Immobilization of histidine-tagged proteins by magnetic nanoparticles encapsulated with nitrilotriacetic acid (NTA)-phospholipids micelle

We described the development of functionalized magnetic nanoparticles (MNPs) with PEG-modification, a phospholipids micelle coating, and their use in manipulating histidine-tagged proteins. Highly monodisperse MNPs were synthesized in an organic solvent and could be phase-transferred into an aqueous solution by encapsulating the nanoparticles with a phospholipids micelle. The phospholipids micelle coating rendered the nanoparticles highly water-soluble, and the functional groups of the phospholipids coating allowed for the bioconjugation of various moieties, such as fluorescent molecules and engineered proteins. Functionalized phospholipids, such as nitrilotriacetic acid (NTA)-phospholipids, caused the MNPs to bind and allowed for manipulation of histidine-tagged proteins. Due to their high surface/volume ratio, the MNPs showed better performance (about 100 times higher) in immobilizing engineered proteins than conventional micrometer-sized beads. This demonstrates that MNPs coated with phospholipids micelle can be a versatile platform for the effective manipulation of various kinds of engineered proteins, which is very important in the field of proteomics. It is expected that a combination of MNPs with optical fluorescent molecules can find applications in bimodal (magnetic and optical) molecular imaging nanoprobes.     

糖纳米材料的研究进展

纳米材料在电子学、光学、磁学和生物医药等方面有着广泛的应用。在过去的20年,金属纳米微粒已经成功地与多肽、蛋白质和DNA结合,但糖类物质直到2001年才被引入到纳米科学中。糖纳米微粒能够很好地构建类似细胞表面糖类表达的生物细胞模型,成为糖生物学、生物药学、材料科学中十分出色的研究工具。随着研究的深入,糖纳米材料由于其制备简便,具有独特的物理、化学和生物性质,其在生物医学成像、诊断及治疗等方面有着广泛的应用前景。     

Polymeric nanoparticles for optical sensing

Nanotechnology is a powerful tool for use in diagnostic applications. For these purposes a variety of functional nanoparticles containing fluorescent labels, gold and quantum dots at their cores have been produced, with the aim of enhanced sensitivity and multiplexing capabilities. This work will review progress in the application of polymeric nanoparticles in optical diagnostics, both for in vitro and in vivo detection, together with a discussion of their biodistribution and biocompatibility.     

Nanoscale Imaging of Whole Cells Using a Liquid Enclosure and a Scanning Transmission Electron Microscope

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.     

纳米材料在生物医学检测中的应用

纳米技术的兴起,对生物医学领域的变革产生了深远的影响。纳米材料是纳米技术发展的重要基础,它具有许多传统材料所不具备的独特的理化性质,因此在生物医学、传感器等重要技术领域有着广泛的应用前景。对几类常见的纳米材料包括纳米金、量子点、磁性纳米粒子、碳纳米管和硅纳米线在蛋白质、DNA、金属离子以及生物相关分子检测方面的应用进行综述。     

Preparation of Gold Nanoparticles and their Applications in Anisotropic Nanoparticle Synthesis and Bioimaging

In this review, we highlight our recent achievements in using colloidal gold nanoparticles as building blocks for fabrication of anisotropic and multicomponent nanoparticles (e.g., nanoshells, semiconductor nanocrystals, and gold nanorods). The tunable optical properties of these nanoparticles are well suited for various biomedical and biophotonic applications.     

