Green fluorescent protein for in situ synthesis of highly uniform Au nanoparticles and monitoring protein denaturation

Purified recombinant green fluorescent protein (GFP) expressed in E. coli was used for single-step synthesis of gold nanoparticles (Au NPs) with extraordinary size specificity in aqueous medium. The fluorescence of GFP offered a probe for concomitant changes in the protein during the course of synthesis, in addition to the monitoring of the time-dependent formation of Au NPs by the surface plasmon resonance. Reaction of AuCl⁻₄ with the protein produced spherical Au NPs having diameters ranging from 5–70 nm. Remarkably, addition of 1.0×10⁻⁵ M AgNO₃ in the medium produced uniform spherical Au NPs with particle diameter of 2.2±0.5 nm. Fluorescence spectroscopic measurements suggest that during synthesis of Au NPs in absence of AgNO₃, partial denaturation of the protein occurred resulting in the lowering of fluorescence intensity. On the other hand, when the NPs were synthesized in the presence of AgNO₃ complete denaturation of the protein with complete loss of fluorescence could be observed, which was further confirmed by native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). However, use of AgNO₃ only resulted neither in the formation of NPs nor had any significant effect on the fluorescence of GFP.     

纳米荧光小球标记在蛋白质微阵列检测中的应用研究

研究了不经分离、一次性制备氨基化联吡啶钌掺杂的双层二氧化硅纳米小球的方法。实验证明该纳米小球尺寸均匀,光稳定性、水溶性好,分散稳定。通过简单的偶联反应后,它能有效的和蛋白质结合,结合后的蛋白能保持其生物活性。以此纳米荧光小球为标记物,应用于蛋白质微阵列的定量检测,结果发现其效果明显优于相同条件下以异硫氰酸荧光素(FITC)为标记物的定量结果,检出限可以达到3.5 mg/L。     

Rhodamine B doped silica nanoparticle labels for protein microarray detection

A core-shell Rhodamine B-doped SiO2 nanoparticle was synthesized and its fluorescent intensity was found to be 1000 times higher than that of individual Rhodamine B molecule. The doped nanoparticles were further conjugated with streptavidin and the resulting nanoparticles were used in the detection of reverse-phase protein microarrays, in which human IgG of various concentrations was first immobilized on aldehyde-modified glass slides and then biotinlyated goat anti human IgG as well as the labeled nanopart...     

Determination of methylene blue by resonance light scattering method using silica nanoparticles as probe

A simple and novel method was developed to determine methylene blue(MB) by resonance light scattering(RLS) using silica nanoparticles(SiO_2NPs) as the probe.It was found that MB could enhance the RLS intensity of SiO_2NPs.Moreover,the increase in RLS intensity was linear with the concentration of MB over the range of 0.01-3.0 μg mL~(-1).The limit of detection(LOD) was as low as 4.36 ng mL~(-1)(3σ) and the relative standard deviation(RSD) was 2.4%(n=6).Under the optimum experimental conditions,this proposed method was successfully applied for the determination of MB in aquaculture samples with recoveries between 96.3% and 107%.Possible mechanisms for the RLS enhancement of SiO_2NPs in the presence of MB were also discussed.     

Determining PPARγ-ligand binding affinity using fluorescent assay with cis-parinaric acid as a probe

Upon the study of small-molecules binding to proteins, the traditional methods for calculating dissociation constants (Kd and Ki) have shortcomings in dealing with the single binding site models. In this paper, two equations have been derived to solve this problem. These two equations are independent of the total concentration or initial degree of saturation of receptor and the activity of the competitive molecule. Through nonlinear fitting against these two equations, Kd value of a probe can be obtained by binding assay, and Ki value of a ligand can be obtained by competitive assay. Moreover, only the total concentrations of receptor([R]t), ligand([L]t) and probe([P]t) are required for the data fitting. In this work, Ki values of some typical ligands of PPARγ were successfully determined by use of our equations, among which the Ki value of PPARγ-LY171883 was reported for the first time.     

