基于上转换纳米材料的免疫检测技术应用

免疫分析法具有简便、快速、准确等特点，广泛应用于医学、食品、环境等领域检测，将免疫分析方法与纳米材料相结合可以提高免疫分析的性能。与传统纳米材料相比，上转换纳米颗粒（upconversion nanoparticles，UCNPs）具有光稳定性好、发光寿命长和狭窄及可调整的发射带等优秀的光学性质，与免疫分析相结合可显著降低背景噪声，提高分析灵敏度。本文简要介绍了UCNPs的发光机制，对UCNPs的合成和表面修饰方法进行了总结，并详细论述荧光共振能量转移、内滤效应、磁分离技术、上转化连接免疫吸附技术和上转换免疫层析技术五种基于UCNPs的免疫检测技术，最后对该技术所面临的挑战和前景进行总结和展望，以期为UCNPs免疫检测技术的发展提供理论指导。     

上转换纳米颗粒介导的无线光遗传学技术的研究进展

光遗传学技术是结合基因工程和光学技术对生物体特定细胞进行精确调控的新兴生物技术,该技术可以特异性地兴奋或抑制靶神经元,成为解析介导特定行为神经环路的强有力的工具.传统技术依赖光纤,对脑组织有损伤且限制了动物的自由活动.新一代上转换纳米颗粒介导的无线光遗传学技术,借助近红外光组织穿透相对深的特性,能够对啮齿类动物脑组织深层核团进行无线调控,克服了传统技术中埋置光纤存在的缺陷.本文总结了上转换纳米颗粒介导的无线光遗传学技术的发展历程及现状,比较分析了这类无线光遗传学技术的优缺点,最后对该技术面临的挑战及未来前景进行了分析和展望.     

Cadmium-containing quantum dots: properties,applications, and toxicity

The marriage of biology with nanomaterials has significantly accelerated advancement of biological techniques, profoundly facilitating practical applications in biomedical fields. With unique optical properties (e.g., tunable broad excitation, narrow emission spectra, robust photostability, and high quantum yield), fluorescent quantum dots (QDs) have been reasonably functionalized with controllable interfaces and extensively used as a new class of optical probe in biological researches. In this review, we summarize the recent progress in synthesis and properties of QDs. Moreover, we provide an overview of the outstanding potential of QDs for biomedical research and innovative methods of drug delivery. Specifically, the applications of QDs as novel fluorescent nanomaterials for biomedical sensing and imaging have been detailedly highlighted and discussed. In addition, recent concerns on potential toxicity of QDs are also introduced, ranging from cell researches to animal models.

     

综述——新型纳米生物材料的研发：稀土上转换荧光纳米材料的制备与生物应用

近几年,稀土上转换荧光纳米材料作为新型的荧光探针受到研究者的广泛关注,其优势在于光化学稳定性好、发射谱带窄、荧光寿命长、Stokes位移大等．同时,它利用近红外激光器作为激发光源,组织穿透能力好、对生物组织的损伤小、几乎没有背景荧光,使其应用于生物活体荧光成像成为可能．本文主要综述了最近稀土上转换荧光纳米材料在制备与生物应用方面的研究进展．     

In vivo molecular imaging using nanomaterials: General in vivo characteristics of nano-sized reagents and applications for cancer diagnosis (Review)

Abstract

Nanoparticles present a new collection of contrast agents for the field of in vivo molecular imaging. This review focuses on promising molecular imaging probes for optical and magnetic resonance imaging based on four representative nanomaterial(s) platforms: quantum dots, upconversion phosphors, superparamagnetic iron oxides, and dendrimer-based agents. Quantum dots are extremely efficient fluorescent nanoparticles with size-tunable emission properties, enabling high sensitivity and greater depth penetration. Their heavy metal composition and long retention in the body, however, pose concerns for clinical translational applications. Upconversion phosphors generate excellent signal-to-background contrast because they emit light with higher energy than the excitation photons and autofluorescence signals. For MRI, iron oxide particles also generate excellent signal and have been used in liver imaging and for cell tracking studies. As they are metabolized through endogenous iron salvage pathways, they have already been introduced as clinical contrast agents. Lastly, dendrimers, a ‘soft’ nanoparticle, can be used as a structural basis for the attachment of small molecule imaging agents and/or targeting groups. This array of nanoparticles should offer insights into the uses and potentials of nanoparticles for the molecular imaging.     

