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. Author manuscript; available in PMC: 2010 Jan 20.
Published in final edited form as: Nano Res. 2008 Dec 1;1(6):450. doi: 10.1007/s12274-008-8047-y

Two-photon Luminescence Imaging of Bacillus Spores Using Peptide-functionalized Gold Nanorods

Wei He 1,‡, Walter A Henne 1,3,‡, Qingshan Wei 1, Yan Zhao 1, Derek D Doorneweerd 1,3, Ji-Xin Cheng 1,2, Philip S Low 1,3, Alexander Wei 1,3,*
PMCID: PMC2808639  NIHMSID: NIHMS166399  PMID: 20098661

Abstract

Bacillus subtilis spores (a simulant of Bacillus anthracis) have been imaged by two-photon luminescence (TPL) microscopy, using gold nanorods (GNRs) functionalized with a cysteine-terminated homing peptide. Control experiments using a peptide with a scrambled amino acid sequence confirmed that the GNR targeting was highly selective for the spore surfaces. The high sensitivity of TPL combined with the high affinity of the peptide labels enables spores to be detected with high fidelity using GNRs at femtomolar concentrations. It was also determined that GNRs are capable of significant TPL output even when irradiated at near infrared (NIR) wavelengths far from their longitudinal plasmon resonance (LPR), permitting considerable flexibility in the choice of GNR aspect ratio or excitation wavelength for TPL imaging.

Keywords: Bionanotechnology, nanorods, nonlinear optics, pathogen detection, peptides

Introduction

Optical modalities for detecting pathogenic microorganisms should be sufficiently sensitive to enable the rapid and accurate identification of bioparticles in natural settings, while discriminating against background signals to minimize false positives. Nonlinear optical modalities such as two-photon luminescence (TPL) are well suited for this purpose because of their intrinsically low autofluorescence under multiphoton excitation conditions, resulting in much higher signal-to-noise than conventional (linear) optical imaging methods [1–4]. TPL and other multiphoton processes can be excited at near infrared (NIR) frequencies below 750 nm, a spectral region which permits photons to penetrate through biological tissues with relatively high transmittivity. These attributes give TPL and related imaging modalities the potential to detect individual pathogens in complex environments, if coupled with a suitably developed agent for targeted imaging.

Plasmon-resonant gold nanorods (GNRs) have recently been shown to produce strong TPL activity using femtosecond-pulsed laser excitation [5–8], and provide excellent contrast for TPL imaging applications [8–12]. GNRs can support a higher absorption cross section at NIR frequencies per unit volume than most other nanostructures [13], are efficiently prepared in micellar surfactant solutions using seeded growth conditions [14, 15], and can be stabilized by mild chemical treatments [16] to facilitate their practical application as TPL contrast agents. GNRs can also be functionalized with chemisorptive ligands such as thiols [17, 18] or dithiocarbamates [10–12, 19] for their targeted delivery to cell surface receptors.

In this report, we demonstrate that GNRs can be used in bacterial detection schemes based on TPL imaging using a high-affinity peptide sequence specific for the spores of Bacillus subtilis, an established and widely used simulant species of Bacillus anthracis, the causative agent of anthrax [20–23]. Current technologies employed for the detection of spores or other pathogenic organisms include the use of traditional antimicrobial culture techniques, antibody-based capture methods, immunoassays, and genomic analysis based on polymerase chain reaction (PCR) amplification [22, 24–27]. All of these methods suffer from some limitations in the time to result or the number of steps involved. TPL imaging with functionalized GNRs is direct and can be complementary or even advantageous for pathogen detection, as its sensitivity has already been demonstrated at the single-particle limit for in vivo applications and also for the targeted labeling of tumor cells in vitro [8, 11, 12].

1. Experimental

TPL imaging was performed using a home-built inverted microscope system (IX-50, Olympus) equipped with a femtosecond-pulsed Ti:sapphire oscillator (Mira 900, Coherent) operating at 77 MHz, with a tunable wavelength output in the range of 700–900 nm. The luminescence was measured by a photomultiplier tube (Hamamatsu) with a bandpass filter of 500–600 nm. The excitation pulses were focused onto the bottom of an optically transparent cover dish (Biosciences, CA) using a 60× water-immersion objective (N.A. = 1.2, Olympus). Images were acquired at a resolution of 256 × 256 pixels (26.2 × 26.2 μm) at a scanning rate of 2 frames per second.

GNRs were synthesized as previously reported [14–16]. Briefly, seeded growth was carried out in a micellar solution of cetyltrimethylammonium bromide (CTAB) with AgNO3 as an additive, followed by treatment with Na2S 15–20 min after injection of the seed solution to arrest further growth. GNRs (ca. 5 mg Au) were precipitated by centrifugation for 15 min at 9,000 rpm (12,500 g) and separated from excess CTAB, then redispersed in 10 mL deionized water to an optical density (O.D.) of ca. 14. The CTAB-stabilized GNRs were then treated with 5 mL of a 1% polystyrenesulfonate (PSS) solution (Mw ~70 kD, sonicated 30 min in 1 mmol/L NaCl before use) and the mixture was allowed to sit overnight, followed by the separation of GNRs from excess PSS by centrifugation. The PSS-stabilized GNRs were resuspended in deionized water to a final O.D. of 0.8. Several batches of GNRs were prepared in this manner, with λmax values of the final dispersions ranging from 685 nm to 875 nm.

