Synthesis and bioconjugation of alkanethiol-stabilized gold bipyramid nanoparticles

Abstract

Gold bipyramid (GBP) nanoparticles are promising for a range of biomedical applications, including biosensing and surface-enhanced Raman spectroscopy, due to their favorable optical properties and ease of chemical functionalization. Here we report improved synthesis methods, including preparation of gold seed particles with an increased shelf life of ~1 month, and preparation of GBPs with significantly shortened synthesis time (< 1 h). We also report methods for the functionalization and bioconjugation of the GBPs, including functionalization with alkanethiol self-assembled monolayers (SAMs) and bioconjugation with proteins via carbodiimide cross-linking. Binding of specific antibodies to the nanoparticle-bound proteins was subsequently observed via localized surface plasmon resonance sensing. Rabbit IgG and goat anti-Rabbit IgG antibodies were used as a model system for antibody-antigen interactions. As-synthesized, SAM-functionalized, and bioconjugated bipyramids were characterized using scanning electron microscopy, UV–vis spectroscopy, zeta potential, and dynamic light scattering.

Introduction

Over the past two decades, considerable work has been put into the development of noble metal nanoparticles for nanomedicine applications. Due to the unique properties of gold, the nanoparticles produced using this metal have been used as drug delivery vehicles [1] as well as enhancement probes for radiation [2] and photothermal therapy [3]. The novelty of gold nanoparticles arises in part from our ability to synthesize anisotropic particles of various sizes tailored for the intended application [4–6]. Gold nanoparticles can be functionalized with a variety of ligands [7]. Many functionalization strategies are made possible by the strong bonds that gold and sulfur form [8]. Ligands bound to gold nanoparticles via the gold-sulfur bond can be the basis for attaching biomolecules to the surface of the nanoparticle to help improve biocompatibility and targeting [9]. The choice of ligand depends on the application, the type of biomolecule and its size, available functional groups, and chemical composition [10].

Gold nanoparticles additionally exhibit unusual optical properties including localized surface plasmon resonance (LSPR) [11, 12]. The electric field of incident light collectively excites the conduction electrons resulting in coherent localized plasma oscillations with a resonant frequency that depends on the composition, size, geometry, dielectric environment, and separation distance of the nanoparticles [13]. When considering non-spherical gold nanoparticles such as nanorods, two plasmon modes can be seen [14, 15]. The resonance along the shorter axis is referred to as the transverse plasmon whereas the resonance along the long axis of the particle is called the longitudinal plasmon. The resonant wavelength of the longitudinal plasmon is highly sensitive to the size and aspect ratio of the particle. By adjusting these parameters, the surface plasmon can be tuned to specific wavelengths.

Gold bipyramids (GBPs) were first synthesized by Liu and Guyot-Sionnest using a citrate stabilized seed mediated synthesis [16]. Due to this particular method of synthesis, nanospheres and smaller nanorods are produced as a byproduct. However, these byproducts are small in overall number and can be removed using centrifugation, diafiltration [17], or gel preparative electrophoresis [18]. Additionally, the growth process can take from two hours as reported by Liu et al [16] up to twelve hours as reported by Navarro et al [19]. The factors required for the synthesis of anisotropic gold nanoparticles such as nanorods and bipyramids have been studied extensively. Factors such as cetyl trimethylammonium bromide (CTAB) and silver nitrate concentration[20, 21], pH [22], and reagent temperature [23, 24] at which the synthesis takes place have been studied extensively in previous works. The role of sodium borohydride in seed-mediated synthesis has also been studied [25].

Several methods for the synthesis of GBPs have been presented [16, 19, 26–29]. Many of these methods utilize a seed mediated synthesis process. Gold seed particles stabilized by sodium citrate as well as by CTAB have been used to seed the growth of GBPs. The biypramids themselves are surfactant-stabilized using CTAB as well as other surfactants such as cetyltriethylammonium bromide (CTEAB). In the latter case, due to larger head groups of the CTEAB, small CTEAB micelles are formed during the synthesis. This leads to a synthesis time nearly 5 times longer [29]. The benefit of a seed mediated synthesis is the ability to produce large quantities of relatively uniform monodisperse particles.