Au nanostructures: an emerging prospect in cancer theranostics

Au nanoparticles have been used in biomedical applications since ancient times. However, the rapid development of nanotechnology over the past century has led to recognition of the great potential of Au nanoparticles in a wide range of applications. Advanced fabrication techniques allow us to synthesize a variety of Au nanostructures possessing physiochemical properties that can be exploited for different purposes. Functionalization of the surface of Au nanoparticles further eases their application in various roles. These advantages of Au nanoparticles make them particularly suited for cancer treatment and diagnosis. The small size of Au particles enables them to preferentially accumulate at tumor sites to achieve in vivo targeting after systemic administration. Efficient light absorption followed by rapid heat conversion makes them very promising in photothermal therapy. The facile surface chemistry of Au nanoparticles eases delivery of drugs, ligands or imaging contrast agents in vivo. In this review, we summarize recent development of Au nanoparticles in cancer theranostics including imaging-based detection, photothermal therapy, chemical therapy and drug delivery. The multifunctional nature of Au nanoparticles means they hold great promise as novel anti-cancer therapeutics.     

Toxicity of inorganic nanomaterials in biomedical imaging

Inorganic nanoparticles have shown promising potentials as novel biomedical imaging agents with high sensitivity, high spatial and temporal resolution. To translate the laboratory innovations into clinical applications, their potential toxicities are highly concerned and have to be evaluated comprehensively both in vitro and in vivo before their clinical applications. In this review, we first summarized the in vivo and in vitro toxicities of the representative inorganic nanoparticles used in biomedical imagings. Then we further discuss the origin of nanotoxicity of inorganic nanomaterials, including ROS generation and oxidative stress, chemical instability, chemical composition, the surface modification, dissolution of nanoparticles to release excess free ions of metals, metal redox state, and left-over chemicals from synthesis, etc. We intend to provide the readers a better understanding of the toxicology aspects of inorganic nanomaterials and knowledge for achieving optimized designs of safer inorganic nanomaterials for clinical applications.     

A novel label-free upconversion fluorescence resonance energy transfer-nanosensor for ultrasensitive detection of protamine and heparin

A novel label-free fluorescence nanosensor was developed for ultrasensitive detection of protamine and heparin based on fluorescence resonance energy transfer (FRET) between NaYF₄:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The FRET system was formed by the electrostatic adsorption of AuNPs on UCNPs, and the fluorescence of UCNPs was significantly quenched. When protamine was added to the mixture of UCNPs–AuNPs, the AuNPs interacted with protamine and then desorbed from the surface of UCNPs and aggregated, resulting in the recovery of the fluorescence of UCNPs. On the addition of both protamine and heparin, the FRET system formed owing to the stronger interaction between heparin and protamine than that with AuNPs, leading to a marked fluorescence quenching of UCNPs. The concentrations of protamine and heparin were proportional to the changes of the fluorescence of UCNPs. The linear response range was obtained over the concentration ranges of 0.02 to 1.2 μg/ml and 0.002 to 2.0 μg/ml with low detection limits of 6.7 and 0.7 ng/ml for protamine and heparin, respectively. Simultaneous measurement of protamine and heparin in human serum can be achieved, suggesting that the nanosensor can be used in a complex biological sample matrix.     

Current state and prospects of the phytosynthesized colloidal gold nanoparticles and their applications in cancer theranostics

The design, development, and biomedical applications of phytochemical-based green synthesis of biocompatible colloidal gold nanoparticles (AuNPs) are becoming an emerging field due to several advantages (safer, eco-friendly, simple, fast, energy efficient, low-cost, and less toxic) over conventional chemical synthetic procedures. Biosynthesized colloidal gold nanoparticles are remarkably attractive in several biomedical applications including cancer theranostics due to small size, unusual physico-chemical properties, facile surface modification, high biocompatibility, and numerous other advantages. Of late, several researchers have investigated the biosynthesis and prospective applications (diagnostics, imaging, drug delivery, and cancer therapeutics) of AuNPs in health care and medicine. However, not a single review article is available in the literature that demonstrates the anti-cancer potential of biosynthesized colloidal AuNPs with detailed mechanistic study. In the present review article, we for the first time discuss the biointerface of colloidal AuNPs, plants, and cancer mainly (i) comprehensive mechanistic aspects of phytochemical-based synthesis of AuNPs; (ii) proposed anti-cancer mechanisms along with biomedical applications in diagnostics, imaging, and drug delivery; and (iii) key challenges for biogenic AuNPs as future cancer nanomedicine.

