Microdosimetric and radiobiological effects of gold nanoparticles at therapeutic radiation energies.

PURPOSE: The purpose of this study was to quantify the microscopic dose distribution surrounding gold nanoparticles (GNPs) irradiated at therapeutic energies and to measure the changes in cell survival in vitro caused by this dose enhancement. METHODS: The dose distributions from secondary electrons surrounding a single gold nanosphere and single gold nanocube of equal volume were both simulated using MCNP6. Dose enhancement factors (DEFs) in the 1 µm3 volume surrounding a GNP were calculated and compared between a nanosphere and nanocube and between 6 and 18 MV energies. This microscopic effect was explored further by experimentally measuring the cell survival of C-33a cervical cancer cells irradiated at 18 MV with varying doses of energy and concentrations of GNPs. Survival of cells receiving no irradiation, a 3 Gy dose, and a 6 Gy dose of 18 MV energy were determined for each concentration of GNPs. RESULTS: It was observed that the dose from electrons surrounding the gold nanocube surpasses that of a gold nanosphere up to a distance of 1.1 µm by 18.5% for the 18 MV energy spectrum and by 23.1% for the 6 MV spectrum. DEFs ranging from ∼2 to 8 were found, with the maximum DEF resulting from the case of the gold nanocube irradiated at 6 MV energy. Experimentally, for irradiation at 18 MV, incubating cells with 6 nM (0.10% gold by mass) GNPs produces an average 6.7% decrease in cell survival, and incubating cells with 9 nM (0.15% gold by mass) GNPs produces an average 14.6% decrease in cell survival, as compared to cells incubated and irradiated without GNPs. CONCLUSION: We have successfully demonstrated the potential radiation dose enhancing effects in vitro and microdosimetrically from gold nanoparticles.

Characterization of thermal and optical properties in porcine pancreas tissue.

BACKGROUND: Photothermal therapies have shown promise for treating pancreatic ductal adenocarcinoma when they can be applied selectively, but off-target heating can frustrate treatment outcomes. Improved strategies leveraging selective binding and localized heating are possible with precision medical approaches such as functionalized gold nanoparticles, but careful control of optical dosage and thermal generation would be imperative. However, the literature review revealed many groups assume liver properties for pancreas tissue or rely on insufficiently rigorous characterization studies. OBJECTIVE: The objective of this study was to determine the thermal conductivity and optical properties at 808/1064 nm wavelengths in healthy samples of fresh and frozen porcine pancreas ex vivo. METHODS: Thermal conductivity of the porcine pancreas tissue was measured by utilizing a hot plate and two K-type thermocouples. Experimental variables such as tissue sample thickness, hot plate temperature, and heat convection coefficient were estimated through the control experiments utilizing specimens with known thermal conductivity. Optical evaluations assessed light attenuation at the 808 and 1064 nm wavelengths (continuous wave, collimated beam) by measuring the light transmittance and reflectance of different tissue thicknesses. In turn, these measurements were input into an inverse adding-doubling program to estimate the optical absorption and reduced scattering coefficients. RESULTS: Interestingly, pancreas tissue thermal conductivity was demonstrated to have no significant difference (p > 0.5) between samples that were fresh, frozen for 7 days, or frozen for 14 days. Conversely, optical property assessment exhibited a significant difference (p < 0.001) between fresh and frozen tissue samples, with increased absorbance and reflectance within the frozen group. However, the optical attenuation values measured were substantially less than that of the liver or reported in previous pancreas studies, suggesting a wide overestimation of these properties. CONCLUSIONS: These thermal and optical properties are critical to the development of novel therapeutic strategies like plasmonic photothermal therapy, but perhaps more importantly, are invaluable towards informing better surgical planning and operative technique among the existing thermal approaches for treating pancreas tissue.

Assessment and Modeling of Plasmonic Photothermal Therapy Delivered via a Fiberoptic Microneedle Device Ex Vivo.

