pH Impacts the Orientation of Antibody Adsorbed onto Gold

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pH Impacts the Orientation of Antibody Adsorbed onto Gold Nanoparticles Guadalupe Ruiz, Kiran Tripathi, Samuel Okyem, and Jeremy D. Driskell Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00123 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 9, 2019

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Bioconjugate Chemistry

pH Impacts the Orientation of Antibody Adsorbed onto Gold Nanoparticles Guadalupe Ruiz‡, Kiran Tripathi‡, Samuel Okyem, and Jeremy D. Driskell* Department of Chemistry, Illinois State University, Normal, IL 61790

ABSTRACT

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

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in anti-HRP-AuNP binding constants are 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, 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.

INTRODUCTION Gold nanoparticles (AuNPs) are an integral part of numerous emerging technologies that aim to meet the increasingly stringent demands placed on diagnostic and clinical tests that are needed for advancements in medicine. Central to the success of these AuNP-enabled technologies is effective surface modification to impart selective binding to specific analytes in diagnostic applications or tissues for imaging and drug delivery. The analytical performance of these assays is governed by the loading density and orientation of the immobilized antibodies, and several previous efforts have demonstrated that the antigen-binding capacity correlates with the number of accessible Fab domains.1-7 Consequently, there is an ongoing effort in search of an optimal immobilization protocol.8-14 Despite extensive efforts to optimize antibody immobilization, a single universally accepted strategy that affords maximum performance has not been identified. The most common approaches to immobilize antibody onto gold nanoparticles include covalent coupling strategies. These strategies often utilize EDC/NHS chemistry15-17 or a


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heterobifunctional crosslinker, such as 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) or 3,3'-dithiobis(succinimidyl propionate) (DSP),11, 18-27 to target the primary amine of lysine residues that are ubiquitous to protein and a terminal thiol for covalent attachment to AuNPs. While covalent coupling results in the formation of stable and robust conjugates, these covalent coupling strategies do not address the need to control the orientation of the immobilized antibody; thus, alternative approaches have been developed to confer proper orientation.2, 10, 12-14, 28-30 Coupling to carbohydrates on the Fc portion of IgG antibodies is one strategy to effectively direct the antigen binding Fab fragment away from the AuNP surface.3, 31, 32 Antibodies can be engineered to contain modifications such as histidine tags, biotin, and “click” chemistries, at site-specific locations to control orientation upon immobilization. 38, 42-44 While effective, these current strategies to control antibody orientation increase the complexity of synthesizing the conjugates by adding additional steps or requiring advanced protein engineering. Despite significant efforts to develop a universal, robust, and controlled coupling chemistry, direct adsorption of antibody onto AuNPs is still routinely used.33-38 Interest in protein-AuNP interactions has been renewed with the significant role of protein coronas in nanoparticle-enabled biotechnologies. Recently, protein-AuNP interactions have been systematically studied to elucidate fundamental mechanisms and provide insight into the formation of protein coronas. Evidence from those works concluded that (1) long-range electrostatic interactions drive the initial interaction10, 12, 39-43 and (2) once in contact, S-Au covalent bonds form between cysteine residues and the AuNP.44, 45 Collectively, those reports suggest that antibody charge can be modulated to impact the region of the protein that is attracted to the AuNP to effectively optimize orientation,10, 12, 41 and robust conjugates can be formed without the need for a covalent linker to mediate immobilization.34, 37, 44 Moreover, direct adsorption, which can result


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in multiple cysteine-Au bonds for large proteins such as IgG antibodies, may lead to a higher affinity interaction and a more robust/stable conjugate than is afforded by covalent strategies relying on linkers that bind through a single thiol moiety.44 Here we systematically investigate antibody adsorption and orientation on AuNPs to characterize the impact of solution pH on antibody-AuNP interactions. Elucidating the mechanistic details of protein adsorption onto nanoparticles is a challenging analytical measurement. Many methods used to study protein-nanoparticle interactions, such as radio-labelled proteins,46 spectrophotometry to measure shifts in plasmon resonance,43, 47, 48 and dynamic light scattering (DLS) to measure hydrodynamic diameter,2, 60-63 require separation of unbound protein form the conjugate prior to analysis, e.g., ex situ, and are therefore not truly a measure of the system at equilibrium. Alternatively, fluorescence correlation spectroscopy (FCS)41,

49

and differential

centrifugal sedimentation (DCS)50-53 have been introduced for the in situ analysis of protein adsorption on nanoparticles; however, each of these techniques have limitations. Fluorescent labeling of proteins in FCS prevents adsorption analysis of the native protein and DCS requires user input of the effective density of the conjugate, which is difficult to know precisely because the density is a composite of the densities of the core material and protein shell. In this work, we use nanoparticle tracking analysis (NTA) to quantitatively assess antibody adsorption onto AuNP as a function of pH. NTA is unique in that it allows analysis of the native unlabeled protein, provides a high-resolution measure of the adsorbed protein layer thickness to give insight into orientation, and does not require the separation of unbound proteins for in situ analysis under equilibrium conditions. After assessment of adsorption via NTA, variations in orientation of the immobilized antibody with respect to pH is further assessed by comparing the number of accessible antigen-binding sites using an enzyme-mediated assay previously developed by our


