analog of α-chymotrypsin (T-ChT) with AuNPs. ChT, which does not present any free thiols, was modified with 2-iminothiolane (Traut's reagent) to synthesize T-ChT consisting of two free thiols. Protein adsorption to AuNPs was monitored with dynamic light scattering and UV-vis spectrophotometry, and fluorescence spectra were acquired to assess changes in protein structure induced by interaction with the AuNP. The biological function of ChT, T-ChT, and respective bioconjugates were compared using a colorimetric enzymatic assay. The thiolated analog exhibited a greater affinity for the AuNP than the unmodified ChT, as determined from adsorption isotherms. The ChT protein formed a soft protein corona in which the enzyme denatures with prolonged exposure to AuNPs and, subsequently, lost enzymatic function. Conversely, the T-ChT formed a robust hard corona on the AuNP and retained structure and function. These data support the hypothesis, provide further insight into protein-AuNP interactions, and identify a simple chemical approach to synthesize robust and functional conjugates.

High-Affinity Points of Interaction on Antibody Allow Synthesis of Stable and Highly Functional Antibody-Gold Nanoparticle Conjugates.

Many emerging nanobiotechnologies rely on the proper function of proteins immobilized on gold nanoparticles. Often, the surface chemistry of the AuNP is engineered to control the orientation, surface coverage, and structure of the adsorbed protein to maximize conjugate function. Here, we chemically modified antibody to investigate the effect of protein surface chemistries on adsorption to AuNPs. A monoclonal anti-horseradish peroxidase IgG antibody (anti-HRP) was reacted with N-succinimidyl acrylate (NSA) or reduced dithiobissuccinimidyl propionate (DSP) to modify lysine residues. Zeta potential measurements confirmed that both chemical modifications reduced the localized regions of positive charge on the protein surface, while the DSP modification incorporated additional free thiols. Dynamic light scattering confirmed that native and chemically modified antibodies adsorbed onto AuNPs to form bioconjugates; however, adsorption kinetics revealed that the NSA-modified antibody required significantly more time to allow for the formation of a hard corona. Moreover, conjugates formed with the NSA-modified antibody lost antigen-binding function, whereas unmodified and DSP-modified antibodies adsorbed onto AuNPs to form functional conjugates. These results indicate that high-affinity functional groups are required to prevent protein unfolding and loss of function when adsorbed on the AuNP surface. The reduced protein charge and high-affinity thiol groups on the DSP-modified antibody enabled pH-dependent control of protein orientation and the formation of highly active conjugates at solution pHs (<7.5) that are inaccessible with unmodified antibody due to conjugate aggregation. This study establishes parameters for protein modification to facilitate the formation of highly functional and stable protein-AuNP conjugates.

Probing the Mechanism of Antibody-Triggered Aggregation of Gold Nanoparticles.

The unique physicochemical properties of gold nanoparticles (AuNPs) provide many opportunities to develop novel biomedical technologies. The surface chemistry of AuNPs can be engineered to perform a variety of functions, including targeted binding, cellular uptake, or stealthlike properties through the immobilization of biomolecules, such as proteins. It is well established that proteins can spontaneously adsorb onto AuNPs, to form a stable and functional bioconjugate; however, the protein-AuNP interaction may result in the formation of less desirable protein-AuNP aggregates. Therefore, it is imperative to investigate the protein-AuNP interaction and elucidate the mechanism by which protein triggers AuNP aggregation. Herein, we systematically investigated the interaction of immunoglobulin G (IgG) antibody with citrate-capped AuNPs as a function of solution pH. We found that the addition of antibody triggers the aggregation of AuNPs for pH < 7.5, whereas a monolayer of antibody adsorbs onto the AuNP to form a stable bioconjugate when the antibody is added to AuNPs at pH ≥ 7.5. Our data identifies electrostatic bridging between the antibody and the negatively charged AuNPs as the mechanism by which aggregation occurs and rules out protein unfolding and surface charge depletion as potential causes. Furthermore, we found that the electrostatic bridging of AuNPs is reversible within the first few hours of interaction, but the protein-AuNP interactions strengthen over 24 h, after which the protein-AuNP aggregate is irreversibly formed. From this data, we developed a straightforward approach to acrylate the basic residues on the antibody to prevent protein-induced aggregation of AuNP over a wide pH range. The results of this study provide additional insight into antibody-nanoparticle interactions and provide a pathway to control the interaction with the potential to enhance the conjugate function.