Plasmonic photothermal therapy (PPTT) has potential as a superior treatment method for pancreatic cancer, a disease with high mortality partially attributable to the currently non-selective treatment options. PPTT utilizes gold nanoparticles infused into a targeted tissue volume and exposed to a specific light wavelength to induce selective hyperthermia. The current study focuses on developing this approach within an ex vivo porcine pancreas model via an innovative fiberoptic microneedle device (FMD) for co-delivering light and gold nanoparticles. The effects of laser wavelengths (808 vs. 1064 nm), irradiances (20-50 mW·mm-2), and gold nanorod (GNR) concentrations (0.1-3 nM) on tissue temperature profiles were evaluated to assess and control hyperthermic generation. The GNRs had a peak absorbance at ~800 nm. Results showed that, at 808 nm, photon absorption and subsequent heat generation within tissue without GNRs was 65% less than 1064 nm. The combination of GNRs and 808 nm resulted in a 200% higher temperature rise than the 1064 nm under similar conditions. A computational model was developed to predict the temperature shift and was validated against experimental results with a deviation of <5%. These results show promise for both a predictive model and spatially selective, tunable treatment modality for pancreatic cancer.

Effects of Conformational Variation on Structural Insights from Solution-Phase Surface-Enhanced Raman Spectroscopy.

Surface-enhanced Raman scattering (SERS) spectra contain information on the chemical structure on nanoparticle surfaces through the position and alignment of molecules with the electromagnetic near field. Time-dependent density functional theory (TDDFT) can provide the Raman tensors needed for a detailed interpretation of SERS spectra. Here, the impact of molecular conformations on SERS spectra is considered. TDDFT calculations of the surfactant cetyltrimethylammonium bromide with five conformers produced more accurate unenhanced Raman spectra than a simple all-trans structure. The calculations and measurements also demonstrated a loss of structural information in the CH2/CH3 scissor vibration band at 1450 cm-1 in the SERS spectra. To study lipid bilayers, TDDFT calculations on conformers of methyl phosphorylcholine and cis-5-decene served as models for the symmetric choline stretch in the lipid headgroup and the C═C stretch in the acyl chains of 1,2-oleoyl-glycero-3-phosphocholine. Conformer considerations enabled a measurement of the distribution of double-bond orientations with an order parameter of SC═C = 0.53.

Synthesis and bioconjugation of alkanethiol-stabilized gold bipyramid nanoparticles.

Gold bipyramid (GBP) nanoparticles are promising for a range of biomedical applications, including biosensing and surface-enhanced Raman spectroscopy, due to their favorable optical properties and ease of chemical functionalization. Here we report improved synthesis methods, including preparation of gold seed particles with an increased shelf life of ∼1 month, and preparation of GBPs with significantly shortened synthesis time (< 1 h). We also report methods for the functionalization and bioconjugation of the GBPs, including functionalization with alkanethiol self-assembled monolayers (SAMs) and bioconjugation with proteins via carbodiimide cross-linking. Binding of specific antibodies to the nanoparticle-bound proteins was subsequently observed via localized surface plasmon resonance sensing. Rabbit IgG and goat anti-Rabbit IgG antibodies were used as a model system for antibody-antigen interactions. As-synthesized, SAM-functionalized, and bioconjugated bipyramids were characterized using scanning electron microscopy, UV-vis spectroscopy, zeta potential, and dynamic light scattering.

A detailed experimental and Monte Carlo analysis of gold nanoparticle dose enhancement using 6 MV and 18 MV external beam energies in a macroscopic scale.

Dose enhancement due to gold nanoparticles (GNPs) has been quantified experimentally and through Monte Carlo simulations for external beam radiation therapy energies of 6 and 18 MV. The highest enhancement was observed for the 18 MV beam at the highest GNP concentration tested, amounting to a DEF of 1.02. DEF is shown to increase with increasing concentration of gold and increasing energy in the megavoltage energy range. The largest difference in measured vs. simulated DEF across all data sets was 0.3%, showing good agreement.

A detailed Monte Carlo evaluation of 192Ir dose enhancement for gold nanoparticles and comparison with experimentally measured dose enhancements.