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lab54 and confirmed using a secondary antibody specific to the Fab fragment of the adsorbed antibody.55 RESULTS AND DISCUSSION pH-Dependent Charge of Antibody. It is well established that immunoglobulin G (IgG) antibodies readily adsorb onto citrate-capped gold nanoparticles.56,

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Electrostatic interactions

between localized regions of positive charge on the antibody with the negatively charged AuNP are largely responsible for driving the adsorption, followed by chemisorption via cysteine residues.42, 44, 45, 58, 59 While the net charge of an IgG molecule can be negative, basic amino acid residues such as lysine and arginine that reside on the surface of the protein can be protonated to carry a positive charge at certain pHs, giving rise to localized regions of positive charge. Thus, the location of the positive patches controls the orientation of the adsorbed antibody. Previously, chemical modification of human serum albumin to manipulate protein surface charge has been shown to impact adsorption and control orientation when adsorbed on quantum dots.41 In this work, we used pH to protonate/deprotonate basic amino acids, thereby altering the localized regions of positive charge on the antibody. The surface charges for an IgG molecule were calculated using molecular simulations.60 An intact monoclonal antibody, subclass IgG1, is used as a model IgG protein because its structure has been fully characterized and is available in the Protein Data Bank (PDB 1IGY). The impact of solution pH on protein surface charge is presented in Figure 1. At pH 7.5, the IgG has the greatest number of surface regions with a positive charge as the basic amino acids are protonated to a greater extent. As the pH increases, the degree to which the basic amino acids are protonated decreases and the number of localized positive regions on the IgG molecule decreases. The


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sequence and structure of the antibody used in our experimental studies presented below is not available; thus, the location of positive and negative regions on the antibody used in the experimental adsorption studies will likely differ from those localized regions on the antibody presented in Figure 1. Nevertheless, the antibody analyzed in Figure 1 serves as a model to demonstrate that protein surface charge varies in this pH range. Based on the results of these calculated surface charges, we hypothesized that the orientation of an antibody adsorbed onto a gold nanoparticle will depend on the solution pH; therefore, pH can be used to control the antigenbinding capacity of antibody-AuNP conjugates.

Figure 1. The surface charges calculated for mouse IgG1 antibody (PDB code 1IGY) at three different pHs. The blue represents positive potential and the red represents negative potential, with a range from -5 kbT/e to +5 kbT/e. The calculations were performed online at http://nbcr222.ucsd.edu/pdb2pqr/.60 Anti-horseradish peroxidase monoclonal antibody (anti-HRP mAb) was prepared in a buffer adjusted to pH 7.5, 8.0, and 8.5. The pH was limited to this range because gold nanoparticles aggregated with the addition of antibody at pHs below 7.5 or greater than 8.5 during antibody adsorption studies. As the pH increases from 7.5 to 8.5, a greater number of solvent accessible basic amino acids are deprotonated, reducing the number of positive patches on the mAb, affecting


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binding and orientation when adsorbed onto AuNPs. As expected, the zeta potential becomes more negative as the pH becomes more basic, measuring -16.2 ± 1.4 mV, -19.8 ± 0.4 mV, and -25.8 ± 1.9 mV at pH 7.5, 8.0, and 8.5, respectively. The hydrodynamic diameter of the mAb at each pH was measured by dynamic light scattering (DLS), resulting in 12.3 ± 0.3 nm, 11.6 ± 0.2 nm, and 13.6 ± 0.4 nm at pH 7.5, 8.0, and 8.5, respectively. The mAb size does not significantly differ as a function of pH; thus, we conclude that the mAb does not denature within this pH range and remains properly folded. These data confirm that solution pH is a suitable means to systematically control the surface charge of the mAb in a predictable manner. Analysis of Antibody Adsorption onto Gold Nanoparticles. To investigate the impact of antibody charge on interactions with AuNPs, we use NTA to quantitatively assess the adsorption of anti-HRP antibody onto AuNPs at various pHs. NTA collects scattered light from individual nanoparticles to measure Brownian motion and to determine the hydrodynamic diameter of each visualized particle. NTA can provide a measurement of AuNP hydrodynamic diameter with subnanometer precision and without bias toward larger particles, as is often the case with dynamic light scattering (DLS).59, 61 NTA is not sensitive to proteins due to insufficient scattering, and therefore, does not require removal of excess, unbound antibodies for accurate analysis of the conjugate. In situ analysis allows for accurate measurement of adsorption dynamics under true equilibrium conditions. Moreover, NTA does not require labeling of the antibody, which alters the antibody interaction with AuNP. In our previous works, we have established NTA provides the requisite size resolution to directly monitor the evolution of antibody adsorption as an increase in hydrodynamic diameter.61