A selective,sensitive and label-free visual assay of fructose using anti-aggregation of gold nanoparticles as a colorimetric probe

A new convenient colorimetric sensor for fructose based on anti-aggregation of citrate-capped gold nanoparticles(Au NPs) is presented. 4-Mercaptophenylboronic acid(MPBA) induces the aggregation of Au NPs, leading to a color change from red to blue. Fructose as a potent competitor has strong affinity for MPBA and a borate ester is formed between MPBA and fructose. There is an obvious color change from blue to red with increasing the concentration of fructose. The anti-aggregation effect of fructose on Au NPs was seen by the naked eye and monitored by UV–vis spectra. Our results showed that the absorbance ratio(A_(519)/A_(640)) was linear with fructose concentration in the range of 0.032–0.96 μmol/L(R~2= 0.996), with a low detection limit of 0.01 μmol/L(S/N = 3). Notably, a highly selective recognition of fructose was shown against other monosaccharide and disaccharide(glucose, mannose, galactose,lactose and saccharose). With anti-aggregation assays higher selectivity is achievable. The results of this work provide a rapid method for evaluating the quantitative analysis of fructose in human plasma at physiologically meaningful concentrations and at neutral pH. The proposed procedure can be used as an efficient method for the precise and accurate determination of fructose.     

Heterogeneous immunoassay for soy protein determination using nile blue-doped silica nanoparticles as labels and front-surface long-wavelength fluorimetry

A long-wavelength fluoroimmunoassay for the determination of soy protein is reported for the first time using a conjugate composed of anti-soy protein antibodies bound to nile blue-doped silica nanoparticles (NPs). These NPs have been synthesized by a reverse-micelle microemulsion method and functionalized by using 3-(aminopropyl)triethoxysilane (APS) and 3-(trihydroxysilyl)propyl methylphosphonate (THPMP) to avoid NP aggregation. The tracer has been obtained by linking the functionalized NPs with anti-soy protein antibodies previously oxidised with sodium periodate. The immunoassay has been developed in 96-well microplates using a heterogeneous competitive format with antibody capture. Soy proteins are immobilised onto the wells and bovine serum albumin is added to block the surface, thus minimising non-specific binding. After washing, the microplates can be stored ready to use. At the analysis time, soy protein standards or sample and tracer are added and incubated and, after the corresponding washing and drying steps, the fluorescence is measured onto the solid surface at λ_ex 620 and λ_em 680 nm. The method features a dynamic range of 0.1–10 mg L⁻¹ and a detection limit of 0.05 mg L⁻¹. The precision of the method has been assayed at 0.5 and 5 mg L⁻¹ protein concentrations, obtaining the values of relative standard deviation of 9.6% and 6.1%, respectively. This new immunoassay has been applied to the analysis of food containing soy protein and the results obtained have been compared to those provided by a commercial ELISA kit with no statistically differing results. Also, a recovery study has been performed, providing percentages in the range of 81.5–111.0%.     

A highly sensitive and selective fluorescent probe for cyanide based on the dissolution of gold nanoparticles and its application in real samples

We report a simple fluorescence method for detection of cyanide sensitively and selectively based on the dissolution of polymer-coated gold nanoparticles by cyanide. The lowest concentration for quantification of cyanide ions was 3.0×10(-7) M, and other common anions nearly have no influence. Furthermore, several real water samples spiked with cyanide, including local groundwater, tap water, boiled water, and lake water, were analyzed, and the experimental results demonstrated that our sensing system worked well in the above water samples, with a good linear correlation.     

Anti-Her-2 monoclonal antibody conjugated polymer fluorescent nanoparticles probe for ovarian cancer imaging

Fluorescent nanoparticles (FNPs) with unique optical properties may be useful as biosensors in living cancer cell imaging and cancer targeting. A novel kind of polymer fluorescent nanoparticles (PFNPs) was synthesized and its application for ovarian cancer imaging with fluorescence microscopy imaging technology was presented in this study. The PFNPs were synthesized with precipitation polymerization by using methacrylic acid (MAA) as monomer, trimethylolpropane trimethacrylate (Trim) as cross-linker, azobisisobutyronitrile (AIBN) as radical initiator and butyl rhodamine B (BTRB) as fluorescent dye. And the fluorescent dye was embedded into the three-dimensional network of the polymer when the polymer was produced. With this method the PFNPs can be prepared easily. And then the PFNPs were successfully modified with anti-Her-2 monoclonal antibody. The fluorescence probe based on anti-Her-2 monoclonal antibody conjugated PFNPs has been used to detect ovarian cancer cells with fluorescence microscopy imaging technology. The experimental results demonstrate that the anti-Her-2 monoclonal antibody conjugated PFNPs can effectively recognize ovarian cancer cells and exhibit good sensitivity and exceptional photostability, which would provide a novel way for the diagnosis and curative effect observation of ovarian cancer cells.     