Gold nanoparticles (GNPs) have been studied extensively as promising radiation dose enhancing agents. In the current study, the dose enhancement effect of GNPs for Ir-192 HDR brachytherapy is studied using Monte Carlo N-Particle code, version 6.2 (MCNP6.2) and compared with experimental results obtained using Burlin cavity theory formalism. The Ir-192 source is verified using TG-43 parameters and dose enhancement factors (DEFs) from GNPs are simulated for three different mass percentages of gold in the GNP solution. These results are compared to DEFs previously reported experimentally by our group (Bassiri et al 2019 Med. Phys.) for a GNP-containing volume in an apparatus designed in-house to measure dose enhancement with GNPs for high dose rate (HDR) Ir-192 brachytherapy. An HDR Ir-192 Microselectron v2 r HDR brachytherapy source was modeled using MCNP6.2 using the TG-43 formalism in water. Anisotropy and radial dose function were verified against known values. An apparatus designed to measure dose enhancement to a 0.75 cm3 volume of GNPs from an Ir-192 brachytherapy seed with average energy of 0.38 MeV was built in-house and modeled using MCNP6.2. Burlin cavity correction factors were applied to experimental measurements. The macroscopic DEF was calculated for GNPs of size 30 nm at mass percentages of gold of 0.28%, 0.56% and 0.77%, using the repeating structures capability of MCNP6.2. DEF was calculated by dividing dose to the GNP solution by dose to water in the same volume. The radial dose function and anisotropy factor values at varying angles and distances were accurate when compared against known values. DEFs of 1.018 ± 0.003, 1.031 ± 0.003, and 1.041 ± 0.003 for GNP solutions containing mass percent of gold of 0.28%, 0.56% and 0.77%, respectively, were computed. These DEFs were within 2% of experimental values with Burlin cavity correction factors applied for all three mass percentages of gold.

Multiphysics Modeling of Plasmonic Photothermal Heating Effects in Gold Nanoparticles and Nanoparticle Arrays.

Induced hyperthermia has been demonstrated as an effective oncological treatment due to the reduced heat tolerance of most malignant tissues; however, most techniques for heat generation within a target volume are insufficiently selective, inducing heating and unintended damage to surrounding healthy tissues. Plasmonic photothermal therapy (PPTT) utilizes light in the near-infrared (NIR) region to induce highly localized heating in gold nanoparticles, acting as exogenous chromophores, while minimizing heat generation in nearby tissues. However, optimization of treatment parameters requires extensive in vitro and in vivo studies for each new type of pathology and tissue targeted for treatment, a process that can be substantially reduced by implementing computational modeling. Herein, we describe the development of an innovative model based on the finite element method (FEM) that unites photothermal heating physics at the nanoscale with the micron scale to predict the heat generation of both single and arrays of gold nanoparticles. Plasmonic heating from laser illumination is computed for gold nanoparticles with three different morphologies: nanobipyramids, nanorods, and nanospheres. Model predictions based on laser illumination of nanorods at a visible wavelength (655 nm) are validated through experiments, which demonstrate a temperature increase of 5 °C in the viscinity of the nanorod array when illuminated by a 150 mW red laser. We also present a predictive model of the heating effect induced at 810 nm, wherein the heating efficiencies of the various morphologies sharing this excitation peak are compared. Our model shows that the nanorod is the most effective at heat generation in the isolated scenario, and arrays of 91 nm long nanorods reached hyperthermic levels (an increase of at least 5 °C) within a volume of over 20 µm3.

Characterization and comparison of imaging contrast enhancement with PEG-functionalized gold nanoparticles in kV cone beam computed tomography and computed tomography imaging.

The purpose of this study is to define a simplified method to accurately predict and characterize kV cone beam computed tomography (kV CBCT) and computed tomography (CT) image contrast enhancement from gold nanoparticles (GNPs). Parameters of the kV CBCT of a Varian Novalis Tx linear accelerator and of a GE LightSpeed 4 Big Bore CT machine were modeled using the MCNP 6.2 Monte Carlo code. A 0.25 × 0.25 cm2 source, defined with a 100 kVp energy spectrum with appropriate filtration, was implemented in the MCNP6.2 model for kV CBCT, which also contained x- and y-blades and a full bowtie filter. A 1 cm3 cube of GNP solution (modeled as a mass percentage of gold in water) was placed 100 cm below the source. For the CT-simulator model, a source was defined with energy spectra for 80 and 140 kVp x-rays with appropriate filtration and angular spectrum. A 1 cm3 GNP solution was modeled as before and a detector was placed 40 cm below that. Attenuation coefficients of four GNP solutions were computed and Hounsfield unit (HU) values were calculated. The computed HU values were compared against experimentally measured values obtained by scanning batches of GNPs of various sizes and concentrations using a GE LightSpeed 4 Big Bore CT scanner at 80 kVp and 140 kVp energies, as well as the kV CBCT capability of a Varian Novalis Tx linear accelerator. HU analysis was carried out using Velocity Medical Solutions clinical CT image analysis software. The MCNP calculated HU values matched the measured values to within ± 5%. Image contrast enhancement analysis showed a total increase in HU of up to 223. The sample having the highest gold mass percentage tested showed the greatest increase in HU number compared to water.

Technical Note: Film-based measurement of gold nanoparticle dose enhancement for 192 Ir.