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Figure 2. Adsorption of anti-HRP mAb onto 60 nm AuNPs. (A) Histograms of hydrodynamic diameters measured for conjugates prepared with varying amounts of mAb at pH 7.5. (B) Plots of the mean hydrodynamic diameter of the conjugate as a function of mAb concentration for the adsorption at pHs 7.5, 8.0, and 8.5. The solid curves represent the best fit to the Hill equation (Equation 1). Figure 2A shows the size distributions for unconjugated AuNPs and AuNPs incubated with antiHRP antibody ranging from 3-310 nM (0.5-50 g/mL) at pH 7.5. As expected, the mean hydrodynamic diameter increases with increasing antibody concentration, until reaching a maximum size that indicates the formation of a monolayer on the AuNP surface. The histograms in Figure 2A show that the AuNP conjugates are highly monodisperse and no detectable aggregates are formed as a result of protein adsorption. Similar size-based histograms are generated for antibody adsorption at pH 8.0 and 8.5 (Figure S1). We note that slight aggregation of the AuNPs at pH 7.5 is occasionally detected when incubated with 3.1 nM antibody, and consistently detected when incubated with 6.2 nM antibody at pH 8.0. It has been demonstrated that proteins with positive charges on opposing sides of the macromolecule act as a bridge to electrostatically


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crosslink two negatively charged gold nanoparticles, e.g., citrate-capped AuNP. 62-64 Thus, at lower pHs, antibodies will carry more positive charge and may trigger AuNP aggregation. Consequently, size measurements for 3.1 nM mAb at pH 7.5 that exhibit aggregation and 6.2 nM mAb at pH 8.0, are not included in subsequent data analysis since it does not accurately reflect the mean hydrodynamic diameter of single AuNP-antibody conjugates. Nevertheless, sufficient adsorption data for a wide range of antibody concentrations that does not induce aggregation is collected to fully and accurately characterize mAb adsorption behavior. Adsorption isotherms are generated at pH 7.5, 8.0, and 8.5 to quantitatively evaluate the impact of protein charge on adsorption behavior. The mean hydrodynamic diameter measured by NTA is plotted as a function of antibody concentration and best-fit to the Hill equation,49, 53, 65, 66

𝐷𝐻 = 𝐷𝐻,𝑖𝑛𝑖𝑡𝑖𝑎𝑙 +

∆𝐷𝐻,𝑚𝑎𝑥[𝑎𝑛𝑡𝑖𝑏𝑜𝑑𝑦]𝑛 𝐾𝑑𝑛 + [𝑎𝑛𝑡𝑖𝑏𝑜𝑑𝑦]𝑛

(1)

where DH is the hydrodynamic diameter, DH,max is the maximum increase in hydrodynamic diameter resulting from a monolayer of adsorbed antibody, n is the Hill coefficient, and Kd is the dissociation constant for antibody-AuNP adsorption (Figure 2B). The Hill-modified Langmuir equation has been previously established and is widely employed to describe the interaction between proteins and nanoparticles which approach monolayer coverage under equilibrium conditions.49, 53, 65, 66 Parameters derived from the best-fit of the experimental data to the Hill equation allows for the determination of maximum size increase (DH, max), which provides insight into the orientation of the adsorbed antibody, a quantitative measure of antibody-AuNP adsorption affinity (Kd), and an estimate of binding cooperativity (n). It should be noted that the antibody concentration used to generate the binding curves refers to the added antibody concentration, while Equation 1 more accurately refers to the free antibody remaining in solution once equilibrium is


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established. However, under our experimental conditions, which include dilute nanoparticles (~40 pM), the more experimentally accessible value for total added antibody can be used to approximate the free antibody concentration in the Hill equation.66 Table 1. Adsorption parameters determined from best-fit of NTA adsorption isotherm to HillLangmuir model. Values represent the average and standard deviation for the analysis of three adsorption datasets at each pH.

pH

DH,max (nm)

