Adsorption properties and activities of lipase on a gold substrate modified by self-assembled monolayers.

The adsorption properties, amount and specific activity of lipase D from Rhizopus delemar were investigated by employing a gold substrate modified with seven kinds of thiol monolayer. Quartz crystal microbalance measurements revealed that the amount of the enzyme adsorbed to the hydrophobic monolayers (e.g. benzenethiol) was much higher than that to the hydrophilic monolayers (e.g. 3-mercaptopropanoic acid). In contrast, lipase D adsorbed to the hydrophilic, 2-amino-1-ethanethiol monolayer showed the highest specific activity, the value being 300-fold higher than for the same enzyme dissolved in an aqueous medium.     

Highly sensitive gold nanoparticles biosensor chips modified with a self-assembled bilayer for detection of Con A

In this paper, an improved method for detection of Concanavalin A (Con A) with label-free optical biosensors is reported. 1-Dodecanethiol (DDT) was self-assembled onto gold nanoparticles which were deposited on glass slides, and then glycolipid molecules were inserted into dodecanethiol by physical interactions only. The recognition between Con A and carbohydrate was observed by UV-vis spectrophotometry. The absorption spectrum shifted when Con A was bound to the sugar residues of glycolipids immobilized onto nanogold slides, while almost no spectrum change was observed when another nonspecific protein molecule met the nanogold slides. The self-assembled bilayer on nanogold substrates had very high sensitivity for Con A, the minimum detection concentration of Con A can be down to 0.1 nM. In addition to the ultra sensitivity for investigating carbohydrate-lectin interaction, the self-assembled bilayer structure, is expected to replace many receptors which require time-consuming organic syntheses for the fixation to the transducer. The simplicity and sensitivity of this biosensor architecture once again show the prospect of nanogold application in biosensor.     

Development of a high analytical performance-tyrosinase biosensor based on a composite graphite-Teflon electrode modified with gold nanoparticles

The design of a new tyrosinase biosensor with improved stability and sensitivity is reported. The biosensor design is based on the construction of a graphite-Teflon composite electrode matrix in which the enzyme and colloidal gold nanoparticles are incorporated by simple physical inclusion. Experimental variables such as the colloidal gold loading into the composite matrix, the enzyme loading and the potential applied to the bioelectrode were optimized. The Tyr-Au(coll)-graphite-Teflon biosensor exhibited suitable amperometric responses at -0.10 V for the different phenolic compounds tested (catechol; phenol; 3,4-dimethylphenol; 4-chloro-3-methylphenol; 4-chlorophenol; 4-chloro-2-methylphenol; 3-methylphenol and 4-methylphenol). The limits of detection obtained were 3 nM for catechol, 3.3 microM for 4-chloro-2-methylphenol, and approximately 20 nM for the rest of phenolic compounds. The presence of colloidal gold into the composite matrix gives rise to enhanced kinetics of both the enzyme reaction and the electrochemical reduction of the corresponding o-quinones at the electrode surface, thus allowing the achievement of a high sensitivity. The biosensor exhibited an excellent renewability by simple polishing, with a lifetime of at least 39 days without apparent loss of the immobilized enzyme activity. The usefulness of the biosensor for the analysis of real samples was evaluated by performing the estimation of the content of phenolic compounds in water samples of different characteristics.     

An enhanced ELISA based on modified colloidal gold nanoparticles for the detection of Pb(II)

A novel probe based on colloidal gold nanoparticles (AuNPs) modified with goat anti-mouse IgG and horseradish peroxidase (HRP) was synthesized and an enhanced enzyme-linked immunosorbent assay (ELISA) based on the probe was developed. In the assay, the synthesized probe is bound with a monoclonal antibody (McAb) which is competitively bound by coated BSA-ITCBE-Pb(II) on plate and Pb(II) in samples. The HRP, used here for signal amplification catalytically oxidize the substrate and generate optical signals that is related to the concentration of Pb(II) and can be measured spectrophotometrically. For the monodisperse AuNPs having high surface areas, it can be conjugated with more amount of HRP than that of IgG. Therefore, compared with traditional ELISA, the signal amplification of catalytically oxidized substrate was enhanced. The detection limit for this novel modified AuNPs probe-based assay was 9 pg mL(-1). The recoveries obtained by standard Pb(II) addition to real samples, including a commercial mineral water, tap water, and lake water were all from 94.9% to 102.9%. And the coefficient of variation (CV) value of all samples was less than 10%. The results indicated that the enhanced assay gave higher sensitivity and reliable reproducibility. It could provide a general detection format for low-molecular weight contaminants.     