Novel fluorescent silica nanoparticle probe for ultrasensitive immunoassays

The preparation and utilization of a novel particulate label based on fluorescent hybrid silica (FHS) nanoparticles are reported in this article. These nanoparticles have shown several unique advantages over existing dye molecules, quantum dots, and latex-based fluorescent particles in easy preparation, good photostability and high sensitivity. A high molar ratio of the fluorescent molecules present in the core to biomolecules on the particle surface was achieved by using the well-developed silica surface immobilization chemistry for biomolecular linking. A fluoroimmunoassay method for detecting trace level Hepatitis B Surface Antigen (HBsAg) was developed. The calibration graph for HBsAg was linear over the range 0.5-220 ng/ml with a detection limit of 0.1 ng/ml. The sensitivity is greatly increased when compared with the corresponding immunoassay performed with direct fluorophore labeling. The present work shows that these FHS nanoparticles are high-quality markers for biochemical assays.     

Identification of live liver cancer cells in a mixed cell system using galactose-conjugated fluorescent nanoparticles

Bio-functioned fluorescent silica nanoparticles have been synthesized for cell labeling and cell differentiation and have shown great promise as novel fluorescent probes. The galactose-conjugated fluorescent nanoparticles (GCFNPs) have been obtained by the conjugation of amino-modified fluorescent silica nanoparticles with lactobionic acid (LA) through EDAC linkage. The GCFNPs retain excellent biological activity and can be used in bioanalysis as an immunofluorescence assay. The specific identification of target cells from the background cells have been directly demonstrated in a simple model system by a laser confocal scanning microscope, because the specific and non-specific labeling can simultaneously visualized in a given microscopic field of view. The flow cytometric analysis has proved that GCFNPs can effectively recognize target cells in the mixed cell system. The demonstration of precise identification of few liver cancer cells in the blood confirmed the excellent capability of GCFNPs in identifying specific cells in a large host cell background. The nanoparticle's excellent photostability, good biocompatibility and significant signal amplification make them well-suited for the identification of individual cells sensitively for a variety of biomedical studies such as cancer metastasis and stem cell progeny in vivo.     

Kinetics of proton transfer in a green fluorescent protein: A laser-induced pH jump study

The sub-millisecond protonation dynamics of the chromophore in S65T mutant form of the green fluorescent protein (GFP) was tracked after a rapid pH jump following laser-induced proton release from the caged photolabile compoundo-nitrobenzaldehyde. Following a jump in pH from 8 to 5 (which is achieved within 2 μs), the fluorescence of S65T GFP decreased as a single exponential with a time constant of ∼90 μs. This decay is interpreted as the conversion of the deprotonated fluorescent GFP chromophore to a protonated non-fluorescent species. The protonation kinetics showed dependence on the bulk viscosity of the solvent, and therefore implicates bulk solvent-controlled protein dynamics in the protonation process. The protonation is proposed to be a sequential process involving two steps: (a) proton transfer from solvent to the chromophore, and (b) internal structural rearrangements to stabilize a protonated chromophore. The possible implications of these observations to protein dynamics in general is discussed     

DNA template-synthesized silver nanoparticles:A new platform for high-performance fluorescent biosensing of biothiols

To develop the high-performance fluorescent bio-sensors, the metal nanoparticles were employed as nanoquenchers and at- tracted reasonable attention in the design of fluorescent biosensors. In this work, silver nanoparticles (AgNPs) were obtained via reduction of Ag+ on FAM-labeled DNA template. For the tight binding between AgNPs and DNA, the tem-plate-ynthesized AgNPs turned out high quenching efficiency and could be applied as super nanoquenchers to establish the biosensing platform for fluorescent detec...     