Recent advances in synthesis and surface modification of lanthanide-doped upconversion nanoparticles for biomedical applications

Lanthanide (Ln)-doped upconversion nanoparticles (UCNPs) with appropriate surface modification can be used for a wide range of biomedical applications such as bio-detection, cancer therapy, bio-labeling, fluorescence imaging, magnetic resonance imaging and drug delivery. The upconversion phenomenon exhibited by Ln-doped UCNPs renders them tremendous advantages in biological applications over other types of fluorescent materials (e.g., organic dyes, fluorescent proteins, gold nanoparticles, quantum dots, and luminescent transition metal complexes) for: (i) enhanced tissue penetration depths achieved by near-infrared (NIR) excitation; (ii) improved stability against photobleaching, photoblinking and photochemical degradation; (iii) non-photodamaging to DNA/RNA due to lower excitation light energy; (iv) lower cytotoxicity; and (v) higher detection sensitivity. Ln-doped UCNPs are therefore attracting increasing attentions in recent years. In this review, we present recent advances in the synthesis of Ln-doped UCNPs and their surface modification, as well as their emerging applications in biomedicine. The future prospects of Ln-doped UCNPs for biomedical applications are also discussed.     

Gold Nanoparticle Uptake by Tumour Cells of B16 Mouse Melanoma

Because of their photo-optical distinctiveness and biocompatibility, gold nanoparticles have proven to be powerful tools in various nanomedical applications. In this article, we discuss the advantage of gold nanoparticles in image diagnostic application of melanoma. It has demonstrated the potential role of gold nanoparticles in the study of tumour tissue architecture and the utility of gold nanoparticles in the hystopathological exam of B16 melanoma with the benefit of fluorescence emission of gold nanoparticles in UV spectrum. The optical properties of colloidal gold nanoparticles allow spectroscopic detection and identification of melanoma cells. The method proposed is easy, inexpensive and reliable for hystopathological analysis of melanoma. The fluorescence images in the cryosections of tissues depicted a strong luminescence property of gold nanoparticles uptaken in melanoma, results that confirm the role of the gold nanoparticles in biological labelling and imaging applications. To emphasize the AuNPs influence over the biological tissues, a study of the chemical bonds configuration was performed using Raman spectrometry.     

Annealing Process in the Refurbishment of the Plasmonic Photonic Structures Fabricated Using Colloidal Gold Nanoparticles

Solution-processible fabrication techniques have been demonstrated with promising features for realizing different types of plasmonic devices, which combine interference lithography, spin-coating of the colloidal gold nanoparticles, and subsequent annealing process at a temperature of 200–300 °C. However, the resultant device needs to be improved in the following considerations: (1) The photoresist master grating needs to be removed for the applications in optoelectronic or sensor devices and (2) each lattice site of the photonic crystals is still composed of closely contacted gold nanoparticles. Actually, these metallic photonic structures can be refurbished through a further annealing process. Using an annealing temperature above 450 °C, we have successfully removed the remaining photoresist and make the gold nanoparticles join into a solid homogenous unit on each lattice site after being fully molten. Thus, high-quality gold nanostructures with excellent plasmonic response can be obtained. This accomplished an improved recipe for the solution-processible fabrication of plasmonic nanostructures. The corresponding devices with improved optical properties become more suitable for biosensors and optoelectronic devices.     

Intracellular Localized Surface Plasmonic Sensing for Subcellular Diagnosis

This paper proposes a method for diagnosing intracellular conditions and organelles of cells with localized surface plasmonic resonance (LSPR) by directly internalizing the gold nanoparticles (AuNPs) into the cells and measuring their plasmonic properties through hyperspectral imaging. This technique will be useful for direct diagnosis of cellular organelles, which have potential for cellular biology, proteomics, pharmaceuticals, drug discovery etc. Furthermore, localization and characterization of citrate-capped gold nanoparticles in HeLa cells were studied, by hyperspectral microscopy and other imaging techniques. Here, we present the method of internalizing the gold nanoparticles into the cells and subcellular organelles to facilitate subcellular plasmonic measurements. An advanced label-free visualization technique, namely hyperspectral microscopy providing images and spectral data simultaneously, was used to confirm the internalization of gold nanoparticles and to reveal their optical properties for possible intracellular plasmonic detection. Hyperspectral technology has proved to be effective in the analysis of the spectral profile of gold nanoparticles, internalized under different conditions. Using this relatively novel technique, it is possible to study the plasmonic properties of particles, localized in different parts of the cell. The position of the plasmon bands reflects the interactions of gold nanoparticles with different subcellular systems, including particle-nucleus interactions. Our results revealed the effect of the different intracellular interactions on the aggregation pattern of gold nanoparticles, inside the cells. This novel technique opens the door to intracellular plasmonics, an entirely new field, with important potential applications in life sciences. Similarly, the characterization of AuNP inside the cell was validated using traditional methods such as light microscopy and scanning electron microscopy. Under the conditions studied in this work, gold nanoparticles were found to be non-toxic to HeLa (cervical cancer) cells.     

