ARL-TN-0755 ● MAY 2016

US Army Research Laboratory

Fluorescent Pressure Response of 

Protein-Nanocluster Polymer Composites

by Alin Cristian Chipara, Mark H Griep, Timothy Walter, and 

Abby L West

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ARL-TN-0755 ● MAY 2016

US Army Research Laboratory

Fluorescent Pressure Response of 

Protein-Nanocluster Polymer Composites

by Alin Cristian Chipara, Mark H Griep, Timothy Walter, and 

Abby L West

Weapons and Materials Research Directorate, ARL

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Fluorescent Pressure Response of Protein-Nanocluster Polymer Composites

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Alin Cristian Chipara, Mark H Griep, Timothy Walter, and Abby L West

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US Army Research Laboratory

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14. ABSTRACT

This research focuses on the uses of polymer gold nanocluster (PNC) composites as pressure sensitive indicators of brain 

damage. The PNC composites are made up of protein coated gold nanoclusters and a styrene-ethylene/butylene-styrene (SEBS)

mineral oil composite, invented at the US Army Research Laboratory. The PNC composites were mechanically tested using a 

custom made setup and an Instron frame under various pressures that match different levels of damage. The stress levels studied 

in this technical note are lower than those previously studied by other groups and provide a unique perspective. The data shows 

that fluorescence quenching is present throughout the PNC samples and can be linked to mineral oil content. This research lays 

the groundwork for future pressure studies and identifies a key issue when using oil inside these PNCs.

15. SUBJECT TERMS

protein stabilized nanoclusters, pressure response, pressure cell, fluorescence, polymer matrices

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Contents

List of Figures iv

Acknowledgments v

1. Introduction and Background 1

2. Synthesis of Materials 2

2.1 Synthesis of BSA-Stabilized Gold Nanoclusters 2

2.2 Pressure Generation Setup 3

2.3 Emission Measurements 4

2.4 Data Analysis 4

3. Results and Discussion 5

4. Summary and Conclusions 7

5. References 8

List of Symbols, Abbreviations, and Acronyms 10

Distribution List 11
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List of Figures

Fig. 1 Instron testing setup for fluorescence change. UV light travels through 

optics and goes to a detector, which can identify the changes in the 

emission of BSA. The acrylic load cell sits in the middle of the path 

between the UV source and detector. In the upper-right-hand corner 

you can see the composition inside the acrylic cell. ..............................3

Fig. 2 This figure shows the emission data of the SEBS composites. a) QD 

control in 15% SEBS, b) BSA:AuNC in 15% SEBS, and c) 

BSA:AuNC in 5% SEBS. Each line represents an averaged 0.78 MPa 

interval. ..................................................................................................4

Fig. 3 The emission spectra of BSA and BSA in SEBS upon excitation at 

370 nm ...................................................................................................5

Fig. 4 Positive pressure response of BSA:AuNCs in 15% SEBS composite. 

Data generated without the use of sandwich pressure cell setup. ..........6
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Acknowledgments

This research was supported in part by an appointment to the Research Participation 

Program at the US Army Research Laboratory (ARL) administered by the Oak 

Ridge Institute for Science and Education through an interagency agreement 

between the US Department of Energy and ARL. This research was funded under 

the ARL Head Protection component of the US Army Natick Soldier RD&E Center

(NSRDEC). We thank NSRDEC for supporting part of this work. 

We would also like to thank Dr Raj Gupta and Dr Shashi Karna for helpful 

conversations relating to the aqueous-phase polymer gold nanocluster (PNC)

fluorescence pressure response. We would like to thank Dr Randy Mrozek for 

providing the styrene-ethylene/butylene-styrene (SEBS):mineral oil composites 

that were developed as a brain tissue surrogate at ARL. Finally, we would like to 

thank Alexis Fakner and Travis Tumlin for their assistance and advice with this 

work.
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1. Introduction and Background

Gold nanoclusters, specifically protein stabilized gold nanoclusters, have been 

heavily researched and have shown a myriad of possible applications including uses 

in sensors,

1,2 bioassays,1,3 and imaging.4,5 Gold nanoclusters are sub 2 nm 

nanoparticles and do not exhibit plasmon resonance like nanoparticles.

