Controlled Bacteria - Gold Nanorod Interactions for Enhancement of Optoacoustic Contrast Anton Liopoa, Paul J. Derryb, Boris Ermolinskyc, Richard Sua, André Conjusteaua, Sergey Ermilova, Eugene R. Zubarevb and Alexander Oraevskya aTomoWave Laboratories Inc., Houston, TX, b Department of Chemistry, Rice University, Houston, TX cUniversity of Texas at Brownsville, TX 1. ABSTRACT Gold-based contrast agents, gold nanorod (GNR), were designed for the enhancement of optoacoustic signal. After synthesis, the GNR-CTAB complexes were modified by pegylation (PEG), or replacement of CTAB (cetyl trimethylammonium bromide) with MTAB (16-mercaptohexadecyl trimethylammonium bromide) for coverage of gold nanorods with heparin (GNR-HP). Modified GNR are purified through centrifugation and filtration. GNR-CTAB can be used as a model of positively charged gold surface for quantitative optoacoustic sensing in GNR-bacteria interactions, whereas GNR-PEG and GNR-HP can be used as negatively charged gold surface models. We studied controlled agglomeration of contrast agents with the bacteria E.Coli and Vibrio Cholerae. For bacterial sensing, the localized plasmon resonance peak shifts as a function of electrostatic binding, which was detected with two different wavelengths through 3D optoacoustic imaging. Key words: CTAB, PEG, MTAB, gold nanorods, optoacoustic imaging and sensing, bacteria particle targeting 2. INTRODUCTION The threat of contaminants such as bacterial, viral, and chemical toxins has long been recognized as a serious public health concern. Foodborne disease has been a persistent and serious threat to public health. During 2011, 47.8 million illnesses caused by foodborne diseases were reported to the CDC. Of those cases, 128,000 resulted in hospitalization and just over 3,000 people died. (CDC 2011*) The most popular detection methods are cell culture and colony counting methods, polymerase chain reaction (PCR), and immunology-based or two-photon Rayleigh scattering methods and biosensors [1-4]. However, these techniques are labor intensive and time consuming often requiring professional operation optically clean media. An alternative method, optoacoustic detection, can be used in optically turbid media with greater sensitivity than existing methodologies such as colorimetric reactions and fluorescence [5]. Furthermore, it is the only viable method of detection when studying heavily light-scattering samples such as milk or ground water. The principle of optoacoustic detection relies on the occurrence of thermal confinement which happens when the laser pulse duration is small compared to the transit time of sound through the penetration depth of the light. In this case, instantaneous heating of the medium can be assumed. The interaction of nanoparticles with biological systems ranging from biomolecules to biological cells is significant for a range of applications such as high-resolution biomedical imaging, gene sequencing for molecular diagnostics, and sensitive electronic devices. [3, 6-8]. Noble metal nanostructures generate significant interest because of their unique properties, including large optical field enhancements resulting in the strong scattering and absorption of light. The absorption spectra of GNRs exhibit two surface plasmon absorption bands, the transverse and the longitudinal surface plasmon resonance (LSPR). The latter is very sensitive to a large number of factors and is often utilized in sensing applications [9]. GNRs have also attracted great interest as a novel platform for nanobiotechnology and biomedicine because of convenient surface bioconjugation with molecular probes and remarkable optical properties related with the LSPR [10-13]. GNRs of various size and aspect ratio are promising for biomedical applications [9,10,14-19]. GNRs can absorb light about thousand times more strongly than an equivalent volume of an organic dye [20,21]. In addition, GNRs are ideal optoacoustic contrast agents because their Photons Plus Ultrasound: Imaging and Sensing 2014, edited by Alexander A. Oraevsky, Lihong V. Wang, Proc. of SPIE Vol. 8943, 894368 · © 2014 SPIE · CCC code: 1605-7422/14/$18 · doi: 10.1117/12.2044628 Proc. of SPIE Vol. 8943 894368-1 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 09/02/2014 Terms of Use: http://spiedl.org/terms optical absorption spectra can be tuned over a broad wavelength range in the near infrared (NIR) spectral region [22-25] and detecting the acoustic waves resulting from the optoacoustic effect [21]. It was previously demonstrated that positively charged GNRs stabilized by CTAB are effective for self-assembling into an electrically percolating monolayer of different nanoshapes on gram-positive bacterium, such as Bacillus cereus [26]. In this report we found that CTAB-capped GNRs can successfully be deposited on gram-negative bacteria, such as E. coli and V. cholerae and this effect has significant