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.     

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.     

Preparation of Nanostructured Film Arrays for Transmission Localized Surface Plasmon Sensing

This article presents a concise review of preparation methods for transparent nanostructured films, with an emphasis on their current applications in transmission-localized surface plasmon resonance (T-LSPR) sensing. One of the first methods used for the fabrication of transparent nanostructured metal films is a direct vacuum evaporation of thin gold films. Self-induced formations of small gold islands result in transparent nanostructured gold arrays. The most well-established method is a nanosphere lithography developed by Van Duyne. Nanotriangular island arrays with controlled size and optical properties can be fabricated by this protocol. A different nanolithography method known as focused ion beam milling is reported and used for the fabrication of nanohole arrays. Simple assembly of solution-phase synthesized nanoparticles has also been utilized for the preparation of nanoparticle arrays capable of T-LSPR sensing. Lastly, this article also describes a new preparation strategy, in which self-assembly/thermolysis of nanoparticle multilayers is employed to obtain transparent nanoisland architectures on glass substrates.     

A new amplification strategy for ultrasensitive electrochemical aptasensor with network-like thiocyanuric acid/gold nanoparticles

An ultrasensitive and highly specific electrochemical aptasensor for detection of thrombin based on gold nanoparticles and thiocyanuric acid is presented. For this proposed aptasensor, aptamerI was immobilized on the magnetic nanoparticles, aptamerII was labeled with gold nanoparticles. The magnetic nanoparticle was used for separation and collection, and gold nanoparticle offered excellent electrochemical signal transduction. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle/thrombin/gold nanoparticle was fabricated, and the signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles. A significant sensitivity enhancement had been obtained, and the detection limit was down to 7.82 aM. The presence of other proteins such as BSA and lysozyme did not affect the detection of thrombin, which indicates a high specificity of thrombin detection could be achieved. This electrochemical aptasensor is expected to have wide applications in protein monitoring and disease diagnosis.     

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.     

Solid and Hollow Gold Nanostructures for Nanomedicine: Comparison of Photothermal Properties

The photothermal properties of solid and hollow gold nanostructures represented by colloidal solutions of spherical nanoparticles, nanoshells, and nanocages upon irradiation with a 100 mW 808 nm continuous-wave laser for the first time were experimentally compared under identical optical density and nanoparticle concentration conditions. Accompanying computer modeling of light absorption by the studied gold nanostructures revealed the general parameters influencing the photothermal efficiency, which is of significance for nanomedical applications. The spectral position of localized plasmonic excitations of the studied nanostructures ranged from 518 nm for solid gold nanoparticles to 718 nm for gold nanocages, which provided a possibility to observe a direct influence of the wavelength proximity between the localized surface plasmon resonance and laser line on the heat generation capability of the nanostructures. As a result, the best photothermal efficiency was registered for gold nanocages, which proves them as an efficient photothermal treatment agent and a possible candidate to build a nanocarrier platform for drug delivery with a controlled release. Light absorption modeling demonstrated an existence of optimal wall thickness for gold nanoshells that should lead to the maximum photothermal efficiency when irradiated with 808 nm light, which varied from about 0.1 to 0.4 in units of external nanoshell radius with an increase of the wall porosity. Additionally, computer modeling results show that increased wall porosity should lead to enhanced photothermal efficiency of polydisperse colloidal solutions of hollow gold nanostructures.     