Kd (nM)

n

7.5 8.0 8.5

13.2 ± 1.1 9.8 ± 1.0 7.4 ± 0.6

11 ± 3 11 ± 3 12 ± 2

0.7 ± 0.1 1.0 ± 0.3 1.3 ± 0.3

Table 1 summarizes the characteristic binding parameters extracted from the adsorption isotherms presented in Figures 2B. Most notably, the thickness of the mAb monolayer differs with pH. At pH 7.5, the protein monolayer increases the DH by 13.2 ± 1.0 nm relative to the unconjugated AuNP, while the DH increases by 9.8 ± 1.0 nm and 7.4 ± 0.6 nm for mAb adsorption at pH 8.0 and 8.5, respectively. As determined above, the hydrodynamic diameter of the free mAb is independent of pH, therefore it can be concluded that the mAb layer thickness is due to protein orientation rather than changes in protein size with pH. The mAb used in this work is an immunoglobulin G (IgG) protein that has a characteristic y-shape with dimensions of 14 × 8.5 × 4.5 nm.67 Thus, the mAb monolayer thickness depends on the mAb orientation, and ranges from a theoretical minimum DH,max of ~9 nm for flat-on orientation to a maximum DH,max of ~28 nm for end-on or head-on orientations (Figure 3). The protein monolayer thickness measured in our experiments suggests a mix of orientations rather than a single orientation at one extreme or the other. However, a clear difference in the DH for the protein monolayers at each pH is observed


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and suggests a repeatable, preferred orientation based on protein charge. Consequently, these differences in mAb orientation could impact the accessibility of the Fab region of the adsorbed mAb to enhance or inhibit antigen-binding activity.

Figure 3. Illustration of the theoretical hydrodynamic diameters of AuNPs saturated with a monolayer of mAb adsorbed with different orientations. Analysis of the adsorption isotherm data with the Hill equation also reveals the adsorption affinity of the mAb for the AuNP is not significantly impacted by the protein charge. We observe Kd values of 11 ± 3, 11 ± 3, and 12 ± 2 nM for the interaction of anti-HRP mAb with AuNP at pH 7.5, 8.0, and 8.5 (Table 1). These values confirm a strong interaction between the mAb and AuNP and are consistent with Kd values reported in literature for IgG-AuNP.48 Hill coefficients were calculated as 0.7 ± 0.1, 1.0 ± 0.3, and 1.3 ± 0.3 for mAb adsorption at pH 7.5, 8.0, and 8.5, respectively (Table 1). A clear trend is found in which the anti-cooperative binding is observed at pH 7.5, and the mAb exhibits more cooperative binding behavior as the pH increases. While detailed interpretation of the Hill coefficient is not possible, we can speculate that at the lower pH the mAb binds in such an orientation that the more abundant regions of localized positive charge inhibit the binding of subsequent molecules through repulsive electrostatic forces. Conversely, at a more basic pH, fewer regions of positive charge exist on the mAb and those positive regions will be directed toward the negatively charged AuNP; thus, leaving the more neutral or negative regions


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of the molecule outward facing, that may not affect or potentially enhance adsorption of subsequent mAbs through attractive electrostatic forces. Quantifying Fab Accessibility of Adsorbed Antibody The orientation of the immobilized mAb will impact both the number of adsorbed mAbs at monolayer coverage and the fraction of the immobilized mAbs with accessible Fab fragments for antigen binding. Our group previously developed a two-tiered analytical scheme to quantify the number of immobilized mAbs and the number of accessible antigen-binding sites.54 Briefly, the number of antibodies is determined by mass difference between the antibodies added to a suspension of AuNPs and the unbound antibodies remaining in the supernatant when separated from the formed mAb-AuNP conjugates. Anti-HRP mAb was selected to form the conjugate to facilitate the measurement of antigen-binding activity of the immobilized antibody. Accessible Fab of the immobilized antibodies is then saturated with HRP, providing a straightforward means to quantify captured antigen via HRP enzymatic activity. We previously established that these two analyses, e.g., antibody loading and captured antigen, provide a quantitative measure of the fraction of accessible Fab fragments to gain insight into the orientation of the adsorbed mAb. Differences in mAb orientation upon immobilization onto AuNP will lead to differences in mAb surface coverage. The cross-sectional footprint of the antibody depends on the orientation of the adsorbed antibody. An end-on or head-on oriented mAb has a smaller footprint than a mAb in the flat-on orientation, thus, a greater number of mAbs can adsorb. A theoretical maximum surface coverage of 296 mAb/AuNP is estimated for end-on or head-on orientation of IgG using the surface area of a 60 nm AuNP, estimating the mAb cross-sectional footprint as 38 nm2 (8.5 nm × 4.5 nm), and assuming close packing of the adsorbed antibodies. Alternatively, we estimate a


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theoretical minimum loading density of 95 mAb/AuNP for flat-on orientation based on a mAb cross-sectional footprint of 119 nm2 (8.5 nm × 14 nm). Experimentally, we find that antibody surface coverages saturate at 171 ± 11 , 227 ± 35, and 240 ±8 mAb/AuNP as the pH decreases from 8.5 to 7.5. These quantities represent the average and standard deviation for >12 independent preparations of conjugates at each pH (Figure 4; Figure S2 and S3). A t-test was performed and the measured surface coverages at each pH differ with statistical significance at the 95% level, with the exception of the coverages at pHs 7.5 and 8.0. Given the similar binding affinities, e.g., Kd, measured with NTA at each pH, and saturating conditions, e.g., 30 g/mL mAb, these differences in antibody loading can be attributed to differences in orientation. Antibody loading increases as the pH decreases from 8.5 to 7.5, and these data indicate more mAbs orient in the endon or head-on orientation with decreasing pH. This result is consistent with the NTA adsorption isotherm data that established the mAb monolayer thickness increases as the pH decreases.