Sensing capabilities of colloidal gold monolayer modified with a phenylboronic acid-carrying polymer brush

A dithiolated random copolymer with pendent phenylboronic acid residues [Cys-poly(3-acrylamidophenylboronic acid-co-N,N-dimethylaminopropyl methacrylamide), Cys-poly(APBA-co-DMAPMA)] that shows the abilities of initiation, transfer, and termination (iniferter) was obtained by using a benzyl N,N-diethyldithiocarbamoyl (BDC) derivative. The obtained disulfide-carrying copolymer was accumulated on a colloidal gold-immobilized glass substrate, and the usefulness of the polymer brush as a sensing element for glycoproteins such as ovalbumin (OVA) was examined by UV-visible spectrophotometry with the help of localized surface plasmon resonance (LSPR). The sensor showed a concentration-dependent binding of OVA with a detection limit of 100 nM, and it had a very high stability at high ionic strength. The sensor chip could be used for a detection of another glycoprotein, avidin, as well. Furthermore, the binding of biotin-modified human serum albumin (biotinylated HSA) to the avidin-phenylboronic acid- (PBA-) carrying polymer brush complex and further specific binding of anti-HSA immunoglobulin G to the biotinylated HSA-avidin-PBA-carrying polymer brush ternary complex could clearly be observed. The polymer-brush-coated device examined here not only was useful as a simple sensor chip, but also is expected to open a new perspective on interfacial phenomena performed by various functional polymer brushes fixed to colloidal gold on glass substrates.     

Immobilization of single-stranded DNA by self-assembled polymer on gold substrate for a DNA chip

We developed a self-assembling polymer based on polyallylamine (PAH) for use in DNA chips. Thioctic acid (TA) was covalently attached to PAH in sidechains to immobilize the polymer on a gold surface by self-assembly. N-hydroxysuccinimide-ester terminated probe single-stranded (ss) DNA is easily covalently immobilized onto a TA-PAH-coated gold surface. Finally, the surface was covered with polyacrylic acid, which formed ion complexes with the TA-PAH, to reduce the cationic charge. This ssDNA on a polymer-coated surface recognized a fully matched DNA sequence and restrained nonspecific adsorption of target DNA. The selectivity and efficiency of hybridization was affected by adjusting the ionic strength of sodium chloride.     

Enhanced immobilization of hexa-arginine-tagged esterase on gold nanoparticles using mixed self-assembled monolayers

Mixed self-assembled monolayers (MSAMs) composed of diverse ligands offer a mechanism for the specific binding of biomolecules onto solid surfaces. In this study, we examined the formation of MSAMs on gold nanoparticles (AuNPs) and the immobilization of hexa-arginine-tagged esterase (Arg₆-esterase) on the surfaces of the resulting particles. The functionalization of AuNPs with MSAMs was achieved by introducing a mixture of tethering and shielding ligands into an AuNP solution. The formation of self-assembled monolayers (SAMs) on the AuNP surface was characterized by UV/visible spectroscopy, transmission electron microscopy, and Fourier-transform infrared spectroscopy. Arg₆-esterase was immobilized in a highly specific manner onto AuNPs treated with mixed SAMs (MSAM–AuNPs) by providing a shielding ligand which reduce the non-specific adsorption of enzymes caused by hydrophobic interaction compared to AuNPs treated with single-component SAMs (SSAM–AuNPs). Moreover, Arg₆-esterase immobilized on MSAM–AuNPs showed substantially enhanced catalytic activity up to an original activity compared to that on SSAM–AuNPs (58%).     

Label-free capacitive immunosensor for microcystin-LR using self-assembled thiourea monolayer incorporated with Ag nanoparticles on gold electrode

A label-free immunosensor based on a modified gold electrode incorporated with silver (Ag) nanoparticles (NPs) to enhance the capacitive response to microcystin-LR (MCLR) has been developed. Anti-microcystin-LR (anti-MCLR) was immobilized on silver nanoparticles bound to a self-assembled thiourea monolayer. Interaction of anti-MCLR and MCLR were directly detected by capacitance measurement. Under optimum conditions, MCLR could be determined with a detection limit of 7.0pgl(-1) and linearity between 10pgl(-1) and 1mugl(-1). The immobilized anti-MCLR on self-assembled thiourea monolayer incorporated with silver nanoparticles was stable and good reproducibility of the signal could be obtained up to 43 times with an R.S.D. of 2.1%. Comparing to the modified electrode without silver nanoparticles it gave 1.7-fold higher sensitivity and lower limit of detection. The developed immunosensor was applied to analyze MCLR in water samples and the results were in good agreement with those obtained by high-performance liquid chromatography (HPLC) (P<0.05).     