PURPOSE: The purpose of this work is to introduce a simple yet accurate technique to measure the dose enhancement factor (DEF) of a citrate-capped gold nanoparticle (GNP) solution using EBT3 film in an 192 Ir setup. METHODS: Dose enhancement factor is the ratio of absorbed dose in a solution compared to absorbed dose in water, assuming identical irradiation parameters. Citrate-capped GNPs were synthesized. An acrylic apparatus was constructed such that the EBT3 film was placed in charged particle equilibrium within the GNP solution with 0.28%, 0.56%, and 0.77% gold by mass. Sets of 12 dose measurements were collected for each GNP concentration as well as for water. The expected value of DEF was also calculated with the effective mass absorption coefficient of the GNP solution and water for an 192 Ir spectrum. Furthermore, Burlin cavity correction factors were calculated and experimentally verified. Experimental verification of the cavity correction was performed by measuring DEF using stacks of 1, 3, and 5 sheets of film and extrapolating the DEF to 0 sheets of film. RESULTS: Experimental cavity corrections agreed with those calculated with the Burlin cavity formalism. The calculated DEF was 1.013, 1.027, and 1.037 for the 0.28%, 0.56%, and 0.77% gold by mass GNP solutions, respectively. The corresponding uncorrected DEF measurement values were 1.013 ± 0.006, 1.024 ± 0.010, and 1.032 ± 0.006, respectively. When applying the Burlin cavity formalism, the final corrected DEF measurement values were 1.016 ± 0.006, 1.029 ± 0.010, and 1.039 ± 0.006, respectively. CONCLUSIONS: The experimental cavity correction results agreed with the theoretical Burlin calculations, which allowed for the Burlin corrections to be performed for all GNP concentrations and measured DEF values. The adjusted DEF values agreed with the theoretical calculations. Thus, these results indicate that a Burlin cavity calculation can be applied to correct film-based DEF measurements for 192 Ir.

Implementation of a simple clinical linear accelerator beam model in MCNP6 and comparison with measured beam characteristics.

Monte Carlo N-Particle 6 (MCNP6) is the latest version of Los Alamos National Laboratory's powerful Monte Carlo software designed to compute general photon, neutron, and electron transport using stochastic algorithms. Here we provide a case study of modeling the photon beam of a Varian 600C Clinical Linear Accelerator (linac), which is used to deliver radiation therapy, along with a comparison to experimentally measured beam characteristics. The source definition parameters in MCNP6, including the energy spectrum and angular spectrum of the photons, secondary and tertiary collimators, and a water phantom that tallied dose delivered at different points along the phantom are included. The experimental data for comparison was in the form of a percent depth dose curve as well as crossline and inline beam profiles. Experimental depth dose curve and beam profiles were acquired using a standard 0.125 cc ion chamber within a water phantom. In the computational model, the simulated depth dose curve was computed by tallying the total energy deposited in a stack of vertical slices down the depth of the phantom for percent depth dose curves. The simulated beam profiles were computed in a similar fashion, by tallying the energy deposited in a horizontal row, both in the x- and y-directions of cubic cells located at various depths. For the percent depth dose curve, a mean absolute percentage difference of 1.02%, 1.07%, and 1.94% were calculated for field sizes of 5 × 5 cm2, 10 × 10 cm2 and 20 × 20 cm2, respectively, between the model and experimental measurements were calculated. We present our model as an example to guide other MCNP6 users in the medical physics community to create similar beam models for biomedical dose estimation and research calculations for predicting dose to newly developed phantoms.

Toward Smaller Aqueous-Phase Plasmonic Gold Nanoparticles: High-Stability Thiolate-Protected ∼4.5 nm Cores.

Most applications of aqueous plasmonic gold nanoparticles benefit from control of the core size and shape, control of the nature of the ligand shell, and a simple and widely applicable preparation method. Surface functionalization of such nanoparticles is readily achievable but is restricted to water-soluble ligands. Here we have obtained highly monodisperse and stable smaller aqueous gold nanoparticles (core diameter ∼4.5 nm), prepared from citrate-tannate precursors via ligand exchange with each of three distinct thiolates: 11-mercaptoundecanoic acid, α-R-lipoic acid, and para-mercaptobenzoic acid. These are characterized by UV-vis spectroscopy for plasmonic properties; Fourier transform infrared (FTIR) spectroscopy for ligand-exchange confirmation; X-ray diffractometry for structural analysis; and high-resolution transmission electron microscopy for structure and size determination. Chemical reduction induces a blueshift, maximally +0.02 eV, in the localized surface plasmon resonance band; this is interpreted as an electronic (-) charging of the monolayer-protected cluster (MPC) gold core, corresponding to a -0.5 V change in electrochemical potential.