Figure 4. Quantitation of antibody loading on AuNP at monolayer coverage. AuNP is saturated with anti-HRP mAb. The adsorbed antibody is determined from the mass difference between the amount of mAb added to the AuNP suspension and the unbound mAb.


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NTA and antibody loading analyses establish pH-dependent orientation; however, these data do not provide insight into potential pH-dependent antigen-binding capacity for the immobilized mAbs. At pH 7.5 the data confirm that more mAbs adsorb onto the AuNP, but the protein layer thickness could indicate either end-on orientation that would enhance antigen-binding capacity or head-on orientation that would inhibit antigen-binding capacity. AuNP-mAb conjugates modified with a monolayer of anti-HRP are incubated with an excess of HRP. The HRP is allowed to bind all accessible Fab antigen-binding sites on the conjugates. HRP catalyzes the oxidation of ABTS to a colored product that can be monitored via UV-visible spectrophotometry (Figure 5A), while minimal oxidation of ABTS is observed in the absence of HRP. Anti-mIgG conjugates serve as a negative control and are incubated with excess HRP to allow non-specific capture of HRP, followed by exposure to ABTS to establish specificity of the enzymatic assay. Figure 5B shows minimal oxidation of ABTS after 15 min by the anti-mIgG conjugates, while a significant color change indicative of ABTS oxidation is observed at t = 15 min for the anti-HRP conjugates. Additional control conjugates were tested in our previous work to thoroughly validate the specificity of the enzymatic assay for anti-HRP conjugates.54 A linear relationship between the reaction rate and HRP concentration allows for the quantitative determination of HRP molecules captured by the anti-HRP conjugates (Figure S4). The anti-HRP conjugates are titrated with increasing concentrations of HRP and the data establish typical ligand binding behavior to reach saturation of antigen binding sites at HRP concentrations greater than 2 g of HRP per 100 L of conjugate (Figure S5). Based on the binding curves, 3 g of HRP are added to conjugates formed at pH 7.5, 8.0, and 8.5 to ensure saturation of all accessible Fab sites and the number of captured HRP molecules are measured. The analysis of four independent preparations of conjugates at each pH shows antigen-binding capacity increases as the pH


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decreases, measuring 63 ± 7, 104 ± 26, and 158 ± 5 HRP/AuNP for conjugates prepared at pH 8.5, 8.0, and 7.5 (Figure 5C). A t-test confirms the differences in antigen binding capacities measured at each pH are statistically significant at the 95% confidence level. While antibody loading on the AuNP increases at lower pHs, this parameter alone does not explain the large increase in antigenbinding capacity observed at lower pHs. The fraction of antigen-binding sites occupied by HRP is calculated from the captured HRP molecules and antibody loading, taking into account the divalency of each antibody. Conjugates formed at pH 7.5 are loaded with 240 ± 8 Ab/AuNP to provide a total of 480 antigen-binding sites per conjugate; however, each conjugate captured 158 ± 5 HRP molecules. Therefore, at pH 7.5, 33 ± 2% of the Fab fragments are oriented in such a way as to allow access for antigen binding (Figure 5D). At pHs 8.0 and 8.5, 23 ± 7% and 18 ± 2% of the Fab fragments are accessible and functional for the immobilized antibodies (Figure 5D). Differences in Fab accessibility was validated for each pH at the 95% confidence level using a ttest, with the exception of the binding capacity for conjugates prepared at pH 8.0 and 8.5 which differed with a confidence level of 90%. These differences in antigen-binding capacity corroborate the NTA adsorption isotherm data to confirm pH-dependent orientation of antibodies adsorbed onto AuNPs. Additionally, the antigen binding capacity enables differentiation between end-on and head-on orientation, and the data suggest greater end-on orientation at lower pHs.


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Figure 5. Quantitation of accessible Fab fragments. (A) Illustration depicting the enzymatic assay used to quantify accessible Fab fragments. Anti-HRP conjugates are saturated with HRP, excess HRP is separated from the conjugates, and ABTS is added to the conjugates. The enzymatic reaction rate at which HRP catalyzes the oxidation of ABTS quantitatively correlates to the number of HRP molecules captured by the conjugates. (B) Photograph of the colored ABTS·+ product immediately after the addition of ABTS to the HRP-saturated conjugates and after 15 min of incubation. Anti-mouse IgG conjugates serve as a negative control to assess non-specific adsorption of HRP to the conjugates. (C) The number of HRP molecules captured per anti-HRP conjugate prepared at pHs 7.5, 8.0, or 8.5. (D) The fraction of Fab fragments that remain accessible for mAbs adsorbed onto AuNPs at pH 7.5, 8.0, or 8.5.