6 Instead, 

gold nanoclusters exhibit fluorescent effects similarly to semiconductor-based 

quantum dots and are not nearly as toxic due to their lack of heavy metals.

7 This 

makes them a prime candidate for biological applications and in-vivo sensing, 

imaging, and labeling.

5,8 Due to their role as fluorophores and desirable

biocompatibility, nanoclusters can take over many possible applications from more 

toxic fluorophores. These applications include mapping protein interactions or 

monitoring drug distribution in a system.2,9

There are several synthesis routes to create nanoclusters; however, we focused on 

the protein stabilized synthesis method10 as the protein shell confers interesting 

mechanical sensing properties as observed in our previously published work and 

work by Zhang et al.11,12,13 In this method, the protein will stabilize and reduce the 

gold nanoclusters while making them more suitable for in vivo uses.2,6 Spurred by 

the research and novel findings of Zhang et al. and previous work from our group, 

which showed a positive linear relationship between fluorescence and pressure; we 

sought to design a composite system that would take advantage of this relationship 

and exploit it to model brain damage. Zhang et al. hypothesized that the pressure

dependence of the bovine serum albumin:gold nanoclusters (BSA:AuNCs) was due 

to protein ligands compressing around the gold nanoclusters. We tried to use a setup 

that retains the same working principle as Zhang et al. while allowing us to use 

solid samples and create a model for brain damage. To this end, we used styreneethylene/butylene-styrene (SEBS) as the matrix to simulate brain tissue and 

examined the emission of BSA. The SEBS system is a system engineered at the US 

Army Research Laboratory (ARL) by Dr Randy Mrozek and colleagues, which was 

designed to specifically mimic the modulus of brain tissue.

14,15

Brain injury, specifically shock-induced brain injury, has been an area of great 

interest. With recent controversies around concussions, this research is becoming 

more important. Previous brain injury models have used a hard outer shell and soft 

gel13 to model brain damage from pressure waves. Our eventual aim is to begin to 

understand brain trauma through the use of synthetic models utilizing brainmimicking polymer composites doped with pressure responsive protein 

nanoclusters at traumatic brain injury-relevant pressures. Recent research by Alley 

et al. shows that their brain model feels an amplification of the force from 
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shockwaves (i.e., shockwaves in the range of 15–108 psig [0.1–0.74 MPa] actually 

feel like an impact in the range of 300–500 psig [2.07–3.44 MPa] on the brain). 

Generally, pressures above 200 psig (1.38 MPa) cause fatality and 29–75 psig (0.2–

0.52 MPa) cause lung damage.

16 In accordance with this data, the range of 0–6 MPa 

is extremely important for understanding concussions and brain damage and has 

not been properly explored. Although, Zhang et al. investigated a wide range of 

pressures they focused more on high pressures (up to 2 GPa) and only investigated 

BSA:AuNC in an aqueous solution.11 More recently, reports by West et al. show a 

similar upward trend in fluorescence in pressures as low as 50 MPa; however, this 

is still an order of magnitude above the targeted pressure range to study brain 

damage.12,13

The purpose of this report is to use BSA:AuNCs in a matrix that closely resembles 

living tissue and to characterize the pressure response of the clusters in this matrix. 

Our data indicates that there is a positive pressure dependence for BSA:AuNCs in 

15% SEBS/85% mineral oil up to 2 MPa, which is consistent with previous 

unpublished data from our group using composites of the same nature. After this 

pressure regime the dependence is lost and a decrease in observed fluorescence can 

be detected, which can be attributed to loss of observed pressure due to an 

inefficient design in the pressure vessel or a quenching interaction between the 

mineral oil in the composite and the BSA:AuNCs. Interestingly, no pressure 

response can be observed with the 5% SEBS/95% mineral oil. We predict that the 

mineral oil is indeed quenching the fluorescence of the clusters from the data 

presented in this technical note. 

2. Synthesis of Materials

2.1 Synthesis of BSA-Stabilized Gold Nanoclusters

To synthesize the BSA:AuNCs, HAuCl4 (5 mL, 10 mM) was mixed with BSA 

(5 mL, 50 mg/mL) at 37 oC and stirred rapidly. The mixture was brought up to a 

pH of approximately 12 through the addition of 0.5 mL 1 M NaOH. The solution 

was left to incubate at 37 °C for 12 h after which it gained a brown tint. When 

illuminated by ultraviolet (UV) the BSA:AuNC glowed red. Centrifugation was 

used to remove any impurities and the BSA:AuNC solution was lyophilized for 

later use. 

