Gold Nanorod Synthesis

Brantley C. Henson, Justin T. Harris, Kimberly A. Homan

NanoHybrids Inc.

Material Matters, 2017, 12.1

kimberly.homan@nanohybrids.net

Introduction

Plasmonic nanoparticles have unique optical properties that can be tailored to suit a variety of applications in the biotechnology^1–8 and electronics^9–16 industries. Gold nanorods (Figure 1), a particular subset of plasmonic nanoparticles, are of interest for biomedical applications due to their tunability in the nearinfrared (NIR) light spectrum and biological inertness.^17–20 There are numerous techniques for the synthesis of gold nanorods, including templating,^21–25 electrochemical growth,^26–28 and reverse micellar systems.^29–30 Here, we focus on seed-mediated colloidal growth methods. While there are multiple published variations on the seed-mediated colloidal growth of gold nanorods,^29,31–38 our discussion will examine the well-known approaches pioneered by Nikoobakht and Murphy.^31,39 Seed-mediated colloidal growth has many advantages over the aforementioned methods,⁴¹ including no need for specialized equipment and straightforward scalability—eventual requirements for clinical translation.^43–44 Although these methods are well understood, their sensitivity to small changes in reaction conditions can lead to a lack of control over the size, shape, purity, and aspect ratio of rods from batch to batch.^{19,33,36–37,44–50} In this review, we briefly discuss seedmediated gold nanorod synthesis, and then present a detailed discussion of the common pitfalls and how best to avoid them.

Figure 1. TEM image of typical gold nanorods.

Synthesis

Basic synthesis follows the work of Nikoobakht and Murphy,^{31,37,39,51–52} where precise reactant concentrations and volumes are discussed. Briefly, a growth solution composed of cetyltrimethylammonium bromide (CTAB, Prod. No. 52370), water, silver nitrate (AgNO₃, Prod. No. 792276), and chloroauric acid (HAuCl₄, Prod. No. 254169) is prepared. If a multisurfactant system is required for longer aspect ratios, benzyldimethyl ammonium chloride (BDAC) is also typically included, though other surfactants such as sodium oleate (Prod. No. O7501) have also been reported.^46,53 Ascorbic acid (Prod. No. A92902) is then added to reduce the Au³⁺ to Au¹⁺. To template further reduction of gold, small gold nanoparticle seeds are then added to the solution. The seeds are synthesized via a rapid reduction of HAuCl₄ by sodium borohydride (Prod. No. 480886) in a CTAB or citrate solution. Upon addition of the seeds, Au¹⁺ is further reduced to metallic Au⁰, with the surfactants and Ag serving to break the symmetry through preferential deposition on the {110} facets of gold, inhibiting growth⁵⁴ and encouraging growth parallel to the {001} plane, resulting in anisotropic gold nanorods.³¹

Six Common Pitfalls in Gold Nanorod Synthesis

The Importance of Good Synthesis Preparation (1)

Slight adjustments in overall concentrations, impurities, and nucleation sites can result in significant differences in particle size, shape, and purity, rendering reproducibility a concern. Thus, successful and repeatable gold nanorod synthesis is highly reliant upon proper and consistent preparation and lab technique. First, to ensure even growth, nucleation sites must be well controlled. As a general requirement for most metallic nanoparticle syntheses, all glassware and stir bars must be thoroughly cleaned with aqua regia⁵⁵ to remove residual metals. Any residual metal will lead to unwanted pre-nucleation in the growth solution, making controlled synthesis impossible. All water used throughout the synthesis process, including glassware cleaning, needs to be 18.2 MΩ・cm ASTM Type I water (commonly referred to as deionized ultrafiltrated, DIUF, or “Nanopure™” water). Likewise, the lab space should be clean, with minimal dust.

Another common cause of variability from lab to lab, or even person to person, is the reagent preparation; even when buying from the same supplier, minor changes in impurities from lot to lot can affect the final product of the nanorod synthesis. For example, Korgel et al. report on the effect of the CTAB source on nanorod synthesis.³³ Interestingly, the results indicate trace impurities in CTAB, particularly iodine, are actually required in order to produce high quality rods.⁵⁶ Similarly, NanoHybrids has found changing the gold acid source can affect the overall size and longitudinal surface plasmon resonance (LSPR) of the resulting rods. For these reasons, documentation and tracking of reagents is necessary for all synthesis. Whenever changing reagent lots or sources, pilot reactions should first be performed to set the new baseline for further size and shape tuning. Other considerations include reagent age and storage conditions. For ease of reaction and to minimize inherent weighing errors, many groups choose to make stock solutions of reactants. Recent work by Murphy et al.³⁸ demonstrated solutions of silver nitrate and ascorbic acid are best prepared fresh due to their susceptibility to photodegradation. Likewise, the age of CTAB solutions has been shown to affect the final product.³⁸ Depending on the quantity and scale of reactions a lab produces, these effects can either be detrimental or potentially leveraged for increased control over reaction kinetics. Again, proper documentation is paramount to success.