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The orientation of the adsorbed antibody is further evaluated using a recently described aggregation-based assay by Zheng et al.55 The hydrodynamic diameter of the anti-HRP conjugate (D1) is measured by dynamic light scattering (DLS). Anti-mouse IgG Fab-specific antibodies are then added to the conjugates and again the hydrodynamic diameter (D2) is measured via DLS. The anti-Fab antibody induces the crosslinking of conjugates in which Fab fragments are oriented towards the solution, resulting in a large value for D2. It was previously established that a greater value for D2 correlates with a greater number of accessible Fab. The ratio of D2/D1 is calculated as the test score and is presented as a semi-quantitative measure of aggregation, e.g., accessible Fab (Figure 6A). The greatest aggregation is observed for conjugates formed at pH 7.5, with minimal aggregation detected for conjugates formed at pH 8.5. Conjugates formed at pHs 7.5, 8.0, and 8.5 yield nanoparticle test scores of 1.50 ± 0.03, 1.31 ± 0.02, and 1.26 ± 0.03, respectively (Figure 6B). This aggregation-based assay provides further evidence that antibody adsorption onto AuNP at pH 7.5 results in an orientation that presents a greater number of accessible Fab antigen-binding sites.


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Figure 6. An aggregation-based assay to assess orientation of mAb adsorbed onto AuNPs. (A) Illustration of the assay. Anti-HRP conjugates are prepared and the hydrodynamic diameter is recorded (D1). Fab-specific antibody is added to the conjugates allowing for crosslinking of the conjugates through accessible Fab domains and the hydrodynamic diameter is measured again (D2). A test score, defined as D2/D1, is calculated. (B) Test scores for anti-HRP conjugates prepared at pHs 7.5, 8.0, and 8.5.

CONCLUSIONS Here, we found solution pH manipulates the surface charge distribution on antibodies to significantly alter the orientation of the protein when adsorbed onto AuNPs. While AuNP-protein adsorption is multi-factorial, involving several types of intermolecular forces, these studies capitalize on electrostatic interactions between the antibody and AuNP to modulate protein adsorption. Both NTA and a quantitative immunometric assay confirmed accessibility of the Fab increased as pH decreased from 8.5 to 7.5. These results support that localized regions of positive charge on the protein are responsible for the initial long-range interaction with AuNPs that govern orientation. Interestingly, adsorption affinity (Kd) was not affected by pH. This finding is


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consistent with other reports that the localized regions of the protein in contact with the AuNP surface may undergo a slight rearrangement/unfolding process to facilitate chemisorption between the cysteine residue and AuNP, independent of pH.68-70 This work underscores the complexity of AuNP-protein interactions and suggests computational analysis of protein surface charge and three-dimensional structure to locate cysteine residues could provide greater insight into the adsorption process. Despite the complexity of protein-AuNP adsorption, we demonstrate solution pH as a pathway to optimize the bioactivity of Ab-AuNP conjugates and maximize the performance of biofunctional nanomaterials. MATERIALS AND METHODS Reagents. All studies were conducted using 60 nm gold nanoparticles (Ted Pella Inc., Redding, CA). Mouse anti-HRP IgG monoclonal antibody (clone 2H11, 5.3 mg/mL) was obtained from MyBioSource.

Borate

buffer,

horseradish

peroxidase

(HRP),

and

2,2’-azino-bis(3-

ethylbenzothiazoline-6-sulphonic acid (1-Step ABTS) were purchased from Thermo Scientific (Rockford, IL). Potassium phosphate monohydrate was purchased from Fisher Scientific (Fair Lawn, NJ). Potassium phosphate dibasic (anhydrous) was obtained from Mallinckrodt Chemicals, Inc. (Paris, KY). Phosphate buffer and borate buffer were adjusted with NaOH and HCl to achieve the desired pH. Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, Inc. Hercules, CA) was used to study the quantity of antibodies conjugated to AuNPs. NANO pure deionized water (18 MΩ) from Barnstead water purification system (Thermo Scientific, Rockford, IL) was used to prepare all aqueous solutions. Antibody Adsorption Isotherm. AuNPs (60 nm, 100 μL) were added into a low binding microcentrifuge tube and 4 μL of either 50 mM phosphate buffer (pH 7.5) or 50 mM of borate buffer (pH 8.0 or pH 8.5) was added to adjust the pH. Varying concentrations of mouse anti-HRP