To manufacture the BSA:AuNC and control quantum dot (QD) composites, 

BSA:AuNCs and QDs were dispersed into SEBS at a temperature of 85 °C. BSA 

solution (250 μL of 25 mg/ml BSA:AuNCs into 10 mL of SEBS matrix) and QD 

(4 μL into 10 mL of SEBS) were doped in various SEBS concentrations 
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(5% and 15%). The composite was left to cool and solidify. Before the test began 

the BSA:AuNC and QD composites were heated to 70 °C to allow transfer into the 

load cell. The cell was loaded in a sandwich configuration where 15% SEBS (85% 

mineral oil 15% SEBS, heated to 120 °C) was drop cast into an acrylic load cell,

then the BSA:AuNC/SEBS or QD/SEBS composite was poured on top, after which 

a final layer of 15% SEBS was dropped to protect the composite. Each layer was 

allowed to cool 5–10 min before the next layer was added to prevent diffusion of 

the BSA:AuNC or QD/SEBS into the top and bottom. This sandwich-based 

pressure setup protected the doped composites from direct contact with the mandrel 

and ensured that there was no loss of doped composite from the load cell due to 

leakage. 

2.2 Pressure Generation Setup

To apply pressure to the BSA:AuNCs, an acrylic load cell was employed for 

hydrostatic pressure testing (Fig. 1).

Fig. 1 Instron testing setup for fluorescence change. UV light travels through optics and 

goes to a detector, which can identify the changes in the emission of BSA. The acrylic load cell 

sits in the middle of the path between the UV source and detector. In the upper-right-hand 

corner you can see the composition inside the acrylic cell.

The pressure testing was carried out using an Instron frame. As previously stated, 

the acrylic load cell used a sandwich structure to protect the BSA:AuNC/SEBS 

composite from direct contact with the mandrel. The sandwich structure can be seen 

in the upper right inset of Fig. 1. The top and bottom are composed of 15% SEBS 

and the middle layer has the BSA:AuNC SEBS composite that needed to be tested. 

The composite was tested up to 400 lbf (6.24 MPa) in 50 lbf (0.78 MPa) increments. 
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The fluorescence was measured using a detector and a UV source was used to excite 

the BSA at approximately 370 nm. The pressure setup itself used a UV light source,

which was collimated to ensure wide field excitation of the gel and passed through 

an OD4 550-nm filter to remove any infrared (IR) emission from the UV source, 

then through the sample and finally passed through an OD4 

575-nm filter to block the UV and hit the detector. 

2.3 Emission Measurements

The measurements were done using a Jaz detector system and an approximately 

370-nm light source for excitation of the clusters. The measurements were done 

with an integration time of 500 ms with 3 averaged scans for each 50 lbf (0.78 MPa) 

increment. Each experiment was repeated 5 times for consistency. 

2.4 Data Analysis

The runs were averaged out and plotted so that each load was represented by one 

spectra (as seen in Fig. 2). The maximum emission of each run was taken and 

plotted to create a trend line representative of the behavior of the composite.

Fig. 2 This figure shows the emission data of the SEBS composites. a) QD control in 15% 

SEBS, b) BSA:AuNC in 15% SEBS, and c) BSA:AuNC in 5% SEBS. Each line represents an 

averaged 0.78 MPa interval.
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3. Results and Discussion

A baseline measurement of the emission spectrum of the BSA:AuNCs in aqueous 

solution and while embedded in the SEBS matrix was taken to observe any matrix 

effects on the emission profile (Fig. 3). The emission spectra shows peaks for 

aqueous BSA:AuNCs in the 670–680 nm range, which shifts to approximately 640 

nm upon incorporation into the SEBS matrix. These spectra are consistent with the 

fluorescence spectra of BSA:AuNCs found in literature.10 Notably, the 

BSA:AuNCs experience peak broadening and blue shifting upon incorporation into 

the polymer matrix. This observation is indicative of an interaction between the 

protein and the SEBS matrix. For example, the peak broadening may be an effect 

of an increased bond length between the nanocluster core and the surrounding 

protein ligands.