Rapid vertical flow immunoassay on AuNP plasmonic paper for SERS-based point of need diagnostics.

SERS based immunoassays for point-of-care diagnostics are a promising tool to facilitate biomarker detection for early disease diagnosis and disease control. The technique is based on a sandwiched system in which antigen is first captured by a selective plasmonic paper substrate and then labeled by an extrinsic Raman label (ERL), consisting of a 60 nm gold nanoparticle (AuNP) functionalized with a mixed monolayer of detection antibody and 4-nitrobenezenethiol (NBT) as a Raman reporter molecule. Here, we report on the use of AuNP modified filter paper as a novel capture membrane in a vertical flow format. This vertical flow configuration affords reproducible flow of sample and label through the capture substrate to overcome diffusion limited kinetics and significantly reduced assay time. The filter paper was selected due to its affordability and availability, while the embedded AuNPs maximized plasmonic coupling with the ERLs and SERS enhancement. Additionally, the embedded AuNP served as a scaffold to immobilize capture antibody to specifically bind antigen. In this work, a SERS-based rapid vertical flow (SERS-RVF) immunoassay for detection of mouse IgG was developed to establish proof-of-principle. Optimization of assay conditions led to a limit of detection of 3 ng/mL, which is comparable to more traditional formats carried out in multi-well plates, and significantly reduced assay time to less than 2 min. Additionally, IgG was accurately quantified in normal mouse serum to validate the SERS-RVF assay for application to the analysis of biological samples. These results highlight the potential advantages of the SERS-RVF platform for point-of-need testing.

Role of Free Thiol on Protein Adsorption to Gold Nanoparticles.

Protein-gold nanoparticle (AuNP) bioconjugates have many potential applications in nanomedicine. A thorough understanding of the interaction between the protein and the AuNP is critical to engineering these functional bioconjugates with desirable properties. In this work, we investigate the role of free thiols presented by the protein on the stability of the protein-AuNP conjugate. Human serum albumin (HSA) was modified with 2-iminothiolane (Traut's reagent) to introduce additional thiols onto the protein surface, and three variants of HSA were synthesized to present 1, 5, and 20 free thiols by controlling the molar excess of the chemical modifier. Protein exchange studies on AuNPs were conducted using these HSA species and an IgG antibody which exhibited 10 free thiols. Antibody-AuNP conjugates were synthesized, purified, and dispersed in solutions containing each of the HSA species. No protein exchange was detected with the HSA or modified HSA containing 5 thiols; however, 85% of the antibody was displaced on the AuNP surface by the extensively thiolated HSA presenting 20 free thiols. Furthermore, the impact of the protein adsorption sequence was probed in which each of the HSA species were preadsorbed onto the AuNP and dispersed in a solution of antibody. The antibody fully displaced the HSA with a single thiol from the AuNP within 3 h, required 24 h to completely displace the modified HSA containing 5 thiols, and was unable to displace the modified HSA containing 20 thiols. These results indicate that the number of Au-S interactions governs the binding interaction between the protein and the AuNP. This work provides further insight into the protein-AuNP binding mechanism and identifies important design principles for engineered proteins to optimize bioconjugates.

Integrating SERS and PSI-MS with Dual Purpose Plasmonic Paper Substrates for On-Site Illicit Drug Confirmation.

Forensic laboratory backlogs are replete with suspected drug samples. Shifting analysis toward the point of seizure would save significant time and public funds. Moreover, a two-tiered identification strategy for controlled substance testing that relies on two independent, discerning methods could entirely circumvent the need for forensic laboratory testing. To this end, we coupled Raman spectroscopy and paper spray ionization mass spectrometry (PSI-MS) on a single instrumental platform. Both methods are capable of ambient analysis with fieldable instruments, yet Raman is often limited to bulk analysis. Critical to this work is the development of a gold nanoparticle (AuNP)-embedded paper swab to extend the capability of Raman spectroscopy to trace evidence via surface-enhanced Raman scattering (SERS). Plasmonic papers are characterized with respect to SERS signals and compatibility with PSI-MS analysis. Proof-of-principle is established with the identification of five representative drugs, and detection limits on the scale of 1-100 ng are achieved for both PSI-MS and SERS. The integrated SERS-PSI-MS system achieved 99.8% accurate chemical identification in a blind study consisting of 500 samples. Additionally, we demonstrate facile discrimination of several JWH-018 isomers via SERS even when MS and MS2 spectra are indistinguishable. Successful coupling of SERS and PSI-MS to enable on-site chemical analysis by two independent methods can potentially lead to a desirable paradigm shift in the handling of drug evidence.