Aggregation Happens: Rod Instability (2)

One of the most common problems during synthesis is nanoparticle aggregation. Knowing when aggregation occurs is just as important as knowing how to prevent it. Aggregation of nanorods with an absorbance in the NIR spectrum can be easily detected by ultraviolet-to-visible (UV/Vis) spectrophotometry. The main indication of aggregation is the presence of a “shoulder” in the extinction spectra. A typical “good” spectrum is shown in Figure 2A. Note the high ratio of the LSPR peak to the transverse surface plasmon resonance (TSPR) peak (labeled as L/T, Figure 2A), and the narrow, symmetric shape of the LSPR peak. Figure 2B shows the result of slight aggregation. Note the obvious lack of symmetry and right-side skew of the LSPR band caused by plasmon coupling in gold nanorod aggregates. Similarly, Figure 2C shows particles made with a dual surfactant system (CTAB + BDAC). While not as obvious as Figure 2B, the asymmetry of the LSPR band and increased full width half maximum (FWHM) indicates problems with the synthesis, such as high polydispersity of size and aspect ratio or slight aggregation. These problems are not inherent to all dual-surfactant reactions and, thus, is an indicator of a failed reaction. Also, note the shape of the TSPR band (BDAC Trans Peak) in Figure 2C. The additional small shoulder is due to subtle differences in the shape of the rods themselves, along with the associated impurities (cubes vs. spheres). Even in a “good” dual-surfactant reaction with a narrow LSPR and high symmetry, this variation in the TSPR band is expected. A spectrum of nanorods with high amounts of shape impurities (large numbers of spheres and cubes) is shown in Figure 2D and discussed further in pitfall #4. Finally, severe aggregation can be easily identified by particle precipitation or a change in appearance from various shades of red to dark gray or purple, such as in Figure 2E.

Figure 2. Spotting nanorod aggregation and shape impurities using normalized UV/Vis spectra. A) Ideal spectra of gold nanorods synthesized with a single surfactant (CTAB) is evidenced by a high LSPR/TSPR peak ratio (L/T) and a narrow symmetric LSPR peak. B) Spectra displaying aggregates in a “shoulder.” C) Spectra of particles synthesized in a binary surfactant (CTAB and BDAC) is shown by the shape of the transverse peak. The spectrum contains a wide asymmetric LSPR peak indicating wide size distribution. D) Spectra of particles exhibiting high amounts of shape impurities. E) Photo of (1) as-synthesized and (2) aggregated gold nanorods.

The root cause of aggregation is often difficult to identify due to the complexity of the synthesis. Unexpected impurities in new reagents, reagent aging, incompatible reagent combinations, contamination, and improper product purification (discussed later) are all potential sources of the problem. To mitigate these problems and maintain general synthesis consistency, it is recommended that regular small-scale pilot studies (20–30 mL reactions) be performed on a monthly to quarterly basis to verify the quality of reagents and identify sources of contamination or degradation. It is important to note that while small-scale reactions are used for verification purposes and reproducibility in comparison to previous studies, the exact peak absorption typically shifts when the reaction is conducted at a larger scale; specifically, a lower LSPR is expected upon scale-up. Moreover, the actual resultant peak is also dependent upon vessel size, shape, spin speed, and heating efficiency. As a result, the expected LSPR shift should be determined for any new glassware and protocol for consistent targeting of LSPR wavelengths.  