Labelling of colloidal gold with IgE

Summary Immunocytochemical markers prepared by labelling colloidal gold with antibodies are gaining wide acceptance both in transmission and scanning electron microscopy. However, detailed information on the process and extent of adsorption of IgG and IgE in particular are still lacking. The adsorption isotherm of mouse monoclonal ¹²⁵I-IgE antibovine milk -lactoglobulin was studied quantitatively with colloidal gold buffered at pH 6.1–8.8 (28 nm in particle diameter). At low coverage of the particles (5 molecules per particle), the isotherm was independant of pH. In the presence of a large excess of IgE, the highest coverage was obtained at pH 6.1 near the pI of IgE (5.2–5.8). The binding constants were higher at low coverage (side-on adsorption) than at high coverage where desorption was observed. IgE-Au markers were unreactive towards the immobilized antigen and did not bind to receptors for IgE of rat basophilic leukemia cells (RBL-1). The reactivity of immobilized anti-IgE antibodies with IgE-Au markers increased as a function of particle coverage. Mappings of RBL-1 cell membrane IgE receptors was achieved by incubating successively IgE-sensitized RBL-1 cells with anti-IgE antibodies and a protein A-gold marker at 4°C. Surface clusters developed when the cells were incubated at 37°C.     

Development of acetylcholinesterase biosensor based on CdTe quantum dots/gold nanoparticles modified chitosan microspheres interface

In this paper, a novel acetylcholinesterase (AChE) biosensor was constructed by modifying glassy carbon electrode with CdTe quantum dots (QDs) and excellent conductive gold nanoparticles (GNPs) though chitosan microspheres to immobilize AChE. Since GNPs have shown widespread use particularly for constructing electrochemical biosensors through their high electron-transfer ability, the combined AChE exhibited high affinity to its substrate and thus a sensitive, fast and cheap method for determination of monocrotophos. The combination of CdTe QDs and GNPs promoted electron transfer and catalyzed the electro-oxidation of thiocholine, thus amplifying the detection sensitivity. This novel biosensing platform based on CdTe QDs-GNPs composite responded even more sensitively than that on CdTe QDs or GNPs alone because of the presence of synergistic effects in CdTe-GNPs film. The inhibition of monocrotophos was proportional to its concentration in two ranges, from 1 to 1000ngmL(-1) and from 2 to 15mugmL(-1), with a detection limit of 0.3ngmL(-1). The proposed biosensor showed good precision and reproducibility, acceptable stability and accuracy in garlic samples analysis.     

Labelling of colloidal gold with protein

Summary Markers prepared by labelling colloidal gold with macromolecules such as lectins, antibodies and protein A are gaining wise acceptance both in transmission and scanning electron microscopy. However detailed information on the process and extent of adsorption of macromolecules onto gold particles are still lacking. The adsorption isotherm of protein onto gold particles was studied quantitatively using goat -lactoglobulin (L) tritiated in vivo. When this protein was modified chemically by iodination with ¹²⁵I, the adsorption isotherm was not significantly different (Langmuir type for monolayer). In the presence of saturating amount of L, a maximum of 13–14 molecules was adsorbed per particle of 12 nm in diameter for a theoretical maximum of 20 (compact monolayer). Ellipsometric measurements on nickel-coated slides indicated that L was adsorbed onto metallic surfaces as a compact monolayer. The molecules were irreversibly adsorbed on gold particles, kept to a large extent their capacity to bind anti-L antibodies and could not be displaced by polyethylene glycol, a stabilizer commonly used in the preparation of gold markers. Only markers labelled with more than 5 L molecules per particle could be completely bound by immobilized anti-L antibodies. Preliminary data indicated that the energetics of adsorption of L onto colloidal gold was in agreement with that expected from the mutual interaction of surface and adsorbate.     

Fabricating upconversion fluorescent nanoparticles modified substrate for dynamical control of cancer cells and pathogenic bacteria

Lanthanide‐doped upconversion nanoparticles (UCNPs) have attracted widespread interests in the field of biomedicine because of their unique upconverting capability by converting near infrared (NIR) excitation to visible or ultraviolet (UV) emission. Here, we developed a novel UCNP‐based substrate for dynamic capture and release of cancer cells and pathogenic bacteria under NIR‐control. The UCNPs harvest NIR light and convert it to ultraviolet light, which subsequently result in the cleavage of photoresponsive linker (PR linker) from the substrate, and on demand allows the release of a captured cell. The results show that after seeding cells for 5 h, the cells were efficiently captured on the surface of the substrate and ?89.4% of the originally captured S. aureus was released from the surface after exposure to 2 W/cm² NIR light for 30 min, and ?92.1% of HepG2 cells. These findings provide a unique platform for exploring an entirely new application field for this promising luminescent nanomaterial.