Base Side of Noble Metal Clusters: Efficient Route to Captamino-Gold, Au n(-S(CH2)2N(CH3)2) p, n = 25-144.

Monolayer-protected clusters (MPCs), typified by the (Au, Ag)-thiolates, share dimensions and masses with aqueous globular proteins (enzymes), yet efficient bioanalytical methods have not proved applicable to MPC analytics. Here we demonstrate that direct facile ESI(+)MS analysis of MPCs succeeds, at the few-picomol level, for aqueous basic amino-terminated thiolates. Specifically, captamino-gold clusters, Au n(SR) p, wherein -R = -(CH2)2N(CH3)2, are prepared quantitatively via a direct one-phase (aq/EtOH) method and are sprayed under weakly acidic conditions to yield intact 6.8 kDa complexes, ( n, p) = (25, 18), with up to 5 H+ adducts, or 34.6 kDa MPCs (144, 60) at charge state z = 8+. These exceed all prior reports of positive charging of MPCs except for those bearing per-cationized (quat) ligands. pH-mediated reversible phase transfer (aqueous to/from DCM-rich phases) are consistent with peripheral exposure of all tertiary amino groups to solutions. This surprising development opens the way to all manner of modifications or extensions, as well as to advanced analyses inspired by those applied to intact biomolecules.

Correlations of salivary biomarkers with clinical assessments in patients with cystic fibrosis.

RATIONALE: Monitoring clinical disease status in cystic fibrosis frequently requires invasive collection of clinical samples. Due to its noninvasive collection process and direct anatomic relationship with the lower airway, saliva shows great potential as a biological fluid for cystic fibrosis monitoring. OBJECTIVES: To measure the levels of multiple protein markers in human saliva supernatants and investigate the possibility of utilizing them to provide a more quantitative measure of disease state for use in research and monitoring of patients with cystic fibrosis clinically. METHODS: Whole saliva samples were collected and processed from cystic fibrosis patients at two distinct time points (2010 and 2013) and measured by two separate platforms. In this cross sectional study, a convenience sample of 71 participants were recruited with samples measured by multiplexed fluorescence microarray (fiber microarray) and another 117 participant samples were measured by an automated, point-of-care, analyzer (SDReader) using a microsphere-based array via fluorescence sandwich immunoassay. For comparison, saliva from 56 and 50 healthy subjects were collected, respectively. The levels of six target proteins were quantified. Various demographic and clinical data, including spirometry, medical history, and clinicians' assessments were also collected from patients with cystic fibrosis on the day of saliva collection. MEASUREMENTS AND MAIN RESULTS: Similar trends were observed with both platforms and compared with healthy subjects, cystic fibrosis patients had significantly elevated levels of VEGF, IP-10, IL-8, and EGF as well as lower levels of MMP-9 (P ≤ 0.005) using fiber microarray and significantly elevated levels of IP-10, IL-8 with lower levels of MMP-9 and IL-1ß (P ≤ 0.02) using the SDReader. The levels of the six proteins correlated with each other significantly, and in some cases, biomarker levels could be used to differentiate between subgroups of patients with different clinical presentations. For example, IP-10 levels significantly correlated with FEV1 and disease severity (as evaluated by clinicians) with both platforms (P < 0.05). CONCLUSIONS: Significant variations of the levels of six proteins in saliva supernatants, and the correlations of these levels with clinical assessments, demonstrated the potential of saliva for cystic fibrosis research and monitoring.

Catalytic kinetics of single gold nanoparticles observed via optical microwell arrays.

Catalytic activities and kinetics are measured at the single-particle level for gold nanoparticles catalyzing a fluorogenic oxidation reaction. This measurement is accomplished by confining the reactions in optically addressable microwell arrays. Citrate-capped gold nanoparticles are isolated in sealed ∼70 fL microwells along with a substrate, and the accumulation of a fluorescent product over time is observed. Thousands of reactions are measured in parallel. Catalytic activities are calculated for each nanoparticle and the activity distribution is analyzed.

An automated integrated platform for rapid and sensitive multiplexed protein profiling using human saliva samples.