Poor Control Over Aspect Ratio (3)

One of the unique properties of gold nanorods is the tunability of their optical peak absorption from the visible to IR range simply by changing their aspect ratio (defined as the ratio between length and width/diameter of the nanorod). Within the standard seed-mediated growth protocols, this requires breaking the symmetrical growth, which is thermodynamically favored, to encourage growth along a single axis. To this end, silver nitrate is a reliable tool for lengthening gold nanorods.³¹ As seen in Figure 3A, generally increasing the silver nitrate concentration increases the aspect ratio and corresponding LSPR, but this method only works up to ~850 nm. For aspect ratios greater than 4.5 or an LSPR greater than 850 nm, Nikoobahkt demonstrated that varying the ratio of BDAC and CTAB in a binary surfactant system yields aspect ratios up to 10 (Figure 3B).³¹ Values as high as 70 have been reported by implementing a multistage addition of growth solution to seeds, as reported by Murphy et al.⁵⁷ Perez–Marzan demonstrated a correlation between increased seed amount and decreased aspect ratio.58 Whichever approach is chosen, the delicate interplay within the multivariable synthetic system illustrates the importance of tight control over the reactant concentrations while making it possible to synthesize a wide range of high quality gold nanorods (Figure 3D). For consistency, massing your reactants versus relying on volumetric techniques is recommended.

It is also worth considering that in all syntheses involving gold nanorods, there is a tendency for the nanorod aspect ratio to decrease over time, which will cause a blue-shift in the LSPR peak. This blue-shift is thought to be a consequence of Ostwald ripening, via a softening of any sharp edges to a more thermodynamically stable arrangement.⁴² You can, therefore, artificially speed up the rate of Ostwald ripening by thermal treatment of the particles in an aqueous solution, lowering their aspect ratio (Figure 3C).

Figure 3. Controlling the aspect ratio of gold nanorods. A) Effect of increasing silver nitrate concentration on the nanorod aspect ratio and resulting optical properties upon synthesis. (1–5) indicate increasing additions of 4 mM AgNO3 from 50–300 μL to identical growth solutions. B) The effect of using single (1) and binary (2) surfactant systems in the synthesis of gold nanorods. C) The effect of heating on the LSPR peak (closed dots) and TSPR peak (open dots) of aqueous solutions of gold nanorods. D) An array of nanorods synthesized using combinations of tuning reactions discussed.

Poor Shape Control (4)

During any nanorod synthesis, the appearance of shape impurities is inevitable. However, these impurities can be monitored and minimized. Figure 2D illustrates a nanorod reaction with poor shape purity. Unwanted spheres in the reaction have a strong absorbance at 510–525 nm, resulting in an increase in the apparent TSPR peak. Thus, monitoring the LSPR/TSPR ratio is a strong indicator of overall shape purity. A “good” ratio of at least 3.5 indicates ~90% shape purity.^59–60 If a high quantity of impurities is observed, this can indicate that reactants are too old, not enough AgNO₃ was used in the reaction, reaction temperature was too high, or too high of a BDAC/CTAB ratio was used, to name a few potential problems. Post-synthesis separations can be performed to remove unwanted shapes, as discussed in the next section. Likewise, several variations of the basic cylindrical shape of nanorods can emerge during their synthesis. Binary surfactant reactions such as those using both BDAC and CTAB generate rods with sharper corners, giving them a more “brick-like” structure (Figure 4A) versus the standard CTAB reaction (Figure 4B). Spectrally, this can be observed as an additional small peak slightly red-shifted from the typical TSPR peak (Figure 2A vs 2C). “Dog bones” or “dumbbells” are another feature that can dramatically affect the spectra of nanorods (Figure 4C). Several groups report these features appear upon secondary additions of ascorbic acid and are pH driven.^32,50–51 We have observed these same features via adding excessive ascorbic acid in a single addition. It is possible to “soften” features like sharp corners and dog bones through thermal treatment, resulting in a reduced intensity of their additional spectral features and an overall blue-shift of their LSPR.  

Figure 4. TEMs of gold nanorods demonstrating a correlation between absorbance spectra and anisotropic geometry of gold nanorods. Insets are typical corresponding UV/Vis spectra for the given shape, spanning 400–1,000 nm. A) Gold nanorods synthesized in binary surfactant system containing CTAB and BDAC. B) Gold nanorods synthesized using only CTAB. C) Dog bone- or dumbbell-shaped nanoparticles.