Antibodies Irreversibly Adsorb to Gold Nanoparticles and Resist Displacement by Common Blood Proteins.

Gold nanoparticles (AuNPs) functionalized with proteins to impart desirable surface properties have been developed for many nanobiotechnology applications. A strong interaction between the protein and nanoparticle is critical to the formation of a stable conjugate to realize the potential of these emerging technologies. In this work, we examine the robustness of a protein layer adsorbed onto gold nanoparticles while under the stress of a physiological environment that could potentially lead to protein exchange on the nanoparticle surface. The adsorption interaction of common blood plasma proteins (transferrin, human serum albumin, and fibrinogen) and anti-horseradish peroxidase antibody onto AuNPs is investigated by nanoparticle tracking analysis. Our data show that a monolayer of protein is formed at saturation for each protein, and the maximum size increase for the conjugate, relative to the AuNP core, correlates with the protein size. The binding affinity of each protein to the AuNP is extracted from a best fit of the adsorption isotherm to the Hill equation. The antibody displays the greatest affinity (Kd = 15.2 ± 0.8 nM) that is ∼20-65 times stronger than the affinity of the other plasma proteins. Antibody-AuNP conjugates were prepared, purified, and suspended in solutions of blood plasma proteins to evaluate the stability of the antibody layer. An enzyme-mediated assay confirms that the antibody-AuNP interaction is irreversible, and the adsorbed antibody resists displacement by the plasma proteins. This work provides insight into the capabilities and potential limitations of antibody-AuNP-enabled technologies in biological systems.

pH Impacts the Orientation of Antibody Adsorbed onto Gold Nanoparticles.

Novel detection strategies that exploit the unique properties of gold nanoparticles (AuNPs) hold great promise for the advancement of diagnostic testing. Fundamental to many of these nanoparticle-enabled techniques is the immobilization of antibodies onto the AuNP surface to afford selective binding to target species. Orientation and loading density of the immobilized antibodies govern Fab accessibility and are critical to the analytical performance. Here, we use pH to systematically control the surface charge distribution on an antibody and investigate the impact of protein charge on adsorption to AuNPs. Nanoparticle tracking analysis (NTA) is used to measure the adsorption dynamics of anti-horseradish peroxidase antibody (anti-HRP) onto AuNPs at different pHs. NTA enables in situ measurement of antibody adsorption on AuNP by measuring the increase in hydrodynamic diameter ( DH) of the AuNPs as a function of antibody concentration. The adsorption affinity, protein layer thickness, and binding cooperativity at each pH are extracted from the best fit of the adsorption isotherms to the Hill-modified Langmuir equation. Our data show a monolayer of antibody is formed at saturation at pHs 7.5, 8.0, and 8.5, and no difference in anti-HRP-AuNP binding constants is observed in this pH range ( Kd ∼11 nM). However, the increase in DH of the AuNPs with adsorbed protein at monolayer coverage is pH-dependent, measuring 13.2 ± 1.1 nm, 9.8 ± 1.0 nm, and 7.4 ± 0.6 nm for pHs 7.5, 8.0, and 8.5, respectively. Moreover, results of an enzyme-mediated assay reveal the antigen-binding capacity of the immobilized anti-HRP antibody is 33 ± 2%, 23 ± 7%, and 18 ± 2% when adsorbed at pHs 7.5, 8.0, and 8.5, respectively. Our data confirm that antibody charge can be altered with pH to modulate and optimize antibody orientation on AuNP. These studies describe our continued efforts to establish design criteria to prepare conjugates with maximum antigen-binding activity that will enhance the performance of biofunctional nanomaterials.

Sandwiching analytes with structurally diverse plasmonic nanoparticles on paper substrates for surface enhanced Raman spectroscopy.