Fig. 3 The emission spectra of BSA and BSA in SEBS upon excitation at 370 nm

Instron mechanical testing revealed an unexpected trend when comparing both 

BSA:AuNC-SEBS matrix concentrations (Fig. 2). The BSA:AuNC-15% SEBS 

shows an initial positive response of up to 2 MPa; however, after this point there is 

a loss of pressure dependence paired with a decrease in fluorescence. The initial 

positive response matches previous unpublished work generated by our group using 

BSA:AuNCs in 15% SEBS without the sandwich structure (Fig. 4). 
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Fig. 4 Positive pressure response of BSA:AuNCs in 15% SEBS composite. Data generated 

without the use of sandwich pressure cell setup.

We hypothesize that the results observed above 2 MPa can either be attributed to a 

leakage of the 15% SEBS sandwich material past the mandrel and out of the cell, 

thus resulting in a loss of pressure or a quenching interaction between the mineral 

oil in the SEBS gel and the BSA:AuNCs. Mineral oil based fluorescence quenching 

is further supported when the 5% SEBS data for BSA:AuNCs is analyzed (Fig. 2c). 

In this data set there is no initial positive response, only a consistent linear decrease 

upon pressure addition. The 5% SEBS is much softer in modulus so that the 15% 

SEBS sample and has 10% higher mineral oil content. Thus, it is much easier for 

the mineral oil in the 5% gel to contact and quench the BSA:AuNC fluorescence. 

Future work will focus on quantifying their pressure response in stiffer gels to 

determine if there is indeed a relationship between pressure response and mineral 

oil content. Furthermore, we are also redesigning the pressure cell to ensure that no 

loss of the sandwich material occurs upon pressurization. We intend to overcome 

this challenge by using a much stiffer sandwich gel (i.e., 30% SEBS as opposed to 

the 15% SEBS that was used in this study).

QDs suspended in SEBS were used as a control to make sure these trends were not 

due to the experimental setup. These changes seen in QDsfurther support the theory 

that quenching is taking place in the BSA:AuNC-SEBS composites and is due to a 

unique interaction between the mineral oil in the composite and the BSA:AuNCs, 

as there is no significant change in fluorescence with the QD control. 
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4. Summary and Conclusions

We have studied the effects of pressure on fluorescence every 50 lbf or 0.78 MPa 

up to 400 lbf or 6.24 MPa. Through the use of a sandwich structure and an acrylic 

load cell we were able see that several variables need to be addressed before any 

tissue injury conclusions can be determined from this type of study. More 

optimization is needed to fully understand the effect of pressure on the P:NC-SEBS 

composite systems. The data showed strong signs of quenching that appeared to be 

dependent upon the mineral oil content in the SEBS polymer composites. We 

predict that the higher mineral oil content paired with the softer modulus of the 5% 

SEBS gels leads to more direct interaction between the mineral oil and the 

BSA:AuNCs. This effect is not observed until above 2 MPa in the 15% SEBS 

composite, as higher pressures are needed in the stiffer gel to force interaction 

between the mineral oil and protein stabilized nanocluster. 

Future studies will focus on changing the SEBS polymer density and BSA:AuNC 

concentrations to determine the effect of mineral oil content on the system. In 

addition, improvements should be made to the setup including the addition of a 

pressure sensor onto the BSA layer to directly measure pressure. Due to the 

pressure being measured at the top of the mandrel it is hard to quantify the loading 

on the BSA itself, except to say that it is under the maximum load experienced by 

the mandrel. A more robust load cell and using the outlined optimized testing 

procedure would be of great use since it would enable researchers to go above 

8 MPa and explore the effect of much larger pressures on the system and compare 

it to previously published data. Different optics would allow testing to be done on 

pure BSA as well, thus leading to better controls. An enclosed, dark box setup 

would give much more control over many of the variables that can cause 

experimental error in the system and would give much more reliable results. 

In conclusion, we have successfully synthesized ARL-developed brain mimicking 

polymer/BSA:AuNC composites and studied their pressure response in the low 

MPa regime. The composites exhibited modulus-based pressure response, wherein 

the stiffer composite showed a positive pressure response up to 2 MPa, and whereas 

the more pliable composite did not show a pressure response.
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5. References

1. Huang CC, Yang Z, Lee KH, Chang HT. Synthesis of highly fluorescent gold 

nanoparticles for sensing Mercury (II). Angew Chem Int Edit. 2007;119:

6948–6952.