GBPs exhibit transverse and longitudinal LSPR modes, similar to gold nanorods. GBPs additionally exhibit an enhanced scattering [30] which can be used for applications in targeted drug delivery [31], diagnostics [32], and photothermal therapy [33]. The longitudinal plasmon mode is tunable over visible and near-infrared wavelengths and is dependent on the size of the particle, the aspect ratio, and the tip sharpness of the particles. By adjusting these parameters, the LSPR can be engineered to be between 600 and 1100 nm [29].

GBPs have also been shown to exhibit improved LSPR immunoassay response over gold nanorods [26]. This improvement comes from the higher refractive index sensitivity and the narrower LSPR linewidth of bipyramids. Additionally, it has been shown that the refractive index sensitivity of the GBPs has a larger tunable range compared to nanorods [34].

Another promising application of GBPs is in surface-enhanced Raman spectroscopy (SERS). Previously SERS has been used with gold nanorods to show the thermophoresis of particle in solutions and how higher excitation laser power decreases SERS signal [35]. Raman spectroscopy provides detailed information about the vibrational modes of molecules. These measurements can be enhanced through the use of metallic nanoparticle substrates due to strong electromagnetic field enhancement near the surface of the particle. Bipyramids are particularly well suited for use as enhancement probes due to their maximum electric field intensity being anywhere from 3 to 6 times greater than nanorods of comparable size [36].

In addition, the high photoluminescence quantum yield of GBPs as compared to nanorods makes them ideal for optical imaging in biology and materials science [37]. Compared to other nanoparticle types with similar surface plasmon resonance wavelengths, GBPs have shown to have nearly double the quantum yield. Nonspherical gold nanoparticles such as bipyramids have also been shown to have high scattering cross-sections for their size [38], making them useful as contrast agents in photoacoustic imaging [39, 40].

Many important medical applications of gold nanoparticles rely on specific targeting of cancer cells. When comparing cancerous cells with healthy cells, specific bio-markers such as epidermal growth factor receptor (EGFR) are overexpressed. Nanoparticle bioconjugates can be used as therapeutic delivery vehicles via antibody targeting [41, 42]. EGFR has been evaluated for antibody targeting with nanoparticle bioconjugates [43, 44]. Gold nanorods have been successfully bioconjugated for use in targeted biomedical applications [45]. The nanorods are often stabilized using a alkanethiol self-assembled monolayer (SAMs) before finally being antibody-conjugated to bind to cancer cells [31]. For example, by using specific antibodies to UM-A9, it is possible to target squamous cell carcinoma cells [46].

Materials and methods

Synthesis of GBPs nanoparticles was carried out using a seed-mediated method similar to that described in Navarro 2012 [18]. This was done with the use of CTAB stabilized seed particles which were introduced to a growth solution as detailed below. This method produces seed particles and subsequently bipyramids that are single crystals [43].

Synthesis of CTAB-capped seed particles

10 ml of 0.2 M CTAB solution was heated to 60 °C and combined with 10 ml of 0.25 mM HAuCl₄ solution and allowed to mix under stirring for several minutes. This produced a solution which was orange in color. 0.6 ml of chilled (~3 °C) 0.1 M NaBH₄ was prepared and added to the solution via dropper. The solution turned from an orange to a light brown color. As the color changed signaling the formation of seed particles, the stirring was stopped and stir bar removed; the solution was then allowed to sit at 40 °C for several days in darkness. After five days of sitting at 40 °C, the seed was stored at room temperature in darkness. This resulted in seeds with a useful shelf life of at least a month as described in the Results and Discussion.