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were added to the AuNP suspensions (0 nM- 310 nM). To adjust for the different volumes in each sample, 2 mM of phosphate buffer (pH 7.5) or 2 mM of borate buffer (pH 8.0 or pH 8.5) was added to achieve a final volume of 114 μL. The anti-HRP-AuNP conjugate suspension was left to incubate for 1 hour at room temperature. After incubation, the functionalized AuNPs were diluted by taking 30 μL of the suspension and adding it to 1.5 mL of 2 mM of phosphate buffer (pH 7.5) or 2 mM of borate buffer (pH 8.0 or pH 8.5). Then, the diluted sample was analyzed using nanoparticle tracking analysis (NTA) to measure the mean hydrodynamic size and standard deviation. This process was repeated for each concentration. Quantifying Antibody Activity. (i). Number of antibodies conjugated onto AuNP. AuNPs (60 nm, 100 μL) and 4 μL of either 50 mM phosphate buffer (pH 7.5) or 50 mM borate buffer (pH 8.0 or pH 8.5) were added into a low binding microcentrifuge tube. Mouse anti-HRP monoclonal antibody (4 μg) was added to the AuNP suspension. To allow for direct adsorption, the antibodyAuNP suspension was incubated for 1 hour at 4 °C. Afterwards, the antibody-AuNP suspension was centrifuged at 5000 g for 5 minutes, the supernatant was removed, and the functionalized nanoparticles were resuspended in 2 mM phosphate buffer (pH 7.5) or 2 mM borate buffer (pH 8.0 or pH 8.5). This process was repeated two additional times to ensure removal of any excess antibody. The supernatant from the first wash was collected to quantify the number of antibodies immobilized onto the AuNP using a modified Bradford assay (Bio-Rad protein assay). Standard solutions from 0 μg/mL to 50 μg/mL of anti-HRP antibody were prepared in 2 mM phosphate buffer (pH 7.5) or 2 mM borate buffer (pH 8.0 or pH 8.5).

The standard solutions and

functionalized AuNPs were centrifuged at 5000 g for 5 minutes. After centrifugation, 90 μL of the sample supernatant or standard solution were placed in a 96 well plate. Then, 70 μL of 2 mM phosphate buffer (pH 7.5) or 2 mM borate buffer (pH 8.0 or pH 8.5), followed by 40 μL of Bio-


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Rad reagent was added to each standard solution and sample well, and left to incubate for 10-15 minutes at room temperature. The number of antibodies present in the supernatant was quantified by the UV-visible absorption at 595 nm. The difference between the quantity of antibodies that was added to the AuNP suspension and the number of antibodies remaining in the supernatant was calculated to determine the total number of antibodies that adsorb onto the AuNPs. (ii). Quantitation of captured HRP per AuNP. HRP (1 mg/mL, 3 μL) was added to the AuNP conjugates and incubated for 1 hour to ensure saturation of all available antigen binding sites on the antibody. A negative control conjugate consisting of goat anti-mouse IgG adsorbed onto AuNP was prepared to assess the non-specific interaction of HRP at pHs 7.5, 8.0, and 8.5. After incubation, the conjugates were centrifuged three times at 5000 g for 5 minutes to remove all the excess HRP. Aliquots of each sample (10 μL) were placed in a 96 well plate and were mixed with 150 μL of 1-step ABTS substrate to produce an enzymatic reaction. The absorbance of the formed product was measured at 10 s intervals for a total of 20 min at 415 nm to determine the enzymatic reaction rate. The enzymatic reaction rate and the concentration of HRP have a linear relationship, allowing the captured HRP on the functionalize AuNP to be quantified through calibration with standard solutions of HRP. Equal concentrations of conjugates were used in each experiment, confirmed via spectrophotometry. This normalization ensured that the differences in enzymatic reaction rate were related to the captured HRP per AuNP conjugate rather than variations in the conjugate concentrations. Anti-Fab Aggregation Assay to Assess Orientation of Adsorbed Antibody. Anti-HRP mAb conjugates were prepared at pH 7.5, 8.0, and 8.5 as described above. The concentration of each of the conjugates was measured via spectrophotometry and adjusted to be equal (O.D. ~1.3). An antiFab aggregation assay was performed to semi-quantitatively evaluate the orientation of the