2. Chevrier DM, Chatt A, Zhang P. Properties and applications of proteinstabilized fluorescent gold nanoclusters: short review. J Nanophotonics. 2012.

3. Triulzi RC, Miodraq M, Giordani S, Serry M, Chiou WA, Leblanc RM. 

Immunoassay based on the andtibody-conjugated PAMAM-dendrimer-gold 

quantum dot complex. Chem Comm. 2006;48:5068–5070.

4. Vosch T, Yasuko A, Hslang JC, Richards CI, Gonzalez JI, Dickson MR. 

Strongly emissive individual DNA-encapsulated Ag nanoclusters as single 

molecule fluorophores. PNAS 31. 2007:12616–12621.

5. Wang F, Tan WB, Zhang Y, Fan X, Wang M. Luminescent nanomaterials for 

biological labelling. Nanotechnology. 2006;17:R1-R13.

6. Lin CJ, Yang T, Lee C, Huang SH, Sperling RA, Zanella M, Li JK, Shen J, 

Wang H, Yeh H, Parak WJ, Chang WH. Synthesis, characterization, and 

bioconjugation of fluorescent gold nanoclusters toward biological labeling

applications. ACSNano. 2009;3:395–401.

7. Kirchner C, Liedl T, Kudera S, Pellegrino T, Javier AM, Gaub HE, Stolzle S, 

Fertig N, Parak WJ. Nano Letters. 2005;l5:331–338.

8. Gao X, Cui Y, Levenson RM, Chung LWK, Nie S. In vivo cancer targeting 

and imaging with semiconductor quantum dots. Nature Biotechnology. 

2004;22:969–976.

9. Medintz IL, Uyeda TH, Goldman ER, Mattoussi H. Quantum dot 

bioconjugates for imaging, labelling and sensing. Nature Materials. 

2005;4:435–446.

10. Xie, J., Zheng, Y., and Ying, J. Y. (2009) Protein-directed synthesis of highly 

fluorescent gold nanoclusters, J Am Chem Soc 131, 888-889.

11. Zhang M, Dang YQ, Liu TY, Li HW, Wu YQ, Li Q, Wang K, Zou B. Pressureinduced fluorescence enhancement of the BSA-protected gold nanoclusters 

and the corresponding conformational changes of protein, J Phys Chem C. 

2013;117:639–647.
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12. West AL, Griep MH, Knoblauch C, Karna SP. Use of an ethanol-driven 

pressure cell to measure hydrostatic pressure response of protein-stabilized 

gold nanoclusters. Aberdeen Proving Ground (MD): Army Research 

Laboratory (US); 2015. Report No.: ARL-TR-7577.

13. West AL, Griep MH, Knoblauch C, Karna SP. Stabilizing protein effects on 

the pressure sensitivity of fluorescent gold nanoclusters; 2016. Report No.: 

ARL-TR-7572.

14. Chantawansri TL, Duncan AJ, Ilavsky J, Stokes KK, Berg MC, Mrozek RA, 

Lenhart JL, Beyer FL, Andzelm JW. Phase behavior of SEBS triblock 

copolymer gels. J. Polym. Sci. B Polym. Phys. 2011;49:1479–1491.

15. Mrozek RA, Leighliter B, Gold CS, Beringer IR, Yu JH, VanLandigham MR, 

Moy P, Foster MH, Lenhart JL. The relationship between mechanical 

properties and ballistic penetration depth in a viscoelastic gel. Journal of 

Mechanical Behavior and Biomedical Materials. 2015;44:109–120.

16. Alley MD, Schimizze BR, Son SF. (2011) Experimental modeling of explosive 

blast-related traumatic brain injuries. NeuroImage. 2011;54:S45–S54.
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List of Symbols, Abbreviations, and Acronyms

ARL US Army Research Laboratory

AuNC gold nanocluster

BSA bovine serum albumin

BSA:AuNC bovine serum albumin:gold nanocluster

IR infrared

NSRDEC US Army Natick Soldier RD&E Center

PNC polymer gold nanocluster

QD quantum dot

SEBS styrene-ethylene/butylene-styrene

UV ultraviolet
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