Preparation of growth solution and growth of bipyramids from CTAB-capped seed

10 ml of 0.1 M CTAB was heated to 60 °C. 0.3 ml of 10 mM AgNO₃ and 0.5 ml of 10 mM HAuCl₄ were added and allowed to heat for 20 min. Once the solution was well combined and heated thoroughly to 60 °C, then the solution was acidified by adding 0.2 ml of 0.1 M HCl and then mixed by inversion. This was followed by adding 80 μl of 100 mM solution of L-ascorbic acid which was mixed by inversion to reduce the HAuCl₄. Prior to use the seed solution was mixed by inversion and sonicated for 20 min. The desired volume (60, 75, or 90 μl) of CTAB-capped seed which was prepared earlier was added to the growth solution and mixed. Then the solution was left on the hot plate covered for approximately 20 min at 60 °C to allow the bipyramids to grow.

The growth solution was cleaned of excess reagents twice using a centrifuge, once at 4500 rpm for 15 min and again at 4000 rpm for 10 min. After each cleaning, the supernatant was removed and replaced with fresh ultrapure water, and the sample was sonicated for 10 min. After the second cleaning the sample was stored at room temperature in the dark until needed for the next stage.

Characterization of GBPs

GPBs were characterized using the Hitachi S 5500 Field-Emission Scanning Electron Microscope located at the Kleberg Advanced Microscopy Center at The University of Texas at San Antonio. Measurements were taken using the ImageJ software application. The UV–vis measurements were taken using an Ocean Optics Flame Mini-Spectrometer.

SAM functionalization of GBPs

20 ml of as-synthesized GBPs in aqueous solution were combined with 2 ml of 20 mM 16-mercaptohexadecanoic acid in methanol. The resulting solution was stirred gently at room temperature for 24 h. Functionalized bipyramids were cleaned by centrifugation at 6500 rpm for 15 min twice with the supernatant being removed and replaced with first ethanol and then ultrapure water successively.

Bioconjugation of GBPs

The bioconjugation of the GBPs was carried out using EDC carbodiimide crosslinking. Rabbit IgG was attached to the carboxyl terminated alkanethiol SAM present on the surface of the GBPs. This method has been used to successfully label gold nanorods with antibodies [44] and was adapted for bioconjugating GBPs.

5 ml of the SAM-functionalized GBPs solution was mixed with 1 ml of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) at 0.4 M and sulfo-NHS (N-hydroxysulfosuccinimide) at 0.1 M. The desired volume (25, 50, or 75 μl) of 200 nM Rabbit IgG solution (corresponding to 150, 300 and 450 ng ml⁻¹ final concentration) was added to the solution of SAM-functionalized GBPs. The solution was sonicated for 1 h at room temperature. After 1 h, the sample was centrifuged for 5 min at 5000 rpm, decanted, and suspended in ultrapure water.

For the antibody-antigen binding experiment, 75 μl of 200 nM Goat Anti-Rabbit IgG solution (450 ng ml⁻¹ final concentration) was added to 5 ml of Rabbit IgG bioconjugated GBP solution. The solution was sonicated for 1 h at room temperature. After 1 h, the sample was centrifuged for 5 min at 5000 rpm, decanted, and suspended in ultrapure water. For the accompanying control experiment, 75 μl of 200 nM Goat Anti-Mouse IgG solution (450 ng ml⁻¹ final concentration) was added to 5 ml of Rabbit IgG bioconjugated GBP solution. The solution was sonicated for 1 h at room temperature. After 1 h, the sample was centrifuged for 5 min at 5000 rpm, decanted, and suspended in ultrapure water.

CTAB was sourced from Sigma Aldrich under product ID H9151 with lot number SLBT9565. Rabbit IgG and Goat Anti-Rabbit IgG antibody (R2004-5X1MG) were purchased from Sigma Aldrich in powder form. Goat Anti-Mouse IgG antibody (M8642-5X1MG) was purchased from Sigma Aldrich in powder form.

