Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.     

Oligonucleotide-coated metallic nanoparticles as a flexible platform for molecular imaging agents

Targeted metallic nanoparticles have shown promise as contrast agents for molecular imaging. To obtain molecular specificity, the nanoparticle surface must be appropriately functionalized with probe molecules that will bind to biomarkers of interest. The aim of this study was to develop and characterize a flexible approach to generate molecular imaging agents based on gold nanoparticles conjugated to a diverse range of probe molecules. We present two complementary oligonucleotide-based approaches to develop gold nanoparticle contrast agents which can be functionalized with a variety of biomolecules ranging from small molecules, to peptides, to antibodies. The size, biocompatibility, and protein concentration per nanoparticle are characterized for the two oligonucleotide-based approaches; the results are compared to contrast agents prepared using adsorption of proteins on gold nanoparticles by electrostatic interaction. Contrast agents prepared from oligonucleotide-functionalized nanoparticles are significantly smaller in size and more stable than contrast agents prepared by adsorption of proteins on gold nanoparticles. We demonstrate the flexibility of the oligonucleotide-based approach by preparing contrast agents conjugated to folate, EGF peptide, and anti-EGFR antibodies. Reflectance images of cancer cell lines labeled with functionalized contrast agents show significantly increased image contrast which is specific for the target biomarker. To demonstrate the modularity of this new bioconjugation approach, we use it to conjugate both fluorophore and anti-EGFR antibodies to metal nanoparticles, yielding a contrast agent which can be probed with multiple imaging modalities. This novel bioconjugation approach can be used to prepare contrast agents targeted with biomolecules that span a diverse range of sizes; at the same time, the bioconjugation method can be adapted to develop multimodal contrast agents for molecular imaging without changing the coating design or material.     

Optimization of immunolabeled plasmonic nanoparticles for cell surface receptor analysis

Noble metal nanoparticles hold great potential as optical contrast agents due to a unique feature, known as the plasmon resonance, which produces enhanced scattering and absorption at specific frequencies. The plasmon resonance also provides a spectral tunability that is not often found in organic fluorophores or other labeling methods. The ability to functionalize these nanoparticles with antibodies has led to their development as contrast agents for molecular optical imaging. In this review article, we present methods for optimizing the spectral agility of these labels. We discuss synthesis of gold nanorods, a plasmonic nanoparticle in which the plasmonic resonance can be tuned during synthesis to provide imaging within the spectral window commonly utilized in biomedical applications. We describe recent advances in our group to functionalize gold and silver nanoparticles using distinct antibodies, including EGFR, HER-2 and IGF-1, selected for their relevance to tumor imaging. Finally, we present characterization of these nanoparticle labels to verify their spectral properties and molecular specificity.     

抗体和寡核苷酸双标记纳米金生物探针的制备及性能分析

基于纳米金粒子与抗体静电吸附作用,与硫醇修饰的寡核苷酸共价结合,建立一种新的双标记纳米金生物探针的制备方法.通过透射电镜（TEM）、紫外光谱、斑点免疫金渗滤法、免疫金银染色光镜观察法、荧光标记法等检测探针表征,及表面抗体活性情况和寡核苷酸的覆盖率,同时采用变性聚丙烯酰胺凝胶电泳（PAGE）检测寡核苷酸的存在.结果表明,纳米金粒子同时连接抗体和寡核苷酸后生物性能良好,且每个纳米金粒子（10±3）nm表面可覆盖寡核苷酸(92±20)条,双标记纳米金生物探针的制备具有简捷、稳定的特点.可作为一种新型探针应用于超微量蛋白质检测.     

Controlled Fabrication of Gold Nanoparticle and Fluorescent Protein Conjugates

The unique optical properties of gold nanoparticles make them attractive for a wide range of applications which require optical detection and manipulation techniques. Here, we experimentally demonstrate the use of single femtosecond pulses at resonance wavelength for a controlled conjugation of gold nanoparticles and fluorescent proteins. This optically driven reaction is rigorously studied and analyzed using a variety of experimental techniques, and a detailed model is proposed which describes the adsorption of the proteins onto the nanoparticles' surface, as well as their subsequent desorption by a reducing agent. Potential applications of the resulting nanoparticle?Cprotein conjugates include controlled delivery of fluorescent markers and local sensing of biochemical processes.     