A cysteine-terminated Bacillus binding oligopeptide (NHFLPKVGGGC) and a scrambled control sequence (LFNKHVPGGGC) were synthesized, purified, and characterized as previously described [22, 28]. 50 μL of a phosphate-buffered solution (PBS) containing oligopeptide (1 mg/mL, pH 7.4) was added to 5 mL of PSS-stabilized GNRs (O.D. 0.8) and allowed to sit at room temperature for at least 5 hours (Fig. 1). The functionalized GNRs were separated from excess ligand by centrifugation for 10 min at 9,000 rpm, and resuspended in 5 mL of a 1 mmol/L NaCl solution (final O.D. 0.5–0.6). Quantitative amino acid and ICP-MS analysis of a concentrated solution of GNRs (71 × 28 nm, based on TEM size analysis) functionalized with the homing oligopeptide (O.D. ca. 12, λex=785 nm) was found to contain 174 pmol/mL peptide and 423 ppm (μg/mL) of Au, corresponding to a GNR concentration of 0.83 nmol/L and a peptide-to-GNR ratio of 210. A similar analysis with the control peptide sequence yielded a somewhat higher peptide-to-GNR ratio, due to an uncertainty in the amount of peptide used.

Figure 1.

Figure 1

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Scheme describing the preparation of peptide-functionalized GNRs and their targeted labeling of Bacillus subtilis spores

Bacillus subtilis sp 168 were cultured for 72 h in sporulation media, purified, and quantified as previously described [22, 28]. In a typical experiment, spores (106–109 per mL) were incubated for 45 minutes in the presence GNRs functionalized with either the homing peptide or negative control (scrambled sequence) at 37 °C (8 fmol/L – 8 pmol/L, or 5×1010–5×1013 GNRs per mL). The spores were subjected to centrifugation (2×5 min at 7000 rpm) with redispersion in fresh PBS to remove unbound GNRs, resulting in a suspension of GNR-labeled spores at final concentrations in the range 106–107 particles/mL. These were deposited onto a cover dish with an optically transparent bottom (Biosciences, CA) and imaged as described above.

2. Results and discussion

We first examined polystyrenesulfonate (PSS)-coated GNRs with different aspect ratios, to evaluate how these might impact their TPL activity at specific excitation wavelengths. The longitudinal plasmon resonance (LPR) responsible for the NIR-absorbing properties of GNRs is well known to be sensitive to particle aspect ratio [29], as well as to changes in the surface dielectric due to chemical adsorption [30]. The range of NIR tunability available by changes in aspect ratio is sufficient to produce GNRs with minimally overlapping LPR modes (Fig. 2a) [14–16]. The peak shifts due to electrostatic adsorption are less pronounced; in our case, only slight changes in LPR are observed with the adsorption of PSS or peptides on the GNR surface (Fig. 2b). Anionic polyelectrolytes such as PSS are often used to coat CTAB-stabilized GNRs to increase their dispersion stability in solutions at physiologically relevant pH and ionic strength, as well as to counteract dilution effects [31–34]. Stabilization issues must be addressed because multiple washes will reduce CTAB to below the critical micelle concentration (ca. 1 mmol/L) [10–12, 35], leading to the eventual flocculation of GNRs. It is worth mentioning that while PSS can help maintain the dispersion stability of GNRs in the short term, its adsorption to the CTAB-coated surface is not stable under shear conditions, indicating a need for more robust alternatives for GNR functionalization [36].

Figure 2.

Figure 2

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(a) Peptide-functionalized GNRs with longitudinal plasmon resonances at 685 nm (black line) and 875 nm (red line). (b) Absorption spectra of as-prepared GNRs (black line), PSS-coated GNRs (red line) and peptide-functionalized GNRs (blue line, λmax= 806 nm). Inset: Transmission electron microscopy (TEM, Philips CM-10, 80 kV) image of peptide-functionalized GNRs

In previous studies, we have shown that the TPL from GNRs is most intense when the excitation wavelength overlaps with the LPR band, which implies a reduction in TPL activity at nonresonant wavelengths [8]. However, the very low autofluorescence background intrinsic to multiphoton imaging may be sufficient to support TPL contrast even under off-resonant excitation conditions. To test this, PSS-stabilized GNRs with well-separated LPRs (λLPR = 715 and 835 nm) were deposited and immobilized onto mercaptopropylsiloxane-coated glass substrates [37], and subsequently exposed to pulsed NIR laser irradiation. As expected, the GNRs produced the maximum TPL contrast when excited at their respective LPR wavelengths, confirming the plasmon-resonant nature of two-photon absorption (Figures 3(a) and 3(d)), but the TPL signals produced at off-peak excitation were also significant (Figures 3(b) and 3(c)). This shows that the position of the LPR mode is not critical for generating TPL contrast with high signal-to-noise from GNRs.