implications for optoacoustic sensing. * http://www.cdc.gov/foodborneburden/PDFs/FACTSHEET_A_FINDINGS_updated4-13.pdf 3. MATERIALS AND METHODS Fabrication of Gold Nanorods (GNRs) We previously described a general strategy for the synthesis and stabilization of GNRs with thiol-terminal polyethylene glycol (mPEG-thiol, or PEG in this report), which displaces the original CTAB surfactant bilayer to provide biocompatibility of the resulting optoacoustic contrast agent [10,12,27-29]. To produce charged, biocompatible GNRs we used a thiolated analogue of CTAB, mercapto-cetyltrimethylammonium bromide (MTAB) as reported by Zubarev in 2012 [10]. These MTAB-GNRs have dramatically lower cytotoxicity; however, they exhibit poor stability in PBS and other high ionic strength buffers. To resolve this problem, we report the preparation of a new heparin-functionalized MTAB-GNRs with significantly improved saline stability. Presented below are the details of our GNR modification protocol adapted from previously reported methodologies [10,27,30,31]. CTAB-GNR Fabrication In a typical synthesis, gold seed particles were prepared by adding 0.250 mL of an aqueous 0.01 M solution of HAuCl4 • 3H2O to 7.5 mL of a 0.1 M CTAB solution in a 15 mL test tube. Afterwards, 0.600 mL of an ice-cold, aqueous 0.01 M NaBH4 solution was added all at once. This seed solution was used 2-4 h after its preparation. Next, a growth solution was prepared consisting of 4.75 mL 0.10 M CTAB, 0.200 mL of 0.01 M HAuCl4 • 3H2O, and 0.030 mL of 0.01 M AgNO3 solutions added in sequence and gently mixed by inversion. The solution at this stage had a bright orange color. Next, 0.032 mL of 0.10 M ascorbic acid was added to the Au(III)/CTAB solution and the combined solution became colorless upon mixing. After 10 min, a solution of gold seed nanoparticles was added to the growth solution and gently mixed for 10 s and left undisturbed for 1-3 h. Afterwards, the solution was left under thermostatic conditions for 24 hours at 30°C. Prior to surface functionalization with mPEG-SH or MTAB and heparin, the CTAB-GNRs were centrifuged at 1000 g for 10 min to separate aggregates. The pellet was removed and only the supernatant fraction was retained. Surface Modification of CTAB-GNRs with mPEG-SH PEGylated-GNRs [27,32] were prepared using previously synthesized CTAB coated GNRs. The GNRs were centrifuged at 14000 g for 10 min and resuspended in 9 mL milli-Q (18.0 MΩ) H2O (optical density ~ 1.0). Next, 0.1 mL of 2 mM K2CO3 was added to 1 mL of aqueous GNR solution and 0.1 mL of 0.1 mM mPEG-SH-5000 (Laysan Bio Inc., Arab, AL). The resulting mixture was kept on a rocking platform at room temperature overnight. Excess mPEG-SH was removed from solution by two rounds of centrifugation (12000×g 10 min) and resuspended in a NaCl solution (pH 7.4) in final concentration 0.5 nM (OD near 2.0). UV-vis extinction spectra were measured by Thermo Scientific Evolution 201 spectrophotometer. Surface Functionalization of CTAB-GNRs with MTAB In a typical synthesis, 40 mL of 0.25 mg/mL CTAB-GNRs, (Liz-Marzan 2011) were centrifuged at 12,000 g for 10 min and resuspended in 40 mL of milli-Q (18.0 MΩ) H2O. In a separate vial, 20 mg of previously prepared MTAB [10] was dissolved in 2 mL milli-Q H2O by gentle heating and then added at once to the CTAB-GNR solution. The solution of CTAB-GNRs was incubated overnight at 30°C and then centrifuged at 12,000 g for 10 min and redispersed in 40 mL milli-Q H2O six times. Secondary Modification of MTAB-GNRs by Heparin Sulfate 20 mg porcine intestine-derived heparin sulfate (HS) (Sigma-Aldrich, St. Louis, MO) was dissolved in 2 mL milli-Q H2O and then added to 10 mL of MTAB-GNR stock. The combined solution was mixed briefly and incubated at 30°C for 1 h. The HS modified MTAB-GNR solution was then centrifuged four times at 12,000 g for 10 min. ζ = -50.9 mV. Proc. of SPIE Vol. 8943 894368-2 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 09/02/2014 Terms of Use: http://spiedl.org/terms Bacterial Cell Culturing Vibrio cholerae classical strain 0395 and E. coli K802 were grown in a Luria-Bertani (LB) broth at 37°C with shaking (200 rpm) overnight. Each bacterial strain was diluted to 1:100 in 3 mL of fresh LB and grown until they reached the mid-logarithmic phase of growth (optical density at 600 nm [OD600] about 0.6). Bacterial cultures were centrifuged at 1500 × g for 5 min to separate cells from supernatant. Incubation both bacteria with modified GNR in final concentration 0.25 nN (OD around 1) were in 0.9% Saline (Sodium Chloride), pH 7.3 from 5 min till 72 hours at room temperature. Optoacoustic (OA) imaging system and measurements bacteria GNR interaction In these studies we used a commercial prototype of a three-dimensional optoacoustic tomography system developed for preclinical research at TomoWave Laboratories (Houston, TX), and introduced in our earlier publications [29,33-37].