ARL-TN-0753 ● APR 2016

US Army Research Laboratory

In-vitro Synthesis of Gold Nanoclusters in 

Neurons

by Maggie Gillan and Nicole Zander

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ARL-TN-0753 ● APR 2016

US Army Research Laboratory

In-vitro Synthesis of Gold Nanoclusters in 

Neurons

by and Nicole Zander

Weapons and Materials Research Directorate, ARL

Maggie Gillan

Oak Ridge Institute for Science and Education, Oak Ridge, TN

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April 2016

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Technical Note

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1 August 2013–31 October 2014

4. TITLE AND SUBTITLE

In-vitro Synthesis of Gold Nanoclusters in Neurons

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6. AUTHOR(S)

Maggie Gillan and Nicole Zander

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US Army Research Laboratory

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Aberdeen Proving Ground, MD 21005-5069

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ARL-TN-0753

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14. ABSTRACT

Gold nanoclusters (AuNCs) synthesized intracellularly have potential for use as in-vivo or in-vitro pressure probes for 

evaluating the effects of traumatic brain injury. AuNCs were grown within NG-108-15 neuroblastoma-glioma hybrid cells, 

indicated by increased fluorescence intensity at 488 nm. Effect of gold salt concentration on fluorescence intensity was 

evaluated. Cell viability at 24-h postgold salt addition was tested via an MTS cell viability assay.

15. SUBJECT TERMS

gold nanoclusters, neurons, in-vitro synthesis, fluorescence, viability

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Nicole Zander

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Contents

List of Figures iv

1. Introduction 1

2. Materials and Methods 2

3. Results and Discussion 2

4. Conclusions 5

5. References 6

Distribution List 7
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List of Figures

Fig. 1 Fluorescence, phase contrast, and combined confocal micrographs of 

NG108-15 cells 24 h after addition of a) 0 mM, b) 0.1 mM, 

c) 1 mM, and d) 10 mM of gold salt......................................................3

Fig. 2 Fluorescence intensity data from NG108-15 cells incubated with 

gold salt for 24 h ....................................................................................4

Fig. 3 Viability as determined by a MTS assay of NG108-15 cells 

incubated with varied concentrations of HAuCl4 salt ............................5
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1. Introduction

Noble metal nanoclusters (NCs) composed of a few hundred atoms are an emerging 

class of luminescent nanomaterials. These NCs typically have diameters less than 

2 nm, approaching the Fermi wavelength of electrons, which leads to very unique 

optical, electrical, and chemical properties compared with nanoparticles.1–3 In 

particular, they have strong photoluminescence and tunable emission wavelengths. 

Conjugation of the NCs with proteins stabilizes the system but also is known to 

alter the fluorescence intensity under pressure due to ligand conformation changes 

for select gold NC (AuNC) systems.4 Thus, these nanomaterials could conceivably 

be used as nano-pressure sensors in a variety of biological and material 

applications. 

One pertinent application is in the study of traumatic brain injury (TBI), which 

affects over 1.7 million people in the United States annually.5 The majority of TBIs 

are classified as mild or mTBIs and account for 80% of all head injuries.

6 mTBIs 

are rarely accompanied with neuroanatomical abnormalities detectable by 

conventional imaging methods such as magnetic resonance imaging.5 Thus,

diagnosis relies heavily on self-reported symptoms and is often undiagnosed even 

though mTBIs and particularly repetitive mTBIs have a significant effect on 

cognitive function. Frequently, Soldiers in particular are unaware that they have 

even suffered a TBI and are often treated for posttraumatic stress disorder, since 

the symptoms at least initially are the same. But mTBI is actually physical brain 

damage and should follow a very different treatment path. One of the reasons for 

this problem is the lack of good pressure sensors on the Soldier to declare when he 

or she has experienced a probable TBI. 

The aforementioned NCs with appropriate ligands could potentially serve as a 

useful tool to diagnose pressure doses. Protein NCs are typically synthesized by 

traditional wet chemistry methods but have recently been shown to be synthesized 

in-vitro by mammalian cell lines.7 These could potentially be isolated from the cells 

or used for in-vitro TBI research and living pressure sensor devices. This work 

details the in-vitro synthesis of AuNCs inside a neuronal and glial co-culture cell 

line (NG108-15) and the effect of gold salt concentration on fluorescence intensity 

and viability.
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2. Materials and Methods

NG108-15 neuroblastoma-glioma hybrid cells (108CC15) (ATCC HB-12317) 

were maintained in culture in Dulbecco’s Modified Eagle Medium supplemented 

with 1% sodium hypoxanthine, aminopterin, and thymidine supplement and 10% 

fetal bovine serum. Cells were incubated at 37 °C and 5% CO2.