     

Streptavidin conjugates with gold nanoparticles for visualization of single DNA interactions on the silicon surface

Applicability of scanning electron microscopy (SEM) for visualization of individual acts of DNA hybridization with oligonucleotide probes has been investigated using gold nanoparticles as a label. DNA or oligonucleotides were labeled with biotin molecules, which were then detected in DNA duplexes using a streptavidin conjugate with gold nanoparticles. Effective imaging of DNA duplexes was possible using the conjugate prepared by covalent binding. The detection limit of the model oligonucleotide of 19 bases was 20 pg.     

基于胶体金的8-羟基-2′-脱氧鸟苷免疫层析试纸条北大核心CSCD

8-羟基-2′-脱氧鸟苷(8-hydroxy-2′-deoxyguanosine,8-OHdG)是评价DNA氧化损伤较灵敏和稳定的生物标志物。文中采用竞争法建立一种快速、灵敏检测8-OHdG的胶体金免疫层析试纸条。将样品垫(玻璃纤维素膜)、结合垫(玻璃纤维素膜)、硝酸纤维素膜和吸水垫依此粘贴在聚氯乙烯(polyvinyl chloride,PVC)底板上,构建试纸条。通过柠檬酸钠还原三水合四氯金酸制备胶体金(gold nanoparticles,AuNPs),8-OHdG配对的抗体(antibody,Ab)包被于AuNPs的外层(Ab coated AuNPs,Ab@AuNPs)作为探针。牛血清蛋白(bovine serum protein,BSA)与8-OHdG用碳二亚胺盐酸盐偶联制备人工抗原,作为检测线的包被抗原。羊抗鼠多抗(imunoglobulin G,IgG)作为质控线的包被抗体。对试纸条的硝酸纤维素膜、上样液的配方、金标抗体喷涂量等实验参数进行了优化。结果表明,硝酸纤维素膜(nitrocellulose film,NC)膜采用CN 95,上样液的最优配方为1%BSA+3%吐温-20+3%蔗糖+0.9%NaCl溶液,最适金标抗体喷涂量为4μL。利用试纸条在可见光下检测8-OHdG,根据检测线(test line,T线)和质控线(control line,C线)的显色强度对比,可初步判断尿液中8-OHdG的含量水平。并通过T线的灰度值计算尿液中的8-OHdG的浓度,检测限为2.55μg/L。该方法简单、快速且有较好的特异性,可检测人体尿液中的8-OHdG含量,以初步评价人体的健康状态。     

Prostate-specific antigen immunosensing based on mixed self-assembled monolayers, camel antibodies and colloidal gold enhanced sandwich assays

Prostate-specific antigen (PSA) is a valuable biomarker for prostate cancer screening. We developed a PSA immunoassay on a commercially available surface plasmon resonance biosensor. Our PSA receptor molecule consists of a single domain antigen-binding fragment, cAbPSA-N7, derived from dromedary heavy-chain antibodies and identified after phage display. It binds PSA with a high k(on) value of 1.9x10(6) M-1 s-1, and was covalently immobilised on a gold substrate via a mixed self-assembled monolayer (SAM) of alkanethiols by using carbodiimide-coupling chemistry in 10mM acetate buffer pH 5.5 to obtain an optimal pre-concentration. The best performing and optimised mixed SAM consisted of (10%) 16-mercapto-1-hexadecanoic acid (16-MHA) for covalent cAbPSA-N7 immobilisation and (90%) 11-mercapto-1-undecanol (11-MUOH) to minimise non-specific adsorption of the analyte. In this way, two advantages are incorporated in a single coupling layer. Up to 28 fmol/mm2 of cAbPSA-N7 could be immobilised and 30% of its binding sites participate actively in PSA interaction. In addition, the optimised layer showed also optimal performance to assess physiological samples. Although PSA concentrations as low as 10 ng/ml could be detected directly, this detection limit could be enhanced to PSA levels in the sub ng/ml range by introducing a sandwich assay involving a biotinylated secondary antibody and streptavidin modified gold nanoparticles. This approach realizes the PSA detection at clinical relevant concentrations.     