Figure 3.

Figure 3

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Two-photon luminescence imaging of PSS-stabilized GNRs immobilized on thiol-derivatized glass coverslips. TPL signals were filtered through a bandpass filter with cutoffs above 500 nm and below 600 nm. (a), (c) λLPR = 715 nm; (b), (d) λLPR = 835 nm. (a), (b) TPL signals from GNRs using unpolarized excitation at 715 nm with an output power of 3 mW. (c), (d) TPL signals from GNRs using unpolarized excitation at 835 nm with an output power of 3 mW.

PSS-coated GNRs were then functionalized with the cysteine-terminated homing peptide (NHFLPKVGGGC), which was recently established as a high-affinity targeting ligand for Bacillus subtilis [22, 28]. Bacillus spores were incubated with the peptide-functionalized GNRs, then washed and examined by TPL microscopy using a confocal laser scanning microscope, with the Ti:sapphire laser tuned to the GNR plasmon resonance with an output power of 1 mW. The excitation beam was aligned for optimal generation of TPL signals, which are displayed as pseudocolor images (Fig. 4a). Spores labeled with the GNR-homing peptide conjugate were easily identified, whereas spores incubated with GNRs conjugated to the control peptide with scrambled sequence (LFNKHVPGGGC) did not produce detectable signals even with an output power of 30 mW, confirming the specific targeting and TPL signaling by the homing peptide and GNR, respectively (Fig. 4b). The TPL signal-to-background ratios are on the order of several hundred, as evaluated from line intensity profiles (Figs. 4c, d).

Figure 4.

Figure 4

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Two-photon luminescence imaging of peptide-functionalized GNRs on Bacillus subtilis spores. TPL signals were filtered through a bandpass filter with cutoffs at 500 and 600 nm. (a) Pseudocolor TPL image of spores incubated with GNRs functionalized with homing peptide, excited by NIR laser pulses. (b) No TPL signals were produced by spores incubated with GNRs functionalized with control peptide, using similar excitation conditions. (c), (d) TPL intensity profiles corresponding to the white lines in TPL images a and b, respectively. Color-coded scalebars in TPL images a and b correspond to the y-axis value in plot c.

With respect to detection, spores at low particle counts (106 and 107 per mL) were dispersed with peptide-conjugated GNRs at concentrations ranging from 8 fmol/L to 8 pmol/L (5×1010 to 5×1013 GNRs per mL), then collected, washed, and examined by TPL and phase-contrast microscopy to determine labeling efficiency (selected images are shown in Fig. 5). A complete correlation between the TPL and brightfield images was observed in every case, demonstrating the high fidelity of targeting by the GNR–peptide labels. The targeting efficiency of the homing peptide for the spore surface compares well with that of folate for its cognate receptor (Kd ~10−10 mol/L) [38, 39]. A comparison of the TPL images reveals that the spore labeling density is quite uniform, suggesting that the spore surfaces are saturated even at the lowest GNR concentrations used.

Figure 5.

Figure 5

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Targeting fidelity of Bacillus subtilis spores by peptide-functionalized GNRs. (a–d) TPL images of GNR-labeled spores isolated from the following suspensions: (a) 106 spores/mL and 5×1010 GNRs/mL; (b) 106 spores/mL and 5×1011 GNRs/mL; (c) 107 spores/mL and 5×1010 GNRs/mL; (d) 107 spores/mL and 5×1011 GNRs/mL. (e–h) Brightfield images of GNR-labeled spores corresponding to TPL images a–d. Bar = 5 μm.

3. Conclusions

Bacterial spores are readily detected by TPL imaging using peptide-functionalized GNRs. The flexibility and high signal-to-background ratios afforded by TPL imaging and the chemical stability of GNRs make this system attractive for further development. Peptide-functionalized GNRs can also be employed as multifunctional imaging and therapeutic agents for the selective detection and photothermal destruction of pathogens, as was recently demonstrated by the targeted delivery of GNRs to parasitic protozoans [40] and other bacteria [41]. The photophysical properties of the GNRs, combined with the efficiency of phage display methods for identifying peptide-based targeting ligands [28], provide the foundations for a new class of imaging agents with potential antibiotic activity.

Acknowledgments

This work is supported by the National Institute of Health (EB-001777) and also by the Department of Defense (W911SR-08-C-0001), administered through the U.S. Army RDECOM (Edgewood Contracting Division) and the Center for Sensing Science and Technology at Purdue University. We gratefully acknowledge Prof. Arthur Aronson for providing B. subtilis spores, Dr. Dorota Inerowicz for quantitative peptide analysis, and Prof. Ron Reifenberger, Prof. Yeong Kim, Dr. Haifeng Wang, and Dr. Alexei Leonov for helpful discussions.

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