     

Targeting of ovarian cancer cell through functionalized gold nanoparticles by novel glypican-3- binding peptide as a ultrasound contrast agents

Gold nanoparticles (AuNPs) are widely studied nanomaterials for their potential employment in advanced biomedical applications, such as selective molecular imaging and targeted drug delivery. AuNPs are generally low cost and highly biocompatible, can be easily functionalized with a wide variety of functional ligands, and have been demonstrated to be effective in enhancing ultrasound contrast at clinical diagnostic frequencies. Therefore, AuNPs might be used as contrast agents in echographic imaging. In this work, we have developed a AuNPs -based system for the in vitro molecular imaging of ovarian carcinoma cells that express high levels of glypican-3 protein (GPC-3) on their surface. In this regard, a novel GPC-3 targeting peptide was designed and conjugated to fluorescent AuNPs nanoparticles. The physicochemical properties, acoustic behavior, and biocompatibility profile of the functionalized AuNPs were characterized. Then, the binding and uptake of both naked and functionalized AuNPs were analyzed by laser scanning confocal microscopy in human HeLa cells (ovarian carcinoma) cell line. The results obtained showed that GPC-3-functionalized fluorescent AuNPs significantly enhanced the ultrasound contrast and were effectively bound and taken up by HeLa cells without affecting their viability.     

Upconversion luminescence resonance energy transfer-based aptasensor for the sensitive detection of oxytetracycline

In this work, a biosensor based on luminescence resonance energy transfer (LRET) from NaYF₄:Yb,Tm upconversion nanoparticles (UCNPs) to SYBR Green I has been developed. The aptamers are covalently linked to UCNPs and hybridized with their complementary strands. The subsequent addition of SYBR Green allows SYBR Green I to insert into the formed double-stranded DNA (dsDNA) duplex and brings the energy donor and acceptor into close proximity, leading to the fluorescence of UCNPs transferred to SYBR Green I. When excited at 980 nm, the UCNPs emit luminescence at 477 nm, and this energy is transferred to SYBR Green I, which emits luminescence at 530 nm. In the presence of oxytetracycline (OTC), the aptamers prefer to bind to its corresponding analyte and dehybridize with the complementary DNA. This dehybridization leads to the liberation of SYBR Green I, which distances SYBR Green I from the UCNPs and recovers the UCNPs' luminescence. Under optimal conditions, a linear calibration is obtained between the ratio of I₅₃₀ to I₄₇₇ nm (I₅₃₀/I₄₇₇) and the OTC concentration, which ranges from 0.1 to 10 ng/ml with a limit of detection (LOD) of 0.054 ng/ml.     

Nanomaterials for cancer therapy and imaging

A variety of organic and inorganic nanomaterials with dimensions below several hundred nanometers are recently emerging as promising tools for cancer therapeutic and diagnostic applications due to their unique characteristics of passive tumor targeting. A wide range of nanomedicine platforms such as polymeric micelles, liposomes, dendrimers, and polymeric nanoparticles have been extensively explored for targeted delivery of anti-cancer agents, because they can accumulate in the solid tumor site via leaky tumor vascular structures, thereby selectively delivering therapeutic payloads into the desired tumor tissue. In recent years, nanoscale delivery vehicles for small interfering RNA (siRNA) have been also developed as effective therapeutic approaches to treat cancer. Furthermore, rationally designed multi-functional surface modification of these nanomaterials with cancer targeting moieties, protective polymers, and imaging agents can lead to fabrication versatile theragnostic nanosystems that allow simultaneous cancer therapy and diagnosis. This review highlights the current state and future prospects of diverse biomedical nanomaterials for cancer therapy and imaging.     