DNAzyme self-assembled gold nanoparticles for determination of metal ions using fluorescence anisotropy assay

Gold nanoparticles can be exploited to facilitate a highly sensitive and selective metal ion detection based on fluorescence anisotropy assay with metal ion-dependent DNA-cleaving DNAzyme. This assay allows rapid and accurate determination of metal ions in aqueous medium at room temperature. The method has been demonstrated for determination of Cu²⁺ and Pb²⁺ ions. The detection sensitivity can be significantly improved to 1 nM by using a “nanoparticle enhancement” approach. Moreover, the assay was also tested in 384-well plates for high-throughput routine determination of toxic metal ions in environmental samples. The method showed distinct advantages over conventional methods in terms of its potential sensitivity, specificity, and ability for rapid response.     

Using self-assembled aptamers and fibrinogen-conjugated gold nanoparticles to detect DNA based on controlled thrombin activity

We have developed a colorimetric probe, based on the aggregation of gold nanoparticles (Au NPs), for the detection of DNA and for the analysis of single-nucleotide polymorphism (SNP); this probe functions through the modulation of the activity of thrombin (Thr) in the presence of bivalent thrombin-binding aptamers (TBAs). The bivalent TBAs were formed from TBA(27') (comprising a 27-base sequence providing TBA(27) functionality, a T(5) linker, and an 11-base sequence for hybridization) and TBA(15') (comprising a 15-base sequence providing TBA(15) functionality, a T(5) linker, and a 12-base sequence for hybridization) through their hybridization with perfectly matched DNA (DNA(pm)). The bivalent TBAs interacted specifically with thrombin, suppressing its activity toward fibrinogen-modified Au NPs (Fib-Au NPs). The potency of the inhibitory effect of TBA(15')-TBA(27')/DNA(pm) toward thrombin - and, thus, the degree of aggregation of the Fib-Au NPs - was highly dependent on the concentration of DNA(pm). Under the optimal conditions (50 pM thrombin, 2 nM TBA(15'), 2 nM TBA(27'), and 38 pM Fib-Au NPs), the linear relationship of the response of the probe toward DNA(pm) extended from 0.1 to 2 nM, with a correlation coefficient of 0.97. The limit of detection (LOD) for DNA(pm) was 20 pM, based on a signal-to-noise ratio of 3. We also applied a corresponding TBA(15″)-TBA(27″)/Thr/Fib-Au NP probe to the detection of the SNP of the Arg249Ser unit in the TP53 gene, with an LOD of 32 pM. Relative to conventional molecular beacon-based and crosslinking aggregation-based Au NP probes, our new approach offers higher sensitivity and higher selectivity toward DNA.     

Thermodynamic and kinetic analysis of the interaction between hepatitis B surface antibody and antigen on a gold electrode modified with cysteamine and colloidal gold via electrochemistry

Hepatitis B surface antibody (HBsAb) was immobilized to the surface of a gold electrode modified with cysteamine and colloidal gold as matrices to detect hepatitis B surface antigen (HBsAg). Differential pulse voltammetry (DPV) method was used for the investigation of the specific interaction between the immobilized HBsAb and HBsAg in solution, which was followed as a change of peak current in DPV with time. With the modified gold electrode, the differences in affinity of HBsAb with HBsAg at the temperatures of 37 and 40 °C were easily distinguished and the kinetic rate constants (k_ass and k_diss) and kinetic affinity constant K were determined from the curves of current versus time. In addition, the thermodynamic constants, ΔG, ΔH and ΔS, of the interaction at 37 °C were calculated, which were −56.65, −64.54 and −25.45 kJ mol⁻¹, respectively.     

An electrophoretic method for selection of conditions for production of electrophoretically uniform protein colloidal gold complexes

An electrophoretic method was developed to determine the conditions required for production of electrophoretically uniform protein-colloidal gold complexes from monodisperse colloidal gold (Au) and electrophoretically uniform protein. The method is based on the electrophoretic migration of protein-Au complexes in agarose. The results demonstrate that two variables, the pH of adsorption and the quantity of protein added, can be manipulated to vary the electrophoretic mobility of the resulting protein-Au complexes. Thus, agarose gel electrophoresis can be used to select the pH of adsorption and the quantity of protein required to produce electrophoretically uniform protein-Au complexes. This new electrophoretic mobility test can be used in place of or in addition to the classical procedure of Zsigmondy and its many variations, both visual and spectrophotometric. The procedure described is also useful for electrophoretic comparison of small quantities of various protein-Au samples.