Innovation: Photoactivatable fluorescent proteins

The fluorescence characteristics of photoactivatable proteins can be controlled by irradiating them with light of a specific wavelength, intensity and duration. This provides unique possibilities for the optical labelling and tracking of living cells, organelles and intracellular molecules in a spatio-temporal manner. Here, we discuss the properties of the available photoactivatable fluorescent proteins and their potential applications.     

Simultaneous synthesis of temperature-tunable peptide and gold nanoparticle hybrid spheres

Hybrid spheres containing peptides and gold nanoparticles have been simultaneously synthesized in water using AG4 (NPSSLFRYLPSD) peptides that acted as a reducing agent to guide the nucleation and growth of gold nanoparticles and a precursor to form sphere-like structure by self-assembly where the size of hybrid spheres is precisely controlled by adjusting the operating temperature. The self-assembled peptide spheres remain stable even after selective removal of the gold nanoparticles by iodide etching. The amino acids containing the aromatic functional group in the peptide sequence significantly affect the construction of sphere structures. The surface of gold nanoparticles containing hybrid spheres has been functionalized using the thiol group linked to biomolecules. The ability to synthesize nanoparticle and self-assembled peptide structures with controlled size and composition in an environmental benign way will allow us to fabricate a new class of multifunctional organic-inorganic hybrid superstructures for various biomedical and electronic applications.     

Resonance Rayleigh-scattering method for the determination of proteins with gold nanoparticle probe

Gold nanoparticles with a 12-nm diameter were used as probes for the determination of proteins by resonance Rayleigh-scattering techniques. In weak acidic solution, large amounts of citrate anions will self-assemble on the surface of positively charged gold nanoparticles to form supermolecular compounds with negative charges. Below the isoelectric point, proteins with positive charges such as human serum albumin (HSA), bovine serum albumin (BSA), and ovalbumin (Ova) can bind gold nanoparticles to form larger volume products (the diameter of the binding product of gold nanoparticles with HSA is 23 nm.) through electrostatic force, hydrogen bonds, and hydrophobic effects, which can result in a red shift of the maximum absorption wavelength, the remarkable enhancement of the resonance Rayleigh-scattering intensity (RRS), and the appearance of the RRS spectra. At the same time, the second-order-scattering (SOS) and frequency-doubling-scattering (FDS) intensities are also enhanced. The binding products of gold nanoparticles with different proteins have similar spectral characteristics and the maximum wavelengths are located near 303 nm for RRS, 540 nm for SOS, and 390 for FDS, respectively. The scattering enhancement (DeltaI) is directly proportional to the concentration of proteins. Among them, the RRS method has the highest sensitivity and the detection limits are 0.38 ng/ml for HSA, 0.45 ng/ml for BSA, and 0.56 ng/ml for Ova, separately. The methods have good selectivity. A new RRS method for the determination of trace proteins using a gold nanoparticle probe has been developed. Because gold nanoparticle probes do not need to be modified chemically in advance, the method is very simple and fast.     

Gold nanostructures absorption capacities of various energy forms for thermal therapy applications

This mini-review has investigated the recent progress regarding gold nanostructures capacities of energy absorption for thermal therapy applications. Unselective thermal therapy of malignant and normal tissues could lead to irreversible damage to healthy tissues without effective treatment on target malignant tissues. In recent years, there has been a considerable progress in the field of cancer thermal therapy for treating target malignant tissues using nanostructures. Due to the remarkable physical properties of the gold nanoparticle, it has been considered as an exceptional element for thermal therapy techniques. Different types of gold nanoparticles have been used as energy absorbent for thermal therapy applications under several types of energy exposures. Electromagnetic, ultrasound, electric and magnetic field are examples for these energy sources. Well-known plasmonic photothermal therapy which applies electromagnetic radiation is under clinical investigation for the treatment of various medical conditions. However, there are many other techniques in this regard which should be explored.