Molecular profiling of single cancer cells and clinical tissue specimens with semiconductor quantum dots

Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive imaging of malignant tumors in living animal models. In this article, we briefly discuss recent developments in bioaffinity QD probes and their applications in molecular profiling of individual cancer cells and clinical tissue specimens.     

Upconversion: road to El Dorado of the fluorescence world

Upconversion nanoparticles (UCNs), in the recent times have attracted attention due to their unique properties, which makes them ideal fluorophores for use in biological applications. There have been various reports on their use for targeted cell imaging, drug and gene delivery and also for diffuse optical tomography. Here we give a brief introduction on what are UCNs and the mechanism of upconversion, followed by a discussion on the biological applications of UCNs and further on what the future holds for UCNs. Copyright © 2010 John Wiley & Sons, Ltd.     

Targeted magnetic iron oxide nanoparticles for tumor imaging and therapy

Magnetic iron oxide (IO) nanoparticles with a long blood retention time, biodegradability and low toxicity have emerged as one of the primary nanomaterials for biomedical applications in vitro and in vivo. IO nanoparticles have a large surface area and can be engineered to provide a large number of functional groups for cross-linking to tumor-targeting ligands such as monoclonal antibodies, peptides, or small molecules for diagnostic imaging or delivery of therapeutic agents. IO nanoparticles possess unique paramagnetic properties, which generate significant susceptibility effects resulting in strong T2 and T*2 contrast, as well as T1 effects at very low concentrations for magnetic resonance imaging (MRI), which is widely used for clinical oncology imaging. We review recent advances in the development of targeted IO nanoparticles for tumor imaging and therapy.     

Magnetic nanobead-based immunoassay for the simultaneous detection of aflatoxin B1 and ochratoxin A using upconversion nanoparticles as multicolor labels

A novel and sensitive immunoassay for the simultaneous detection of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) in food samples was developed by using artificial antigen-modified magnetic nanoparticles (MNPs) as immunosensing probes and antibody functionalized upconversion nanoparticles (UCNPs) as signal probes. NaY_0.78F₄:Yb_0.2, Tm_0.02 and NaY_0.28F₄:Yb_0.7,Er_0.02 UCNPs were prepared and functionalized, respectively, with immobilized monoclonal anti-AFB₁ antibodies and anti-OTA antibodies as signal probes. Based on a competitive immunoassay format, the detection limit for both AFB₁ and OTA under optimal conditions was as low as 0.01 ng mL⁻¹, and the effective detection range was from 0.01 to 10 ng mL⁻¹. The proposed method was successfully applied to measure AFB₁ and OTA in naturally contaminated maize samples and compared to a commercially available ELISA method. The high sensitivity and selectivity of this method is due to the magnetic separation and concentration effect of the MNPs, the high sensitivity of the UCNPs, and the different emission lines of Yb/Tm and Yb/Er doped NaYF₄ UCNPs excited by 980 nm laser. Multicolor UCNPs have the potential to be used in other applications for detecting toxins in the field of food safety and other fields.     

Selective cellular imaging with lanthanide‐based upconversion nanoparticles

Upconversion nanoparticles (UCNPs) with sodium yttrium fluoride, NaYF₄ (host lattice) doped with Yb³⁺ (sensitizer) and Er³⁺ (activator) were synthesized via hydrothermal route incorporating polyethyleneimine (PEI) for their long‐term stability in water. The cationic PEI‐modified UCNPs with diameter 20 ± 4 nm showed a zeta potential value of +36.5 mV and showed an intense, visible red luminescence and low‐intensity green emission with 976 nm laser excitation. The particles proven to be nontoxic to endothelial cells, with a 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, showing 90% to 100% cell viability, across a wide range of UCNP concentrations (0.3 ng/mL‐0.3 mg/mL) were used in multiphoton imaging. Multiphoton cellular imaging and emission spectroscopy data reported here prove that the UCNPs dispersed in cell culture media are predominantly concentrated in the cytoplasm than the cell nucleus. The energy transfer from PEI‐coated UCNPs to surrounding media for red luminescence in the biological system is also highlighted with spectroscopic measurements. Results of this study propose that UCNPs can, therefore, be used for cytoplasm selective imaging together with multiphoton dyes (eg, 4′,6‐diamidino‐2‐phenylindole (DAPI)) that are selective to cell nucleus.      