Review of Some Interesting Surface Plasmon Resonance-enhanced Properties of Noble Metal Nanoparticles and Their Applications to Biosystems

Noble metal, especially gold (Au) and silver (Ag) nanoparticles exhibit unique and tunable optical properties on account of their surface plasmon resonance (SPR). In this review, we discuss the SPR-enhanced optical properties of noble metal nanoparticles, with an emphasis on the recent advances in the utility of these plasmonic properties in molecular-specific imaging and sensing, photo-diagnostics, and selective photothermal therapy. The strongly enhanced SPR scattering from Au nanoparticles makes them useful as bright optical tags for molecular-specific biological imaging and detection using simple dark-field optical microscopy. On the other hand, the SPR absorption of the nanoparticles has allowed their use in the selective laser photothermal therapy of cancer. We also discuss the sensitivity of the nanoparticle SPR frequency to the local medium dielectric constant, which has been successfully exploited for the optical sensing of chemical and biological analytes. Plasmon coupling between metal nanoparticle pairs is also discussed, which forms the basis for nanoparticle assembly-based biodiagnostics and the plasmon ruler for dynamic measurement of nanoscale distances in biological systems.     

A real-time PCR-based method for determining the surface coverage of thiol-capped oligonucleotides bound onto gold nanoparticles

Here we report a real-time PCR-based method for determining the surface coverage of dithiol-capped oligonucleotides bound onto gold nanoparticles alone and in tandem with antibody. The detection of gold nanoparticle-bound DNA is accomplished by targeting the oligonucleotide with primer and probe binding sites, amplification of the oligonucleotide by PCR, and real-time measurement of the fluorescence emitted during the reaction. This method offers a wide dynamic range and is not dependant on the dissociation of the oligonucleotide strands from the gold nanoparticle surface; the fluorophore is not highly quenched by the gold nanoparticles in solution during fluorescence measurements. We show that this method and a fluorescence-based method give equivalent results for determining the surface coverage of oligonucleotides bound onto 13 or 30 nm gold nanoparticles alone and in tandem with antibody. Quantifying the surface coverage of immobilized oligonucleotides on metallic nanoparticle surfaces is important for optimizing the sensitivity of gold nanoparticle-based detection methods and for better understanding the interactions between thiol-functionalized oligonucleotides and gold nanoparticles.     

Aptamer-targeted gold nanoparticles as molecular-specific contrast agents for reflectance imaging

Targeted metallic nanoparticles have shown potential as a platform for development of molecular-specific contrast agents. Aptamers have recently been demonstrated as ideal candidates for molecular targeting applications. In this study, we investigated the development of aptamer-based gold nanoparticles as contrast agents, using aptamers as targeting agents and gold nanoparticles as imaging agents. We devised a novel conjugation approach using an extended aptamer design where the extension is complementary to an oligonucleotide sequence attached to the surface of the gold nanoparticles. The chemical and optical properties of the aptamer-gold conjugates were characterized using size measurements and oligonucleotide quantitation assays. We demonstrate this conjugation approach to create a contrast agent designed for detection of prostate-specific membrane antigen (PSMA), obtaining reflectance images of PSMA(+) and PSMA(-) cell lines treated with the anti-PSMA aptamer-gold conjugates. This design strategy can easily be modified to incorporate multifunctional agents as part of a multimodal platform for reflectance imaging applications.     

One-step preparation of antibody and oligonucleotide dual-labeled gold nanoparticle bio-probes and their properties

A novel method of one-step preparation of dual-labeled gold nanoparticle bio-probes was established by the electrostatic adsorption and the covalent bonding of gold nanoparticles with antibodies and thiol-modified oligonucleotides, respectively. Characterization of probes, the coverage and activity of antibodies and oligonucleotides on probe surfaces were detected. The results indicated that the gold nanoparticles labeled with antibodies and oligonucleotides possess good bioactivity and the coverage of oligonucleotide and antibody on a dual-labeled gold nanoparticle bio-probe was (92 ± 20) and (8 ± 3), respectively. The preparative method is simple and stable. The dual-labeled gold nanoparticle bio-probes have an application value in detection of ultramicro protein.     

Synthesis of 64Cu-labeled magnetic nanoparticles for multimodal imaging

Complementary imaging modalities provide more information than either method alone can yield and we have developed a dual-mode imaging probe for combined magnetic resonance (MR) and positron emission tomography (PET) imaging. We have developed dual-mode PET/MRI active probes targeted to vascular inflammation and present synthesis of (1) an aliphatic amine polystyrene bead and (2) a novel superparamagnetic iron oxide nanoparticle targeted to macrophages that were both coupled to positron-emitting copper-64 isotopes. The amine groups of the polystyrene beads were directly conjugated with an amine-reactive form (isothiocyanate) of aza-macrocycle 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA). Iron oxide nanoparticles are dextran sulfate coated, and the surface was modified to contain aldehyde groups to conjugate to an amine-activated DOTA. Incorporation of chelated Cu-64 to nanoparticles under these conditions, which is routinely used to couple DOTA to macromolecules, was unexpectedly difficult and illustrates that traditional conjugation methods do not always work in a nanoparticle environment. Therefore, we developed new methods to couple Cu-64 to nanoparticles and demonstrate successful labeling to a range of nanoparticle types. We obtained labeling yields of 24% for the amine polystyrene beads and 21% radiolabeling yield for the anionic dextran sulfate iron oxide nanoparticles. The new coupling chemistry can be generalized for attaching chelated metals to other nanoparticle platforms.     

Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen-printed carbon electrodes

Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient's samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon-metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool.     

Optical Absorption in Overcoats of Nanoparticle Arrays on a Metallic Substrate

Surface plasma oscillations in metallic particles as well as in thin metallic films have been studied extensively in the past decades. New features regarding surface plasma excitations are, however, constantly discovered, leading, for example, to surface-enhanced Raman scattering studies and enhanced optical transmission though metal films with nanohole arrays. In the present work, the role of a metallic substrate is examined in two cases, one involving an overcoat of dielectric nanoparticles and the other an overcoat of metallic nanoparticles. Theoretical results are obtained by modeling the nanoparticles as forming a two-dimensional, hexagonal lattice of spheres. The scattered electromagnetic field is then calculated using a variant of the Green function method. Comparison with experimental results is made for nanoparticles of tungsten oxide and tin oxide deposited on either gold or silver substrates, giving qualitative agreement on the extra absorption observed when the dielectric nanoparticles are added to the metallic surfaces. Such absorption would be attributed to the mirror image effects between the particles and the substrate. On the other hand, calculations of the optical properties of silver or gold nanoparticle arrays on a gold or a silver substrate demonstrate very interesting features in the spectral region from 400 to 1,000 nm. Interactions between the nanoparticle arrays surface plasmons and their images in the metallic substrate would be responsible for the red shift observed in the absorption resonance. Moreover, effects of particle size and ambient index of refraction are studied, showing a great potential for applications in biosensing with structures consisting of metallic nanoparticle arrays on metallic substrates.     

DNA gold nanoparticle conjugates incorporating thiooxonucleosides: 7-deaza-6-thio-2'-deoxyguanosine as gold surface anchor

A new protocol for the covalent attachment of oligonucleotides to gold nanoparticles was developed. Base-modified nucleosides with thiooxo groups were acting as molecular surface anchor. Compared to already existing conjugation protocols, the new linker strategy simplifies the synthesis of DNA gold nanoparticle conjugates. The phosphoramidite of 7-deaza-6-thio-2'-deoxyguanosine (6) was used in solid-phase synthesis. Incorporation of the sulfur-containing nucleosides can be performed at any position of an oligonucleotide; even multiple incorporations are feasible, which will increase the binding stability of the corresponding oligonucleotides to the gold nanoparticles. Oligonucleotide strands immobilized at the end of a chain were easily accessible during hybridization leading to DNA gold nanoparticle network formation. On the contrary, oligonucleotides immobilized via a central position could not form a DNA-AuNP network. Melting studies of the DNA gold nanoparticle assemblies revealed sharp melting profiles with a very narrow melting transition.     

Femtomolar DNA detection by parallel colorimetric darkfield microscopy of functionalized gold nanoparticles

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0×10(-14) M without the need for a spectrometer or a scanning stage.     

Two ways to inactivate the Ki‐67 protein—Fragmentation by nanoparticles,crosslinking with fluorescent dyes

Light can manipulate molecular biological processes with high spatial and temporal precision and optical manipulation has become increasingly popular during the last years. In combination with absorbing dyes or gold nanoparticles light is a valuable tool for cell and protein inactivation with high precision. Here we show distinct differences in the underlying mechanisms whether gold nanoparticles or fluorescent dyes are used for the inactivation of the Ki‐67 protein. The proliferation‐associated protein Ki‐67 was addressed by the antibody MIB‐1. In vitro studies showed a fragmentation of the Ki‐67 protein after laser irradiation of 15 nm gold nanoparticle antibody conjugates with nanosecond pulsed laser, while continuous wave (cw) irradiation of fluorescein isothiocyanate (FITC)‐ and Alexa 488‐labeled antibodies led to specific crosslinking of Ki‐67. The irradiation energy for the gold nanoparticles was above cavitation bubble formation threshold. We observed a fragmentation of the target protein and also of the gold particles. The understanding of the underlying inactivation mechanisms is important for the application and further development of these two techniques, which can harness nanotechnology to introduce molecular selectivity to biological systems.     