Zeta potential and hydrodynamic diameter measurements

Zeta potential and hydrodynamic diameter measurements were conducted using the Malvern Zetasizer Nano Range. The as-synthesized GBPs were diluted to 5 percent of a starting concentration that provided a UV–vis absorbance value of OD 1 at the longitudinal LSPR peak. The pH of each sample was adjusted by addition of HCl or NaOH. pH values from 5 to 9 were used to measure the stability of the particles. Zeta potential measurements of functionalized and bioconjugated nanoparticles were taken with all samples at pH 7.

Results and discussion

GBPs synthesis and characterization

Synthesis techniques for producing GBPs generally require either long growth periods, (>12 h). In addition, the seed particles used in seed mediated synthesis methods generally have a short shelf life. The ability to produce large volumes of GBPs in a short time (<1 h) with specifically tailored LSPRs would be of great benefit for nanomedicine applications. Once the GBPs are synthesized, their surface chemistry can be modified for biomedical targeting applications.

First, CTAB stabilized seed particles were synthesized using the method outlined in Navarro et al [19]. The seed solution was allowed to age for 5 d in the dark at 40 °C. During this time, the seed solution changed color from a dark brown color to a light purple color. Following the aging of the seed particles, the growth stage of the nanoparticle can begin.

GBPs synthesis was conducted as outlined in the methods section. During this process, it is important to note the color change of the growth solution from clear to a light purple and to ultimately a dark purple color. This process takes roughly 20–25 min. It is after this color change was complete that the UV–vis spectra of the growth solutions presented in figure 1 were collected. All spectra were normalized to an absorbance value of 1 at the maximum peak wavelength for each spectrum. The growth solution displays two characteristic plasmonic peaks corresponding to the longitudinal and transverse plasmon resonances of the GBPs. The nanospheres produced as a byproduct may also contribute to the lower-wavelength peak. A large blue shift in the longitudinal LSPR is observed as the seed volume is increased. By increasing the number of seed particles, the available gold ions in solution are spread across a greater number of seeds. This leads to a larger number of particles that are smaller in size.

Figure 1.

UV–vis absorbance spectra of three aqueous solutions of gold bipyramid nanoparticles synthesized with varied seed solution volumes from 60 to 90 μl.

The GBP preparations from figure 1 were also characterized by scanning electron microscopy (SEM). SEM images of these GBPs are shown in figure 2. These images confirm that as the seed volume is increased, the nanoparticle size decreases. This is consistent with the blue shift seen in the spectra in figure 1. In all three preparations, along with bipyramid type particles, nanospheres and nanorods were produced as byproducts, as can be seen in figure 2. SEM images were analyzed for particle dimensions using ImageJ. Particle statistics can be seen in table 1.

Figure 2.

SEM images of each of the gold bipyramid growth solutions with different seed volumes used: (a) 60 μl, (b) 75 μl, (c) 90 μl.

Table 1.

Particle dimensions of gold bipyramids synthesized using varying seed volumes.

Particle statistics from SEM images
Seed volume	60 μl	75 μl	90 μl
Average length	85.04 nm	70.64 nm	62.72 nm
Average width	32.32 nm	31.08 nm	27.16 nm
Aspect ratio	2.63	2.27	2.31
Tip radius	6.28 nm	6.18 nm	5.66 nm

Stability of gold seed solution

In order to determine the long-term viability of using the same seed solution to produce consistent nanoparticles, the seed solution was examined over the course of one month. The initial seed solution was aged for five days prior to being used. After the initial five days elapsed, the seed solution was aged an additional month. After the initial growth trial was conducted using the seed solution, additional growth trials were conducted one week apart. Figure 3 show the results of this seed stability trial with an average longitudinal peak wavelength of 697 ± 5.4 nm. For all the trials, a seed volume of 75 μl was used. The average transverse LSPR peak position of these trails was 697 ± 6.04 nm. In most seed-mediated synthesis methods the seed solution has average lifespan of only a few days. This seed solution provides consistent results for at least a month. UV–vis spectra, TEM images, and photographs of as-synthesized and aged seed solutions are available in the supporting information (available online at stacks.iop.org/NANO/32/225601/mmedia).

Figure 3.