Engineering of galectin-3 for glycan-binding optical imaging

Galectin-3 (Gal-3) is a multifunctional glycan-binding protein that participates in many pathophysiological events and has been described as a biomarker and potential therapeutic target for severe disorders, such as cancer. Several probes for Gal-3 or its ligands have been developed, however both the pathophysiological mechanisms and potential biomedical applications of Gal-3 remain not fully assessed. Molecular imaging using bioluminescent probes provides great sensitivity for in vivo and in vitro analysis for both cellular and whole multicellular organism tracking and target detection. Here, we engineered a chimeric molecule consisting of Renilla luciferase fused with mouse Gal-3 (RLuc-mGal-3). RLuc-mGal-3 preparation was highly homogenous, soluble, active, and has molecular mass of 65,870.95 Da. This molecule was able to bind to MKN45 cell surface, property which was inhibited by the reduction of Gal-3 ligands on the cell surface by the overexpression of ST6GalNAc-I. In order to obtain an efficient and stable delivery system, RLuc-mGal-3 was adsorbed to poly-lactic acid nanoparticles, which increased binding to MKN45 cells in vitro. Furthermore, bioluminescence imaging showed that RLuc-mGal-3 was able to indicate the presence of implanted tumor in mice, event drastically inhibited by the presence of lactose. This novel bioluminescent chimeric molecule offers a safe and highly sensitive alternative to fluorescent and radiolabeled probes with potential application in biomedical research for a better understanding of the distribution and fate of Gal-3 and its ligands in vitro and in vivo.     

Selection of quantum dot wavelengths for biomedical assays and imaging

Fluorescent semiconductor nanocrystals (quantum dots [QDs]) are hypothesized to be excellent contrast agents for biomedical assays and imaging. A unique property of QDs is that their absorbance increases with increasing separation between excitation and emission wavelengths. Much of the enthusiasm for using QDs in vivo stems from this property, since photon yield should be proportional to the integral of the broadband absorption. In this study, we demonstrate that tissue scatter and absorbance can sometimes offset increasing QD absorption at bluer wavelengths, and counteract this potential advantage. By using a previously validated mathematical model, we explored the effects of tissue absorbance, tissue scatter, wavelength dependence of the scatter, water-to-hemoglobin ratio, and tissue thickness on QD performance. We conclude that when embedded in biological fluids and tissues, QD excitation wavelengths will often be quite constrained, and that excitation and emission wavelengths should be selected carefully based on the particular application. Based on our results, we produced near-infrared QDs optimized for imaging surface vasculature with white light excitation and a silicon CCD camera, and used them to image the coronary vasculature in vivo. Taken together, our data should prove useful in designing fluorescent QD contrast agents optimized for specific biomedical applications.     

Fluorescent Nanocrystals Reveal Regulated Portals of Entry into and Between the Cells of Hydra

Initially viewed as innovative carriers for biomedical applications, with unique photophysical properties and great versatility to be decorated at their surface with suitable molecules, nanoparticles can also play active roles in mediating biological effects, suggesting the need to deeply investigate the mechanisms underlying cell-nanoparticle interaction and to identify the molecular players. Here we show that the cell uptake of fluorescent CdSe/CdS quantum rods (QRs) by Hydra vulgaris, a simple model organism at the base of metazoan evolution, can be tuned by modifying nanoparticle surface charge. At acidic pH, amino-PEG coated QRs, showing positive surface charge, are actively internalized by tentacle and body ectodermal cells, while negatively charged nanoparticles are not uptaken. In order to identify the molecular factors underlying QR uptake at acidic pH, we provide functional evidence of annexins involvement and explain the QR uptake as the combined result of QR positive charge and annexin membrane insertion. Moreover, tracking QR labelled cells during development and regeneration allowed us to uncover novel intercellular trafficking and cell dynamics underlying the remarkable plasticity of this ancient organism.