A 25-mM stock solution of chloroauric acid (HAuCl4, Sigma ) was prepared in 

phosphate buffered saline. The gold salt solution was sterilized by filtering through 

a syringe filter before adding to cells in the desired amount. Imaging was carried 

out with a Zeiss LSM 700 fluorescent confocal microscope equipped with a 10×

objective. Data were quantified in Zeiss ZEN Black software.

Cell viability at 24 h after gold salt addition was evaluated using an MTS cell 

viability assay from Promega. Cells were grown to approximately 80% confluence 

in a 24-well plate. Stock gold salt solution was added to each well according to the 

desired gold salt concentration. The MTS assay was carried out at 24 h after 

addition of the gold salt. Cell absorbance measurements were collected using a plate 

reader set to 490 nm (Biotek).

3. Results and Discussion

The NG 108-15 cells were found to synthesize AuNCs intracellularly over a period 

of 24 h. This was confirmed via fluorescent confocal microscopy. Phase contrast 

and fluorescent micrographs for each gold salt concentration (0.1–10 mM) are 

shown in Fig. 1. Higher concentrations of salt solution led to an increase in 

fluorescence but did not appear to affect cellular morphology. There is evidence of 

similar amounts of cell spreading at all concentrations. A 100-mM gold salt 

concentration was attempted but immediately led to cell death and no data are

included in this report. Quantification of the fluorescence data confirms that there 

is a significant increase in fluorescence intensity with increasing concentration of 

gold salt in the media for all concentrations (except between 0 and 0.1 mM) (Fig. 2). 

This is attributed to production of AuNCs within the cells. 
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Fig. 1 Fluorescence, phase contrast, and combined confocal micrographs of NG108-15 cells 

24 h after addition of a) 0 mM, b) 0.1 mM, c) 1 mM, and d) 10 mM of gold salt
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Fig. 2 Fluorescence intensity data from NG108-15 cells incubated with gold salt for 24 h

An MTS cell viability assay was performed on the cells to test viability in the 

presence of the gold salt. The MTS dye, in the presence of living cells, is reduced 

to a purple formazan product with an absorption maximum at about 490 nm. The 

viability was reduced at all concentrations of gold salt, compared with the control, 

but there was no significant difference in cell death between the concentrations 

evaluated (Fig. 3). The evaluation of cell death at time points beyond 24 h might 

yield different results, as it is expected that higher concentrations may be more toxic 

to the cells based on the results from the 100-mM solutions discussed previously. 

Potentially, other cell lines might yield brighter clusters with less cell death.
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Fig. 3 Viability as determined by a MTS assay of NG108-15 cells incubated with varied 

concentrations of HAuCl4 salt

4. Conclusions

It was shown that NG108-15 cells synthesize AuNCs when incubated in the 

presence of a gold salt. Since AuNCs are sensitive to pressure, this shows promise 

as a method to study the effects of blast trauma on neuronal cells. There is a 

decrease in cell viability as concentration of gold salt increases, but approximately 

25% of the seeded cells remain viable even at concentrations as high as 10 mM. 
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5. References

1. Xavier PL, Chaudhari K, Baksi A, Pradeep T. Protein-protected luminescent 

noble metal quantum clusters: an emerging trend in atomic cluster 

nanoscience. Nano Rev. 2012;3:14767−14783.

2. Maity P, Xie S, Yamauchiab M, Tsukuda T. Stabilized gold clusters: from 

isolation toward controlled synthesis. Nanoscale. 2012;4:4027−4037.

3. Xie J, Zheng Y, Ying JY. Protein-directed synthesis of highly fluorescent gold 

nanoclusters. J Am Chem Soc. 2009;131(3):888−889.

4. Zhang M, Dang Y-Q, Liu T-Y, Li H-W, Wu Y, Li Q, Wang K, Zou BJ. 

Pressure-induced fluorescence enhancement of the BSA-protected gold 

nanoclusters and the corresponding conformational changes of protein. Phys 

Chem. 2013;117(1):639–647.

5. Kane MJ, Hatic H, Delic V, Dennis JS, Butler CL, Saykally JN, Citron BA.

Modeling the pathobiology of repetitive traumatic brain injury in immortalized 

neuronal cell lines. Brain Res. 2011;1425:123–131.

6. Meaney DF, Smith DH. Biomechanics of concussion. Clin Sports Med. 

2011;30(1):19–31.

7. Wang J, Zhang G, Li Q, Jiang H, Liu C, Amatore C, Wang X. In vivo self-bioimaging of tumors through in situ biosynthesized fluorescent gold 

nanoclusters. Sci Rep. 2013;3:1157–1162.
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