Insufficient Purification (5)

After any synthesis, purification is required to remove excess reactants and reduce the overall concentration of CTAB for storage. If left in the original reactants for 48 h or more, nanorods have a tendency to aggregate. Thus, purification is typically accomplished by pelleting via centrifugation, and the main pitfall is aggregation post-centrifugation. Since different sizes of particles will require different spin speeds to adequately drop out of solution, optimization for your particular nanorods is necessary. Centrifuging particles at too high of a speed or for too long can lead to the formation of aggregates, while under-centrifuging results in nanorod losses. This is especially true for nanorods with higher aspect ratios with absorbance outside of the visible spectra. Counterintuitively, higher aspect ratio rods are more stable in solution and, thus, require higher spin speeds for precipitation. Therefore, it is recommended that you check the UV/Vis spectra of supernatants before discarding. It is also important to be mindful of the final concentration of CTAB in your solution, as the removal of too much CTAB can cause the nanorods to become unstable. Typically, we recommend maintaining >10 mM surfactant through all purification steps. Particles can then be safely diluted to 1 mM CTAB for final storage. As seen in Figure 5, centrifugation can also be used for additional separation and purification of a sample with high shape impurities. While the majority of particles will remain together in a pellet (3), you can see on the fringes a differentiation in color (1 and 2) indicative of sphere and rod separation. While the method is inefficient, centrifugal separation can be utilized to increase purity and has been reported previously.⁶¹ Other groups have also explored multiphase centrifugal separations⁶² and flocculation63 techniques with some success.

Figure 5. A) Top-down view of centrifuge tube containing gold nanoparticle pellet. Particles partially separate according to size and particle geometry: 1) Very high shape purity nanorod population. 2) Primarily nanosphere population. 3) Mix of both populations. B) Photo of (1) highly purified gold nanorods and (2) primarily spheres from pellet in A).

Incomplete Characterization (6)

In order to properly characterize nanorods, it is important to use multiple complimentary techniques to assess their quality. The most commonly used characterization tools are UV/Vis spectrophotometers, transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), and zeta potential. As discussed previously, UV/Vis spectroscopy is the most useful tool for measuring aspect ratio, shape purity, and rod quality for an ensemble of nanoparticles. However, UV/Vis provides little information on actual size. TEM and SEM are the most robust tools for verifying particle dimension, but without taking a proper number of images and doing so at multiple magnifications, it is easy to accidently bias the true average dimension of your particles. Figure 6 illustrates just how different the quality of a lot of nanorods can look based on what section of a TEM grid you choose to observe.⁴⁷ Figure 6C demonstrates that particles can have a tendency to “self-sort” when drying on a grid. Groups of impurities such as spheres or cubes can separate from sections of rods leaving the false impression of a high shape purity synthesis.

Figure 6. TEM’s of gold nanorods illustrating selection bias. A) Section of TEM grid indicates high purity rod synthesis. B) Different section of same grid shown by (A) indicates a large number of impurities in sample. C) TEM demonstrates shape segregation drying pattern on TEM grid.

Techniques such as dynamic light scattering (DLS), which are typically employed for the characterization of spherical nanoparticles, rely on algorithms that are not designed for detection and sizing of nanorods due to their anisotropic geometry. However, measurement of the zeta potential can still act as a good marker of stability for nanorods stabilized by CTAB. Typically, stable as-synthesized gold nanorods after purification have a zeta potential >+30 mV; lower values indicate particle instability and a high likelihood of aggregation with time. Alternatively, gold nanorods can undergo ligand exchanges to be sterically stabilized by neutral moieties, such as polyethylene glycol (PEG). This method is often employed to remove CTAB and reduce particle toxicity, resulting in a zeta potential of ±5 mV.

Conclusions

The synthesis of gold nanorods via colloidal seed-mediated growth offers the user tunability over a wide range of aspect ratios and corresponding LSPR optical peaks, with the opportunity for easy volumetric synthesis scaling. However, this tunability comes at a price; a synthesis that requires precise control over a number of variables in multiple phases of the process. The extensive list of variables leads to difficulty in reproducibility and consistency of reactions, potentially frustrating researchers who are moving toward large-scale in vivo studies. Because of these challenges, small-scale manufacture can be costly to maintain.⁶⁴ Therefore, many researchers opt to purchase nanorods rather than produce them in-house, as many companies now provide them as products with good quality control and standardized characterization.