Highly sensitive electrochemical detection of immunospecies based on combination of Fc label and PPD film/gold nanoparticle amplification

A highly sensitive electrochemical immunoassay strategy based on the combination of ferrocene (Fc) label and poly(o-phenylenediamine) (PPD) film/gold nanoparticle (GNP) amplification for the detection of immunospecies is proposed using human IgG as the model analyte. A gold electrode is firstly modified with an electropolymerized film of poly(o-phenylenediamine), which provides a stable matrix with abundant amino-groups for the fabrication of sensing interface. Using glutaraldehyde as a cross-linker, cystamine is coupled onto the modified electrode. Subsequently, gold nanoparticle monolayer is assembled onto the resulting surface. Making use of the unique properties of gold nanoparticles, antibodies can be self-assembled onto the surface-confined gold nanoparticles via amine-Au affinity with a high loading amount and reserve high immunological activity. After the introduction of model analyte, the ferrocene (Fc)-labeled antibody is immobilized on the sensing interface by antibody-antigen specific reaction, resulting in a redox current signal. The peak current is proportional to the amount of the analyte. Under the optimized experimental conditions, the proposed sensing strategy provides a wide linear dynamic range from 25 to 1000pg/mL with a low detection limit of 10pg/mL. In addition, good reproducibility, high selectivity and stability are achieved. In particular, the extremely high stability of both poly(o-phenylenediamine) and gold nanoparticle monolayer allows the designed biosensing interface to withstand harsh regeneration treatment, making it reusable.     

Photothermal nanotherapeutics and nanodiagnostics for selective killing of bacteria targeted with gold nanoparticles

We describe a new method for selective laser killing of bacteria targeted with light-absorbing gold nanoparticles conjugated with specific antibodies. The multifunctional photothermal (PT) microscope/spectrometer provides a real-time assessment of this new therapeutic intervention. In this integrated system, strong laser-induced overheating effects accompanied by the bubble-formation phenomena around clustered gold nanoparticles are the main cause of bacterial damage. PT imaging and time-resolved monitoring of the integrated PT responses assessed these effects. Specifically, we used this technology for selective killing of the Gram-positive bacterium Staphylococcus aureus by targeting the bacterial surface using 10-, 20-, and 40-nm gold particles conjugated with anti-protein A antibodies. Labeled bacteria were irradiated with focused laser pulses (420-570 nm, 12 ns, 0.1-5 J/cm(2), 100 pulses), and laser-induced bacterial damage observed at different laser fluences and nanoparticle sizes was verified by optical transmission, electron microscopy, and conventional viability testing.     

Ambiguous Refractive Index Sensitivity of Fano Resonance on an Array of Gold Nanoparticles

We investigate the optical response to refractive index changes of a Fano resonance occurring in a random array of gold nanoparticles supported on a glass substrate. The Fano resonance results from the interference between localized surface plasmon on a gold nanoparticle and the light reflected at the boundary of the glass substrate. We demonstrate that the sensitivity of the resonance to the refractive index of the surrounding medium is highly dependent on the excitation geometry and can assume either positive or negative values. We furthermore present a theoretical analysis explaining this behavior based on the rigorous coupled wave analysis (RCWA) as well as the island film theory.

     

A novel electroconductive interface based on Fe3O4 magnetic nanoparticle and cysteamine functionalized AuNPs: Preparation and application as signal amplification element to minoring of antigen‐antibody immunocomplex and biosensing of prostate cancer

In this study, a novel electroconductive interface was prepared based on Fe₃O₄ magnetic nanoparticle and cysteamine functionalized gold nanoparticle. The engineered interface was used as signal amplification substrate in the electrochemical analysis of antibody‐antigen binding. For this purpose, biotinilated‐anti‐prostate‐specific antigen (PSA) antibody was bioconjugated with iron oxide magnetic nanoparticles (Fe₃O₄) and drop‐casted on the surface of glassy carbon electrode (GCE). Also, secondary antibody (HRP‐Ab2) encapsulated on gold nanoparticles caped by cysteamine was immobilized on the surface of GCE modified electrode. A transmission electron microscopy images shows that a sandwich immunoreaction was done and binding of Ab1 and Ab2 performed successfully. Various parameters of immunoassay, including the loading of magnetic nanoparticles, the amount of gold nanoparticle conjugate, and the immunoreaction time, were optimized. The detection limit of 0.001 μg. L^?1 of PSA was obtained under optimum experimental conditions. It is found that such magneto‐bioassay could be readily used for simultaneous parallel detection of multiple proteins by using multiple inorganic metal nanoparticle tracers and are expected to open new opportunities for early stage diagnosis of cancer in near future.     

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture and detection of circulating tumor cells (CTCs), and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.