UV–vis absorption spectra from the seed solution stability study. The seed solution was aged initially for 5 d before use. Growth trials were conducted 1 week apart.

Bioconjugation of GBPs

After synthesis, the GBPs can be processed for functionalization and bioconjugation. As-synthesized GBPs are coated with a CTAB surfactant layer which acts to stabilize the nanoparticles in solution; however, this surfactant layer is not biocompatible and does not allow the particles to be easily bioconjugated. In the functionalization process, the as-synthesized GBPs were combined with 16-mercaptohexadecanoic acid, an alkanethiol ligand, in methanol. The CTAB layer, which is loosely bound to the surface, is replaced by the alkanethiol ligand which forms a SAM. The alkanethiol layer allows the particle to move between aqueous and organic phases without precipitating. If CTAB coated nanoparticles were present after the functionalization process, they would aggregate and precipitate when the solution was moved into an organic phase and thus would not be present during the bioconjugation stage.

Bioconjugation of the nanoparticles was achieved by activating the carboxyl-terminated SAM using a combination of EDC and sulfo-NHS. During this process the EDC reacts with a carboxylic acid group at the end of the alkanethiol ligand forming an amine-reactive O-acyl isourea intermediate. The sulfo-NHS stabilizes the amine-reactive intermediate by converting it to an amine-reactive sulfo-NHS ester. This increases the chances of EDC-mediated coupling of an protein to the nanoparticle [47–50]. Once this is done, the protein of choice can be bioconjugated to the surface of the SAM. In this study, SAM-functionalized GBPs were bioconjugated with Rabbit IgG. The GBP bioconjugates were then exposed to Goat anti-Rabbit IgG antibodies, in order to confirm that antibody-antigen binding on the nanoparticle surface is preserved. This Rabbit IgG system was chosen as a proof-of-concept model which can stand in for any nanoparticle bioconjugation scheme which relies on antibody-antigen interaction.

UV–vis absorbance spectra collected from the GBPs at each stage of the functionalization and bioconjugation process are seen in figure 4. LSPR sensing was used to verify the specific antibody-antigen binding at the nanoparticle surface, in which the specific antibody is Goat anti-Rabbit IgG. Rabbit IgG, in this case can be considered as the antigen. The LSPR wavelength of the GBPs red shifted as each successive layer was coated on to the surface of the particles. This is an expected result as the local refractive index of the particle is increasing through the deposition of layers. The longitudinal LSPR peak for the CTAB-coated GPBs was at 704 nm. For SAM-coated GBPs, the peak was at 723 nm. For SAM and Rabbit IgG coated GBPs, the peak was at 729 nm. Finally, for GBPs with SAM, Rabbit IgG, and Anti-Rabbit IgG antibody with Horseradish Peroxidase (HRP) enzyme, the peak was at 731 nm.

Figure 4.

UV–vis absorbance spectra of SAM-functionalized and bioconjugated gold bipyramids. The peak position of the longitudinal LSPR is highlighted. The CTAB layer on the as-synthesized gold bipyramids (black) was replaced with an alkanethiol (16-mercaptohexadecanoic acid) SAM (red). Subsequently, the bipyramids were bioconjugated with Rabbit IgG (blue). Finally, goat anti-Rabbit IgG antibodies were added to test antibody-antigen binding on the bipyramid surface (magenta).

In order to verify the specificity of the antibody binding results seen in figure 4, a control experiment was performed in which a non-specific antibody, namely Goat anti-Mouse IgG antibody, was used instead of the specific Goat anti-Rabbit IgG antibody. The results of this can be seen in figure 5. The Anti-Mouse IgG did not produce an LSPR red shift in the longitudinal peak.

Figure 5.

Specificity of antibody-antigen interaction. Rabbit IgG is bound to the surface of GBPs. Specific (Goat Anti-Rabbit IgG) and nonspecific (Goat Anti-Mouse IgG) antibodies are then added. The peak position value given is for the longitudinal LSPR peak.