References

1. Lee K, El-Sayed MA. 2005. Dependence of the Enhanced Optical Scattering Efficiency Relative to That of Absorption for Gold Metal Nanorods on Aspect Ratio, Size, End-Cap Shape, and Medium Refractive Index. J. Phys. Chem. B. 109(43):20331-20338. https://doi.org/10.1021/jp054385p

2. Jain P, Huang X, El-Sayed I, El-Sayed MA. 2008, 41 (12), 1578–86. . Acc. Chem. Res.. https://doi.org/10.1021/ar7002804

3. Loo C, Lee M, Hirsch L, West J, Halas N, Drezek R. 2004. Proc. SPIE 2004, 5327, 1–4. https://doi.org/10.1117/12.553184

4. Huang X, El-Sayed IH, Qian W, El-Sayed MA. 2007. Cancer Cells Assemble and Align Gold Nanorods Conjugated to Antibodies to Produce Highly Enhanced, Sharp, and Polarized Surface Raman Spectra:  A Potential Cancer Diagnostic Marker. Nano Lett.. 7(6):1591-1597. https://doi.org/10.1021/nl070472c

5. ELSAYED I, HUANG X, ELSAYED M. 2006. Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles. Cancer Letters. 239(1):129-135. https://doi.org/10.1016/j.canlet.2005.07.035

6. Aslan K, Leonenko Z, Lakowicz JR, Geddes CD. 2005. Annealed Silver-Island Films for Applications in Metal-Enhanced Fluorescence: Interpretation in Terms of Radiating Plasmons. J Fluoresc. 15(5):643-654. https://doi.org/10.1007/s10895-005-2970-z

7. Aslan K, Lakowicz JR, Geddes CD. 2005. Plasmon light scattering in biology and medicine: new sensing approaches, visions and perspectives. Current Opinion in Chemical Biology. 9(5):538-544. https://doi.org/10.1016/j.cbpa.2005.08.021

8. Travis K, Aaron J, Harrison N, Sokolov K. 2008. Plasmonics in Biology and Medicine V, VoDinh, T.; Lakowicz, J. R., Eds. 2008; . Vol. 6869, pp H8690–H8690

9. Mubeen S, Lee J, Lee W, Singh N, Stucky GD, Moskovits M. 2014. On the Plasmonic Photovoltaic. ACS Nano. 8(6):6066-6073. https://doi.org/10.1021/nn501379r

10. Moran CE, Steele JM, Halas NJ. 2004. Chemical and Dielectric Manipulation of the Plasmonic Band Gap of Metallodielectric Arrays. Nano Lett.. 4(8):1497-1500. https://doi.org/10.1021/nl049635c

11. Maier S, Brongersma M, Kik P, Meltzer S, Requicha A, Atwater H. 2001. Adv. Mater. . 13 (19), 1501–1505 https://doi.org/10.1002/1521-4095(200110)13:19<1501::AID-ADMA1501>3.0.CO;2-Z

12. Jackson JB, Halas NJ. 2004. Surface-enhanced Raman scattering on tunable plasmonic nanoparticle substrates. Proceedings of the National Academy of Sciences. 101(52):17930-17935. https://doi.org/10.1073/pnas.0408319102

13. Gryczynski I, Malicka JB, Gryczynski Z, Nowaczyk K, Lakowicz JR. 2004. Surface plasmon-coupled directional fluorescence emission. https://doi.org/10.1117/12.530048

14. Cole JR, Halas NJ. 2006. Optimized plasmonic nanoparticle distributions for solar spectrum harvesting. Appl. Phys. Lett.. 89(15):153120. https://doi.org/10.1063/1.2360918

15. Charnay C, Lee A, Man S, Moran CE, Radloff C, Bradley RK, Halas NJ. 2003. Reduced Symmetry Metallodielectric Nanoparticles:  Chemical Synthesis and Plasmonic Properties?. J. Phys. Chem. B. 107(30):7327-7333. https://doi.org/10.1021/jp034309r

16. Xu X, Du Q, Peng B, Xiong Q, Hong L, Demir HV, Wong TKS, Ko Kyaw AK, Sun XW. 2014. Effect of shell thickness on small-molecule solar cells enhanced by dual plasmonic gold-silica nanorods. Appl. Phys. Lett.. 105(11):113306. https://doi.org/10.1063/1.4896516

17. Mackey MA, Ali MRK, Austin LA, Near RD, El-Sayed MA. 2014. The Most Effective Gold Nanorod Size for Plasmonic Photothermal Therapy: Theory and In Vitro Experiments. J. Phys. Chem. B. 118(5):1319-1326. https://doi.org/10.1021/jp409298f