This report describes the systematic combination of structurally diverse plasmonic metal nanoparticles (AgNPs, AuNPs, Ag core-Au shell NPs, and anisotropic AuNPs) on flexible paper-based materials to induce signal-enhancing environments for surface enhanced Raman spectroscopy (SERS) applications. The anisotropic AuNP-modified paper exhibits the highest SERS response due to the surface area and the nature of the broad surface plasmon resonance (SPR) neighboring the Raman excitation wavelength. The subsequent addition of a second layer with these four NPs (e.g., sandwich arrangement) leads to the notable increase of the SERS signals by inducing a high probability of electromagnetic field environments associated with the interparticle SPR coupling and hot spots. After examining sixteen total combinations, the highest SERS response is obtained from the second layer with AgNPs on the anisotropic AuNP paper substrate, which allows for a higher calibration sensitivity and wider dynamic range than those of typical AuNP-AuNP arrangement. The variation of the SERS signals is also found to be below 20% based on multiple measurements (both intra-sample and inter-sample). Furthermore, the degree of SERS signal reductions for the sandwiched analytes is notably slow, indicating their increased long-term stability. The optimized combination is then employed in the detection of let-7f microRNA to demonstrate their practicability as SERS substrates. Precisely introducing interparticle coupling and hot spots with readily available plasmonic NPs still allows for the design of inexpensive and practical signal enhancing substrates that are capable of increasing the calibration sensitivity, extending the dynamic range, and lowering the detection limit of various organic and biological molecules.

Quantifying Bound and Active Antibodies Conjugated to Gold Nanoparticles: A Comprehensive and Robust Approach To Evaluate Immobilization Chemistry.

Gold nanoparticles (AuNPs) functionalized with antibodies have the potential to improve biosensing technology because of the unique optical properties of AuNPs and the specificity of antibody-antigen interactions. Critical to the development and optimization of these AuNP-enabled sensing technologies is the immobilization of the antibody onto the AuNP. The development of novel immobilization strategies that optimize antibody loading and orientation in an effort to enhance antibody activity, and therefore assay performance, has been the focus of many recent studies. However, few analytical methods exist to accurately quantify the activity of conjugated antibodies and reliably compare different immobilization strategies. Herein, we describe an enzyme-mediated assay to quantify the fraction of the immobilized antibodies that is accessible for antigen binding. Anti-horseradish peroxidase (anti-HRP) antibody is mixed with AuNPs to allow for conjugation, and the unbound, excess antibody is quantified with a modified Bradford assay to determine antibody loading onto AuNPs. The conjugates are then mixed with excess HRP to saturate all accessible binding sites, and bound HRP is quantified based on enzymatic reaction rate. This analytical scheme was used to compare two common immobilization strategies, nonspecific adsorption and protein A-mediated immobilization. We found that the antibody surface coverage is greater for direct adsorption than protein A-mediated binding; however, 23 ± 6% of the directly adsorbed antibodies were active, whereas 91 ± 19% of the antibodies bound through protein A were active. In addition to establishing this method as quantitatively precise and accurate, our results emphasize the need to quantify both antibody loading and antibody activity upon conjugation to gain greater insight into differences in immobilization chemistries and identify optimum protein conjugation strategies to maximize immunoassay performance.

Chemical modification of antibodies enables the formation of stable antibody-gold nanoparticle conjugates for biosensing.

Antibody-modified gold nanoparticles (AuNPs) are central to many novel and emerging biosensing technologies due to the specificity provided by antibody-antigen interactions and the unique properties of nanoparticles. These AuNP-enabled assays have the potential to provide significant improvements in sensitivity and multiplexed analysis compared to conventional immunoassays. However, a major challenge for these AuNP platform technologies is the synthesis of stable antibody-AuNP conjugates that resist aggregation in high salt environments and biological matrices. Moreover, synthetic strategies to form stable conjugates often require different solution conditions, e.g., pH, for each unique antibody. Herein we describe our effort to develop an approach to chemically modify lysine residues on antibodies to facilitate the formation of stable antibody-AuNP conjugates over a wide pH range. In this work, we systematically investigated the immobilization of native and chemically modified antibodies to 60 nm citrate-capped AuNPs as a function of pH and evaluated the stability of the antibody-AuNP conjugate in a saline environment. We have developed a method to chemically modify the lysine residues on an antibody prior to conjugation to the AuNP that results in stable conjugates over a wide pH range (6.0-8.5). Amino acid analysis and zeta potential measurements of native and modified antibodies reveal that the requisite modification correlates with the number of lysine residues, and a reduction in positive charge contribution from protonated lysine is required to form stable, pH-independent conjugates. Furthermore, we demonstrate that the chemically modified antibodies maintain antigen-binding capabilities. We apply this novel conjugation strategy to develop a surface-enhanced Raman spectroscopy (SERS)-based assay for the accurate subtyping of avian influenza viruses.

Structural characterization and photoluminescence in the rare earth-free oxy-fluoride anti-perovskites Sr3-xBi2x/3AlO4F and Sr3-xBi2x/3GaO4F.