The LSPR response of the GBPs is a function of the local refractive index of the particles. The as-synthesized GBPs are stabilized in solution by a loosely bound CTAB layer. During the ligand exchange process, this CTAB layer is replaced with an alkanethiol SAM which changes the local refractive index of the particles. The change in the local refractive index on the surface has a direct effect on the measured extinction spectra. As seen in figure 4, this results in a roughly 19 nm shift of the LSPR. This observation is in agreement with previous studies [26, 51]. Figure 6 shows the peak position of the LSPR at differing indices of refraction. As the index of refraction increases, the peak position red shifts.

Figure 6.

The LSPR sensitivity to the refractive index of the GBPs was measured in various environments. The peak LSPR position of both the longitudinal and transverse peak in each medium is (A) Air 632 and 503 nm, (B) Methanol 700 and 527 nm, (C) Water 704 and 528 nm, (D) DMSO 728 and 541 nm, (E) Toluene 736 and 546 nm respectively. The slope of each of the lines yields a sensitivity of 206 nm/RIU and 82 nm/RIU.

It was seen that the sensitivity of the of each peak differed. The longitudinal peak displays greater sensitivity over the transverse peak over a similar RI range. The linear fit of both of these peak positions results in a LSPR sensitivity of 206 nm/RIU for the longitudinal peak and 82 nm/RIU for the transverse peak. Using the longitudinal peak, it is reasonable to infer that as the CTAB layer is replaced with the alkanethiol SAM, the local refractive index has increased 0.092 RIU as evidenced by the LSPR redshift. Subsequent changes in the LSPR have been noted as the protein and antibody layers are added. The broadening in the LSPR peak observed at longer red shifts is likely due to some contribution from nanoparticle aggregates.

The number of proteins that can be conjugated to the surface of the nanoparticle is limited by the nanoparticle size (surface area). Up to this limit, the number of proteins on the surface of the GBPs can be adjusted by changing the concentration of proteins in solution. By adjusting the number of proteins on the surface of the nanoparticles, the position of the longitudinal peak is tuned. In order to determine the optimum concentration of protein with which to conjugate the bipyramids, the concentration was varied. Figure 7 shows UV–vis absorbance spectra of bioconjugated bipyramids where the concentration of Rabbit IgG during the bioconjugation step was 450, 300 and 150 ng ml⁻¹. It was seen that beyond 450 ng ml⁻¹ concentration, the amount of unbound protein left in solution would cause a drop in the absorbance. This is most likely due to nanoparticle aggregation. For the GBPs functionalized with Rabbit IgG at 450 ng ml⁻¹, the longitudinal plasmon resonance peak was at 732 nm. For the GBPs functionalized with Rabbit IgG at 300 ng ml⁻¹, the peak was at 729 nm. Finally, for the GBPs functionalized with Rabbit IgG at 150 ng ml⁻¹, the peak was at 727 nm.

Figure 7.

UV–vis absorbance spectra of gold bipyramids with varied Rabbit IgG concentrations.

Zeta potential and hydrodynamic diameter

Zeta potential measurements were conducted to test the stability of the particles at varying pH values as well as provide additional confirmation of the surface modification of the nanoparticles. The ability of the particles to remain stable at varying pH values would be beneficial for biomedical and nanomedicine applications.

In order to understand the zeta potential behavior of GBPs, examining the zeta potential of gold nanorods will allow us to approximate the behavior of GBPs. It is expected that CTAB capped gold nanorods should have a positive zeta potential, with each subsequent layer making the zeta potential more negative [52]. GBPs share a similar crystal structure and starting surface chemistry with gold nanorods.

Table 2 shows the results of the zeta potential measurements on the as-synthesized CTAB stabilized GBPs. At a pH of 7, the zeta potential of the particle is around 34 mV. The variation of zeta potential with pH shown in figure 8 is similar to that observed for gold nanorods [53]. As expected, zeta potential decreases with increasing pH.

Table 2.

Zeta potential measurements of the as-synthesized CTAB stabilized gold bipyramids.