18. Dickerson EB, Dreaden EC, Huang X, El-Sayed IH, Chu H, Pushpanketh S, McDonald JF, El-Sayed MA. 2008. Gold nanorod assisted near-infrared plasmonic photothermal therapy (PPTT) of squamous cell carcinoma in mice. Cancer Letters. 269(1):57-66. https://doi.org/10.1016/j.canlet.2008.04.026

19. Zhang L, Xia K, Lu Z, Li G, Chen J, Deng Y, Li S, Zhou F, He N. 2014. Efficient and Facile Synthesis of Gold Nanorods with Finely Tunable Plasmonic Peaks from Visible to Near-IR Range. Chem. Mater.. 26(5):1794-1798. https://doi.org/10.1021/cm403109k

20. Jia H, Fang C, Zhu X, Ruan Q, Wang YJ, Wang J. 2015. Synthesis of Absorption-Dominant Small Gold Nanorods and Their Plasmonic Properties. Langmuir. 31(26):7418-7426. https://doi.org/10.1021/acs.langmuir.5b01444

21. Pan S, Chen M, Li H. 2001. Aqueous gold sols of rod-shaped particles prepared by the template method. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 180(1-2):55-62. https://doi.org/10.1016/s0927-7757(00)00759-7

22. Sun Y. 2002. Shape-Controlled Synthesis of Gold and Silver Nanoparticles. 298(5601):2176-2179. https://doi.org/10.1126/science.1077229

23. Mock JJ, Oldenburg SJ, Smith DR, Schultz DA, Schultz S. 2002. Composite Plasmon Resonant Nanowires. Nano Lett.. 2(5):465-469. https://doi.org/10.1021/nl0255247

24. Loweth C, Caldwell W, Peng X, Alivisatos A, Schultz P. 1999. Angew. Chem. Int. Ed. . 38 (12), 1808–1812 https://doi.org/10.1002/(SICI)1521-3773(19990614)38:12<1808::AID-ANIE1808>3.0.CO;2-C

25. Li Z, Chung S, Nam J, Ginger DS, Mirkin CA. 2003. Living Templates for the Hierarchical Assembly of Gold Nanoparticles. Angew. Chem. Int. Ed.. 42(20):2306-2309. https://doi.org/10.1002/anie.200351231

26. Yu, Chang S, Lee C, Wang CRC. 1997. Gold Nanorods:  Electrochemical Synthesis and Optical Properties. J. Phys. Chem. B. 101(34):6661-6664. https://doi.org/10.1021/jp971656q

27. Ghoreishi SM, Behpour M, Khoobi A. 2012. Central composite rotatable design in the development of a new method for optimization, voltammetric determination and electrochemical behavior of betaxolol in the presence of acetaminophen based on a gold nanoparticle modified electrode. Anal. Methods. 4(8):2475. https://doi.org/10.1039/c2ay25268f

28. Huang S, Ma H, Zhang X, Yong F, Feng X, Pan W, Wang X, Wang Y, Chen S. 2005. Electrochemical Synthesis of Gold Nanocrystals and Their 1D and 2D Organization. J. Phys. Chem. B. 109(42):19823-19830. https://doi.org/10.1021/jp052863q

29. Alekseeva AV, Bogatyrev VA, Khlebtsov BN, Mel?nikov AG, Dykman LA, Khlebtsov NG. 2006. Gold nanorods: Synthesis and optical properties. Colloid J. 68(6):661-678. https://doi.org/10.1134/s1061933x06060019

30. Zhu J, Yong K, Roy I, Hu R, Ding H, Zhao L, Swihart MT, He GS, Cui Y, Prasad PN. 2010. Additive controlled synthesis of gold nanorods (GNRs) for two-photon luminescence imaging of cancer cells. Nanotechnology. 21(28):285106. https://doi.org/10.1088/0957-4484/21/28/285106

31. Nikoobakht B, El-Sayed MA. 2003. Preparation and Growth Mechanism of Gold Nanorods (NRs) Using Seed-Mediated Growth Method. Chem. Mater.. 15(10):1957-1962. https://doi.org/10.1021/cm020732l

32. Wang C, Wang T, Ma Z, Su Z. 2005. pH-tuned synthesis of gold nanostructures from gold nanorods with different aspect ratios. Nanotechnology. 16(11):2555-2560. https://doi.org/10.1088/0957-4484/16/11/015

33. Smith DK, Korgel BA. 2008. The Importance of the CTAB Surfactant on the Colloidal Seed-Mediated Synthesis of Gold Nanorods. Langmuir. 24(3):644-649. https://doi.org/10.1021/la703625a