A comparative study on the preparation methods and properties of coal-based fluorescent carbon nanoparticles

Fluorescent carbon nanoparticles (FCNPs) have broad application prospects in the fields of bioimaging, ion detection, and photocatalysis. In this paper, coal-based FCNPs were prepared by using mixed acid oxidation, hydrogen peroxide etching, and organic solvent extraction methods (marked as FCNPs-AO, FCNPs-HE, and FCNPs-OS, respectively), and the structures and properties of the as-prepared products were compared. It was found that the coal-based FCNPs obtained by three kinds of methods are all aromatic structural nanomaterials linked with oxygen-containing groups. Among them, FCNPs-AO is a kind of hollow annular spherical particles and FCNPs-HE and FCNPs-OS are solid spherical particles. These FCNPs not only have similar fluorescence properties as traditional quantum dots, but also can be photoexcited to generate photogenerated electrons and holes, and it can also suppress the recombination of photogenerated electrons and holes by using its own surface defects. In particular, the electron transport capability of the FCNPs-AO is stronger than that of FCNPs-HE and FCNPs-OS because of its lower charge transfer impedance, so it can be excited to generate more photogenerated electrons and has the best photogenerated carrier separation efficiency.     

Determining PPARγ-ligand binding affinity using fluorescent assay with cis -parinaric acid as a probe

Upon the study of small-molecules binding to proteins, the traditional methods for calculating dissociation constants (K _d and K _i ) have shortcomings in dealing with the single binding site models. In this paper, two equations have been derived to solve this problem. These two equations are independent of the total concentration or initial degree of saturation of receptor and the activity of the competitive molecule. Through nonlinear fitting against these two equations, K _d value of a probe can be obtained by binding assay, and K _i value of a ligand can be obtained by competitive assay. Moreover, only the total concentrations of receptor([R]_t), ligand([L]_t) and probe([P]_t) are required for the data fitting. In this work, K _i values of some typical ligands of PPARγ were successfully determined by use of our equations, among which the K _i value of PPARγ-LY171883 was reported for the first time.     

多肽荧光探针在蛋白质检测中的应用

荧光探针法是痕量蛋白质检测的重要方法,其中多肽荧光探针得到了广泛的应用.本文综述了3种主要类型多肽荧光探针,即单荧光标记探针、双荧光标记探针和与其他材料形成复合物的探针的结构特点、检测原理以及不同类型多肽荧光探针在蛋白质定性、定量检测和酶活性测定等方面的应用,并对多肽荧光探针的未来发展方向进行了展望.     

Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 × 10⁻⁸ to 2.0 × 10⁻⁶ M with a detection limit of 1.6 × 10⁻⁸ M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications.     

Label-free detection of adenosine based on fluorescence resonance energy transfer between fluorescent silica nanoparticles and unmodified gold nanoparticles

A sensitive and convenient strategy was developed for label-free assay of adenosine. The strategy adapted the fluorescence resonance energy transfer property between Rhodamine B doped fluorescent silica nanoparticles (SiNPs) and gold nanoparticles (AuNPs) to generate signal. The different affinities of AuNPs toward the unfolded and folded aptamers were employed for the signal transfer in the system. In the presence of adenosine, the split aptamer fragments react with adenosine to form a structured complex. The folded aptamer cannot be adsorbed on the surface of AuNPs, which induces the aggregation of AuNPs under high ionic concentration conditions, and the aggregation of AuNPs leads to the decrease of the quenching ability. Therefore, the fluorescence intensity of Rhodamine B doped fluorescent SiNPs increased along with the concentration of adenosine. Because of the highly specific recognition ability of the aptamer toward adenosine and the strong quenching ability of AuNPs, the proposed strategy demonstrated good selectivity and high sensitivity for the detection of adenosine. Under the optimum conditions in the experiments, a linear range from 98 nM to 100 μM was obtained with a detection limit of 45 nM. As this strategy is convenient, practical and sensitive, it will provide a promising potential for label-free aptamer-based protein detection.     

Microwave-assisted synthesis of biofunctional and fluorescent silicon nanoparticles using proteins as hydrophilic ligands

Protective shell: A microwave-assisted method allows rapid production of biofunctional and fluorescent silicon nanoparticles (SiNPs), which can be used for cell labeling. Such SiNPs feature excellent aqueous dispersibility, are strongly fluorescent, storable, photostable, stable at different pH values, and biocompatible. The method opens new avenues for designing multifunctional SiNPs and related silicon nanostructures.