Zeta potential of as-synthesized CTAB stabilized gold bipyramids
	pH 5	pH 6	pH 7	pH 8	pH 9
Average zeta potential	63.4 ± 2.45 mV	37.6 ± 2.16 mV	33.4 ± 1.50 mV	21.6 ± 0.55 mV	5.67 ± 1.29 mV

Figure 8.

Zeta potential versus pH curve of the CTAB stabilized gold bipyramids.

Because the GBPs are not spherical particles, but rather oblate particles, the hydrodynamic diameter reported in table 3 can be considered an average diameter for all axes of the particle. During the substitution of the CTAB layer for the SAM, the diameter measured remains nearly the same. As the bioconjugation and antibody-antigen binding take place, the diameter can be seen to increase with each successive layer. This can be illustrated in figure 9.

Table 3.

Zeta potential measurements for functionalized and bioconjugated gold bipyramids.

Zeta potential of functionalized and bioconjugated gold bipyramids at pH 7
Outermost layer	16-Mercaptohexadecanoic acid	Rabbit IgG	Anti-rabbit IgG w/HRP enzyme
Average zeta potential	−32.96 ± 1.19 mV	21.83 ± 0.47 mV	−10.06 ± 0.70 mV

Figure 9.

Schematic diagram of GBPs at each stage of functionalization and bioconjugation. As each successive layer is added, the effective hydrodynamic radius increases.

Tables 3 and 4 display the zeta potential and hydrodynamic diameter measurements, respectively, of the functionalized and bioconjugated GBPs after the addition of each layer. All measurements were carried out at a pH of 7. It can be seen that after CTAB is displaced by the alkanethiol SAMs, the zeta potential shifts to a negative value due to the deprotonation of the carboxyl group at the end of the ligand. After the Rabbit IgG layer is bound, the zeta potential attains a positive value. After the antibody-antigen binding step takes place with the addition of enzyme-labeled anti-Rabbit IgG antibodies, the zeta potential takes on a negative value of lower magnitude. The shift in zeta potential measurements provides confirmation of the surface modification of the GBPs.

Table 4.

Hydrodynamic diameter of functionalized and bioconjugated gold bipyramids.

Hydrodynamic diameter of functionalized and bioconjugated gold bipyramids
Outermost layer	CTAB (as-synthesized)	16-Mercaptohexadecanoic acid	Rabbit IgG	Anti-rabbit IgG w/HRP enzyme
Average diameter	106.6 ± 3.20 nm	113.1 ± 5.74 nm	293.0 ± 5.10 nm	384.8 ± 19.9 nm

In addition to the zeta potential, the hydrodynamic diameter was measured. Due to the bipyramids elongated shape, the hydrodynamic diameter provides a rough size that is averaged over all orientations. After the initial layer replacement, the nanoparticle bioconjugate diameter increases as each layer is added. Between the CTAB layer and SAM, the hydrodynamic diameter does not change substantially. This is to be expected as the SAM is replacing the CTAB layer and not deposited on the surface. As the protein layers are added the hydrodynamic radius increases substantially.

Conclusion

GBP nanoparticles were synthesized using a seed mediated method using various seed volumes. CTAB molecules bound to the surface of the as-synthesized nanoparticles were replaced with an alkanethiol ligand. By replacing the surfactant layer with a carboxy-terminated SAM, the GBPs were made ready for EDC coupling with proteins. Synthesis, functionalization, and bioconjugation were characterized using SEM, UV–vis spectroscopy, zeta potential, and dynamic light scattering measurements.

The synthesis methods described herein make it possible to produce GBPs consistently in a shortened synthesis time (<1 h) from a seed solution with a long shelf life. As nanoparticle therapeutics is becoming a growing area of interest, it is advantageous to produce consistent nanoparticles in a relatively short time. The bioconjugate complex described above as well as other like it can be developed for nanomedical applications such as biosensing applications, drug delivery, and targeted radiation therapy.

Data availability statement

All data that support the findings of this study are included within the article (and any supplementary files).