34. Ye X, Zheng C, Chen J, Gao Y, Murray CB. 2013. Using Binary Surfactant Mixtures To Simultaneously Improve the Dimensional Tunability and Monodispersity in the Seeded Growth of Gold Nanorods. Nano Lett.. 13(2):765-771. https://doi.org/10.1021/nl304478h

35. Ye X, Gao Y, Chen J, Reifsnyder DC, Zheng C, Murray CB. 2013. Seeded Growth of Monodisperse Gold Nanorods Using Bromide-Free Surfactant Mixtures. Nano Lett.. 13(5):2163-2171. https://doi.org/10.1021/nl400653s

36. Koeppl S, Ghielmetti N, Caseri W, Spolenak R. 2013. Seed-mediated synthesis of gold nanorods: control of the aspect ratio by variation of the reducing agent. J Nanopart Res. 15(3): https://doi.org/10.1007/s11051-013-1471-2

37. Johnson CJ, Dujardin E, Davis SA, Murphy CJ, Mann S. 2002. Growth and form of gold nanorods prepared by seed-mediated, surfactant-directed synthesis. J. Mater. Chem.. 12(6):1765-1770. https://doi.org/10.1039/b200953f

38. Burrows ND, Harvey S, Idesis FA, Murphy CJ. 2017. Understanding the Seed-Mediated Growth of Gold Nanorods through a Fractional Factorial Design of Experiments. Langmuir. 33(8):1891-1907. https://doi.org/10.1021/acs.langmuir.6b03606

39. Murphy CJ, Sau TK, Gole AM, Orendorff CJ, Gao J, Gou L, Hunyadi SE, Li T. 2005. Anisotropic Metal Nanoparticles:  Synthesis, Assembly, and Optical Applications. J. Phys. Chem. B. 109(29):13857-13870. https://doi.org/10.1021/jp0516846

40. Alex SA, Satija J, Khan MA, Bhalerao GM, Chakravarty S, Kasilingam B, Sivakumar A, Chandrasekaran N, Mukherjee A. Etching-based transformation of dumbbell-shaped gold nanorods facilitated by hexavalent chromium and their possible application as a plasmonic sensor. Anal. Methods. 7(13):5583-5592. https://doi.org/10.1039/c5ay00935a

41. Murphy CJ, Thompson LB, Chernak DJ, Yang JA, Sivapalan ST, Boulos SP, Huang J, Alkilany AM, Sisco PN. 2011. Gold nanorod crystal growth: From seed-mediated synthesis to nanoscale sculpting. Current Opinion in Colloid & Interface Science. 16(2):128-134. https://doi.org/10.1016/j.cocis.2011.01.001

42. Zou R, Zhang Q, Zhao Q, Peng F, Wang H, Yu H, Yang J. 2010. Thermal stability of gold nanorods in an aqueous solution. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 372(1-3):177-181. https://doi.org/10.1016/j.colsurfa.2010.10.012

43. Jana N. 2005. Gram-Scale Synthesis of Soluble, Near-Monodisperse Gold Nanorods and Other Anisotropic Nanoparticles. Small. 1(8-9):875-882. https://doi.org/10.1002/smll.200500014

44. Vigderman L, Zubarev ER. 2013. High-Yield Synthesis of Gold Nanorods with Longitudinal SPR Peak Greater than 1200 nm Using Hydroquinone as a Reducing Agent. Chem. Mater.. 25(8):1450-1457. https://doi.org/10.1021/cm303661d

45. Perez-Juste J, Pastoriza-Santos I, Liz-Marzan L, Mulvaney P. 2005. Coord. Chem. Rev. 2005, . 249 (17–18), 1870–1901

46. Ye X, Jin L, Caglayan H, Chen J, Xing G, Zheng C, Doan-Nguyen V, Kang Y, Engheta N, Kagan CR, et al. 2012. Improved Size-Tunable Synthesis of Monodisperse Gold Nanorods through the Use of Aromatic Additives. ACS Nano. 6(3):2804-2817. https://doi.org/10.1021/nn300315j

47. Scarabelli L, Sánchez-Iglesias A, Pérez-Juste J, Liz-Marzán LM. 2015. A ?Tips and Tricks? Practical Guide to the Synthesis of Gold Nanorods. J. Phys. Chem. Lett.. 6(21):4270-4279. https://doi.org/10.1021/acs.jpclett.5b02123