During the last decade, saliva has emerged as a potentially ideal diagnostic biofluid for noninvasive testing. In this paper, we present an automated, integrated platform useable by minimally trained personnel in the field for the diagnosis of respiratory diseases using human saliva as a sample specimen. In this platform, a saliva sample is loaded onto a disposable microfluidic chip containing all the necessary reagents and components required for saliva analysis. The chip is then inserted into the automated analyzer, the SDReader, where multiple potential protein biomarkers for respiratory diseases are measured simultaneously using a microsphere-based array via fluorescence sandwich immunoassays. The results are read optically, and the images are analyzed by a custom-designed algorithm. The fully automated assay requires as little as 10 µL of saliva sample, and the results are reported in 70 min. The performance of the platform was characterized by testing protein standard solutions, and the results were comparable to those from the 3.5 h lab bench assay that we have previously reported. The device was also deployed in two clinical environments where 273 human saliva samples collected from different subjects were successfully tested, demonstrating the device's potential to assist clinicians with the diagnosis of respiratory diseases by providing timely protein biomarker profiling information. This platform, which combines noninvasive sample collection and fully automated analysis, can also be utilized in point-of-care diagnostics.

Nanostructure shape effects on response of plasmonic aptamer sensors.

A localized surface plasmon resonance (LSPR) sensor surface was fabricated by the deposition of gold nanorods on a glass substrate and subsequent immobilization of the DNA aptamer, which specifically bind to thrombin. This LSPR aptamer sensor showed a response of 6-nm λ(max) shift for protein binding with the detection limit of at least 10 pM, indicating one of the highest sensitivities achieved for thrombin detection by optical extinction LSPR. We also tested the LSPR sensor fabricated using gold bipyramid, which showed higher refractive index sensitivity than the gold nanorods, but the overall response of gold bipyramid sensor appears to be 25% less than that of the gold nanorod substrate, despite the approximately twofold higher refractive index sensitivity. XPS analysis showed that this is due to the low surface density of aptamers on the gold bipyramid compared with gold nanorods. The low surface density of the aptamers on the gold bipyramid surface may be due to the effect of shape of the nanostructure on the kinetics of aptamer monolayer formation. The small size of aptamers relative to other bioreceptors is the key to achieving high sensitivity by biosensors on the basis of LSPR, demonstrated here for protein binding. The generality of aptamer sensors for protein detection using gold nanorod and gold nanobipyramid substrates is anticipated to have a large impact in the important development of sensors toward biomarkers, environmental toxins, and warfare agents.

Ground state depletion microscopy for imaging interactions between gold nanowires and fluorophore-labeled ligands.

Ground state depletion with individual molecule return (GSDIM) is used to interrogate the location of individual fluorescence bursts from fluorophore-labelled DNA molecules on gold nanowire surfaces. Carboxytetramethyl rhodamine (TAMRA)-labelled double-stranded DNA molecules were bound to the surface of gold nanowires via gold-thiol linkages. Individual fluorescence bursts were spatially localized using point spread function fitting and used to reconstruct the image of the underlying nanowire. While the reconstructed images reproduce the size and shape of the nanowire structures, plasmonic coupling between the nanowire and fluorophore is observed, indicating that the location of the observed fluorescence may not precisely correlate with the location of the emitting fluorophore. Thus, plasmonic coupling is an important factor when using super-resolution imaging techniques to study plasmonic nanostructures.

A single molecule immunoassay by localized surface plasmon resonance.

Noble metal nanoparticles exhibit sharp spectral extinction peaks at visible and near-infrared frequencies due to the resonant excitation of their free electrons, termed localized surface plasmon resonance (LSPR). Since the resonant frequency is dependent on the refractive index of the nanoparticle surroundings, LSPR can be the basis for sensing molecular interactions near the nanoparticle surface. However, previous studies have not yet determined whether the LSPR mechanism can reach the ultimate sensing limit: the detection of individual molecules. Here we demonstrate single molecule LSPR detection by monitoring antibody-antigen unbinding events through the scattering spectra of individual gold bipyramids. Both experiments and finite element simulations indicate that the unbinding of single antigen molecules results in small, discrete < 0.5 nm blue-shifts of the plasmon resonance. The unbinding rate is consistent with antibody-antigen binding kinetics determined from previous ensemble experiments. According to these results, the effective refractive index of a single protein is approximately 1.54. LSPR sensing could therefore be a powerful addition to the current toolbox of single molecule detection methods since it probes interactions on long timescales and under relatively natural conditions.