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adsorbed anti-HRP mAb following a previously published method.55 Briefly, the mean hydrodynamic diameter of each conjugate suspension was measured using dynamic light scattering (DLS) and recorded as D1 using intensity distribution to calculate the size. A total of 50 g of antimouse IgG (Fab-specific) was added to 100 L of the conjugates and allowed to react. After 1 hour, the mean hydrodynamic of the conjugates were measured via DLS and recorded as D2. A test score was calculated as D2/D1. The test score increases with increasing aggregation that arises from a greater number of accessible Fab fragments on the conjugates. Instrumentation. (i) Nanoparticle Tracking Analysis (NTA). Hydrodynamic diameters of the anti-HRP-AuNP conjugates were measured using a NanoSight LM10 NTA system configured with an LM14 532 nm laser module and a high sensitivity sCMOS camera. Analysis was performed under a constant flow rate of 15 μL/min to improve sampling. The mean hydrodynamic diameter and standard deviation results from each sample were obtained from the analysis of approximately 10,000 individual functionalized AuNPs. NTA analyzed five 60 second videos, using camera level 9 and detection threshold 5. (ii). UV-Visible Spectrophotometer. A Cary 1 Bio UV-visible dual-beam spectrophotometer, with the slit set at 0.2 nm, was used to obtain spectra of the conjugates. The measurements were collected over a range of 400-900 nm at 0.5 nm increments. For the Bio-Rad protein assay and HRP-ABTS enzymatic reaction, absorbance was collected with an iMark Microplate Reader (BioRad). The absorbance for the Bio-Rad assay was measured at 595 nm and the HRP- 1-step ABTS reaction absorbance was collected at 415 nm for 20 minutes at 10 s intervals. (iii) Dynamic Light Scattering (DLS) and Zeta Potential. A Malvern Zetasizer Nano ZSP was used to analyze the zeta potential of the anti-HRP mAb as a function of pH. A Zeba spin desalting column was used following the manufacturer protocols for buffer exchange to suspend the


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antibody in buffer with the appropriate pH. The anti-HRP mAb concentration was adjusted to ~0.5 mg/mL for analysis. The samples were loaded into a folded capillary cell and the diffusion barrier technique was used to analyze the protein zeta potential. Additionally, the Zetasizer Nano ZSP was used in to measure the mean hydrodynamic diameter of conjugates and aggregates formed in the anti-Fab aggregation assay.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. NTA histograms for conjugates at pHs 8.0 and 8.5 (Figure S1) Calibration curve for Bio-Rad antibody assay (Figure S2) Saturation curve for antibody adsorption on to AuNP (Figure S3) HRP calibration curve (Figure S4) Saturation curve for HRP molecules captured by anti-HRP conjugates (Figure S5) AUTHOR INFORMATION Corresponding Author *[email protected]


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Author Contributions ‡These

authors contributed equally. The manuscript was written through contributions of all

authors. All authors have given approval to the final version of the manuscript. ORCID Jeremy D. Driskell: 0000-0001-5082-898X

ACKNOWLEDGEMENTS This work was supported by the Defense Threat Reduction Agency, Basic Research Award # HDTRA1-13-1-0028 and the National Science Foundation through the Macromolecular, Supramolecular and Nanochemistry Program, Award # CHE-1807126. REFERENCES (1) Joshi, P. P., Yoon, S. J., Hardin, W. G., Emelianov, S., and Sokolov, K. V. (2013) Conjugation of Antibodies to Gold Nanorods through Fc Portion: Synthesis and Molecular Specific Imaging. Bioconjugate Chem. 24, 878-888. (2) Kausaite-Minkstimiene, A., Ramanaviciene, A., Kirlyte, J., and Ramanavicius, A. (2010) Comparative Study of Random and Oriented Antibody Immobilization Techniques on the Binding Capacity of Immunosensor. Anal. Chem. 82, 6401-6408. (3) Lin, P. C., Chen, S. H., Wang, K. Y., Chen, M. L., Adak, A. K., Hwu, J. R. R., Chen, Y. J., and Lin, C. C. (2009) Fabrication of Oriented Antibody-Conjugated Magnetic Nanoprobes and Their Immunoaffinity Application. Anal. Chem. 81, 8774-8782. (4) Puertas, S., Moros, M., Fernandez-Pacheco, R., Ibarra, M. R., Grazu, V., and de la Fuente, J. M. (2010) Designing novel nano-immunoassays: antibody orientation versus sensitivity. J. Phys. D Appl. Phys. 43, 474012. (5) Saha, B., Evers, T. H., and Prins, M. W. J. (2014) How Antibody Surface Coverage on Nanoparticles Determines the Activity and Kinetics of Antigen Capturing for Biosensing. Anal. Chem. 86, 8158-8166. (6) Saha, B., Songe, P., Evers, T. H., and Prins, M. W. J. (2017) The influence of covalent immobilization conditions on antibody accessibility on nanoparticles. Analyst 142, 42474256. (7) Song, H. Y., Zhou, X. D., Hobley, J., and Su, X. D. (2012) Comparative Study of Random and Oriented Antibody Immobilization as Measured by Dual Polarization Interferometry and Surface Plasnnon Resonance Spectroscopy. Langmuir 28, 997-1004.


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pH Impacts the Orientation of Antibody Adsorbed onto Gold

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