48. Scarabelli L, Grzelczak M, Liz-Marzán LM. 2013. Tuning Gold Nanorod Synthesis through Prereduction with Salicylic Acid. Chem. Mater.. 25(21):4232-4238. https://doi.org/10.1021/cm402177b

49. Jana NR, Gearheart L, Murphy CJ. 2001. Wet Chemical Synthesis of High Aspect Ratio Cylindrical Gold Nanorods. J. Phys. Chem. B. 105(19):4065-4067. https://doi.org/10.1021/jp0107964

50. Ratto F, Matteini P, Rossi F, Pini R. 2010. Size and shape control in the overgrowth of gold nanorods. J Nanopart Res. 12(6):2029-2036. https://doi.org/10.1007/s11051-009-9712-0

51. Gou L, Murphy CJ. 2005. Fine-Tuning the Shape of Gold Nanorods. Chem. Mater.. 17(14):3668-3672. https://doi.org/10.1021/cm050525w

52. Jana NR, Gearheart L, Murphy CJ. 2001. Wet Chemical Synthesis of High Aspect Ratio Cylindrical Gold Nanorods. J. Phys. Chem. B. 105(19):4065-4067. https://doi.org/10.1021/jp0107964

53. Lohse SE, Murphy CJ. 2013. The Quest for Shape Control: A History of Gold Nanorod Synthesis. Chem. Mater.. 25(8):1250-1261. https://doi.org/10.1021/cm303708p

54. Wang H, Wu Y, Lassiter B, Nehl CL, Hafner JH, Nordlander P, Halas NJ. 2006. Symmetry breaking in individual plasmonic nanoparticles. Proceedings of the National Academy of Sciences. 103(29):10856-10860. https://doi.org/10.1073/pnas.0604003103

55. Low A, Bansal V. A visual tutorial on the synthesis of gold nanoparticles. Biomed. Imaging Interv. J.. 6(1): https://doi.org/10.2349/biij.6.1.e9

56. Meena SK, Celiksoy S, Schäfer P, Henkel A, Sönnichsen C, Sulpizi M. The role of halide ions in the anisotropic growth of gold nanoparticles: a microscopic, atomistic perspective. Phys. Chem. Chem. Phys.. 18(19):13246-13254. https://doi.org/10.1039/c6cp01076h

57. Chen HM, Peng H, Liu R, Asakura K, Lee C, Lee J, Hu S. 2005. Controlling the Length and Shape of Gold Nanorods. J. Phys. Chem. B. 109(42):19553-19555. https://doi.org/10.1021/jp053657l

58. Pérez-Juste J, Correa-Duarte MA, Liz-Marzán LM. 2004. Silica gels with tailored, gold nanorod-driven optical functionalities. Applied Surface Science. 226(1-3):137-143. https://doi.org/10.1016/j.apsusc.2003.11.013

59. Link S, Mohamed MB, El-Sayed MA. 1999. Simulation of the Optical Absorption Spectra of Gold Nanorods as a Function of Their Aspect Ratio and the Effect of the Medium Dielectric Constant. J. Phys. Chem. B. 103(16):3073-3077. https://doi.org/10.1021/jp990183f

60. Huang X, Neretina S, El-Sayed MA. 2009. Gold Nanorods: From Synthesis and Properties to Biological and Biomedical Applications. Adv. Mater.. 21(48):4880-4910. https://doi.org/10.1002/adma.200802789

61. Sharma V, Park K, Srinivasarao M. 2009. Shape separation of gold nanorods using centrifugation. Proceedings of the National Academy of Sciences. 106(13):4981-4985. https://doi.org/10.1073/pnas.0800599106

62. Akbulut O, Mace CR, Martinez RV, Kumar AA, Nie Z, Patton MR, Whitesides GM. 2012. Separation of Nanoparticles in Aqueous Multiphase Systems through Centrifugation. Nano Lett.. 12(8):4060-4064. https://doi.org/10.1021/nl301452x

63. Park K, Koerner H, Vaia RA. 2010. Depletion-Induced Shape and Size Selection of Gold Nanoparticles. Nano Lett.. 10(4):1433-1439. https://doi.org/10.1021/nl100345u

64. 2017. NanoHybrids Make or Buy Gold Nanoparticles: A Quick Economic Analysis. [Internet]. NanoHybrids. Available from: https://nanohybrids.net/pages/make-or-buy-gold-nanoparticles-a-quick-economic-analysis