The activator Bi3+ has been successfully incorporated into the anti-perovskite oxy-fluoride host lattice Sr3MO4F (M = Al, Ga) to form rare earth-free phosphors of the composition Sr3-xBi2x/3AlO4F, 0 ≤ x ≤ 0.1, and Sr3-xBi2x/3GaO4F, 0 ≤ x ≤ 0.048. These phases absorb in the UV region (λex = 240-326 nm) and exhibit broad emission in the blue region of the visible spectrum (λem = 446.5-455 nm). The optimum compositions for maximum photoluminescent intensity were determined to be Sr2.976Bi0.016AlO4F and Sr2.976Bi0.016GaO4F before concentration quenching occurs. Full structural characterization based upon PXRD and NPD data were performed with DFT calculations suggesting that Bi3+ ions are preferentially incorporated on the ten coordinate Sr(1) site.

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.

A fluorescence-based method to directly quantify antibodies immobilized on gold nanoparticles.

The ability to evaluate antibody immobilization onto gold nanoparticles is critical for assessing coupling chemistry and optimizing the sensitivity of nanoparticle-enabled biosensors. Herein, we developed a fluorescence-based method for directly quantifying antibodies bound onto gold nanoparticles. Antibody-modified gold nanoparticles were treated with KI/I2 etchant to dissolve the gold nanoparticles. A desalting spin column was used to recover the antibody released from the nanoparticles, and NanoOrange, a fluorescent dye, was used to quantify the antibody. We determined 309 ± 93 antibodies adsorb onto a 60 nm gold nanoparticles (2.6 × 10(10) NP mL(-1)), which is consistent with a fully adsorbed monolayer based on the footprint of an IgG molecule. Moreover, the increase in hydrodynamic diameter of the conjugated nanoparticle (76 nm) compared to that of the unconjugated nanoparticle (62 nm) confirmed that multilayers did not form. A more conventional method of indirectly quantifying the adsorbed antibody by analysis of the supernatant overestimated the antibody surface coverage (660 ± 87 antibodies per nanoparticle); thus we propose the method described herein as a more accurate alternative to the conventional approach.

SERS immunoassay based on the capture and concentration of antigen-assembled gold nanoparticles.

A simple, rapid, and sensitive immunoassay has been developed based on antigen-mediated aggregation of gold nanoparticles (AuNP) and surface-enhanced Raman spectroscopy (SERS). Central to this platform is the extrinsic Raman label (ERL), which consists of a gold nanoparticle modified with a mixed monolayer of a Raman active molecule and an antibody. ERLs are mixed with sample, and antigen induces the aggregation of the ERLs. A membrane filter is then used to isolate and concentrate the ERL aggregates for SERS analysis. Preliminary work to establish proof-of-principle of the platform technology utilized mouse IgG as a model antigen. The effects of membrane pore diameter and AuNP size on the analytical performance of the assay were systematically investigated, and it was determined that a pore diameter of 200 nm and AuNP diameter of 80 nm provide maximum sensitivity while minimizing signal from blank samples. Optimization of the assay provided a detection limit of 1.9 ng/mL, 20-fold better than the detection limit achieved by an ELISA employing the same antibody-antigen system. Furthermore, this assay required only 60 min compared to 24h for the ELISA. To validate this assay, mouse serum was directly analyzed to accurately quantify IgG. Collectively, these results demonstrate the potential advantages of this technology over current diagnostic tests for protein biomarkers with respect to time, simplicity, and detection limits. Thus, this approach provides a framework for prospective development of new and more powerful tools that can be designed for point-of-care diagnostic or point-of-need detection.

Effect of hydration on plasmonic coupling of bioconjugated gold nanoparticles immobilized on a gold film probed by surface-enhanced Raman spectroscopy.

Gold nanoparticle (AuNP)-Au film constructs were prepared using antibody-antigen interactions or a small organic cross-linker to systematically control the gap between the AuNP and Au film. Surface-enhanced Raman spectroscopy (SERS), scanning electron micrsocopy (SEM), and atomic force microscopy (AFM) were used to characterize each construct and elucidate structure-activity relationships. Interestingly, plasmonic coupling and SERS intensity were reversibly modulated with wetting/drying cycles for the protein immobilized AuNP, and this effect was attributed to changes in protein size with hydration state. This work provides insight into fundamental limitations of AuNP-enabled SERS bioassays and will facilitate rational design of novel biospecific ligands that maximize SERS sensitivity.