Enzymatic Biosensor Based on Carbon Paste Electrodes Modified with Gold Nanoparticles and Polyphenol Oxidase

The results presented here demonstrate the important catalytic effect of a carbon paste electrode modified by dispersion of gold nanoparticles towards different electroactive compounds. The oxidation of hydrogen peroxide starts at potentials 400 mV less positive than at bare carbon paste, while the reduction, almost negligible at bare carbon paste, starts at 0.100 V. The influence of the size and amount of gold nanoparticles in the composite matrix on the response of the electrode is discussed. The incorporation of albumin within the carbon paste facilitates the dispersion of gold nanoparticles, improving substantially the catalytic effects. At carbon paste modified with gold nanoparticles and albumin, the peak potential separation for hydroquinone decreases from 0.385 V to 0.209 V while the reduction current increases from 16.6 to 75.2 μA. The immobilization of polyphenol oxidase within the carbon paste electrode modified with nanoparticles has allowed us to obtain a very sensitive biosensor for dopamine even in the presence of large excess of ascorbic acid.     

生物大分子自组装膜及其应用研究进展

本文主要介绍了酶，蛋白质、DNA等生物大分子自组装膜的研究进展，并对生物大分子膜在生物传感器，分子器件，高效催化材料，医用生物材料等方面的应用前景进行了展望。     

Highly sensitive amplified electronic detection of DNA by biocatalyzed precipitation of an insoluble product onto electrodes

The amplified detection of a target DNA, based on the alkaline phosphatase oxidative hydrolysis of the soluble 5-bromo-4-chloro-3-indoyl phosphate to the insoluble indigo product as an amplification path, is addressed by two different sensing configurations. The accumulation of the insoluble product on Au electrodes or Au/quartz crystals alters the interfacial electron-transfer resistance at the Au electrode or the mass associated with the piezoelectric crystal, thus enabling the quantitative transduction of the DNA sensing by Faradaic impedance spectroscopy or microgravimetric quartz crystal microbalance measurements, respectively. One sensing configuration involves the association of a complex consisting of the target DNA and a biotinylated oligonucleotide to the functionalized transducers. The binding of the avidin/alkaline phosphatase conjugate to the sensing interface followed by the biocatalyzed precipitation provides the amplification path for the analysis of the target DNA. This analysis scheme was used to sense the target DNA with a sensitivity limit that corresponds to 5 x 10(-14) M. The second amplified detection scheme involves the use of a nucleic-acid-functionalized alkaline phosphatase as a biocatalytic conjugate for the precipitation of the insoluble product. Following this scheme, the functionalized transducers are interacted with the analyzed sample that was pretreated with the oligonucleotide-modified alkaline phosphatase, followed by the biocatalyzed precipitation as the amplification route for the analysis of the target DNA. By the use of this configuration, a detection limit corresponding to 5 x 10(-13) M was achieved. Real clinical samples of the Tay-Sachs genetic disorder were easily analyzed by the developed detection routes.     

Construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors

A new general method, post-photoaffinity-labeling modification (PPALM), for constructing fluorescent saccharide biosensors based on naturally occurring saccharide-binding proteins, lectins, is described in detail. An active-site-directed incorporation of a masked reactive site into a lectin was conducted by using a photoaffinity labeling technique followed by demasking and then chemical modification to yield a fluorescent lectin. Two photoaffinity labeling reagents were designed and synthesized in this study. The labeling reagent with a photoreactive site appended through a disulfide link to a mannoside unit was bound to the saccharide-binding pocket of the lectin concanavalin A (Con A). After light irradiation, the mannoside unit was cleaved by reduction. The unique thiol group thus produced was site-specifically modified with various fluorescent groups (dansyl, coumarin, or dimethylaminobenzoate derivatives) to afford fluorescent Con As. The labeling site was characterized by protease-catalyzed digestion followed by HPLC, MALDI-TOF MS, and tandem mass-mass spectrometry; these methods indicated that the photolabeling step is remarkably site specific. Strong fluorescence was observed in the engineered Con A with a fluorophore, and the emission changed sensitively upon saccharide complexation. The binding constants for various saccharides were determined by fluorescence titration and demonstrated that the binding selectivity and affinity of the engineered Con As are comparable to those of native Con A. The red shift of the emission maximum, the decrease in the fluorescence anisotropy of the dansyl unit, and the increase in the twisted intramolecular charge transfer emission caused by sugar binding to the engineered Con A explicitly indicate that the microenvironment of the appended fluorophores changes from a restricted and relatively hydrophobic environment into a rather freely mobile and hydrophilic environment.     

Antigen-antibody binding kinetics for biosensor applications

The diffusion-limited binding kinetics of antigen (or antibody) in solution to antibody (or antigen) immobilized on a biosensor surface is analyzed within a fractal framework. The fit obtained by a dual-fractal analysis is compared with that obtained from a single-fractal analysis. In some cases, the dual-fractal analysis provides an improved fit when compared with a single-fractal analysis. This was indicated by the regression analysis provided by Sigmaplot (San Rafael, CA). These examples are presented. It is of interest to note that the state of disorder (or the fractal dimension) and the binding rate coefficient both increase (or decrease, a single example is presented for this case) as the reaction progresses on the biosensor surface. For example, for the binding of monoclonal antibody MAb 49 in solution to surface-immobilized antigen, a 90.4% increase in the fractal dimension (D_f1 toD _f2 ) from 1.327 to 2.527 leads to an increase in the binding rate coefficient (k₁ to k₂) by a factor of 9.4 from 11.74 to 110.3. The different examples analyzed and presented together provide a means by which the antigen-antibody reactions may be better controlled by noting the magnitude of the changes in the fractal dimension and in the binding rate coefficient as the reaction progresses on the biosensor surface.     

Integrated nanoparticle-biomolecule hybrid systems: synthesis, properties, and applications

Nanomaterials, such as metal or semiconductor nanoparticles and nanorods, exhibit similar dimensions to those of biomolecules, such as proteins (enzymes, antigens, antibodies) or DNA. The integration of nanoparticles, which exhibit unique electronic, photonic, and catalytic properties, with biomaterials, which display unique recognition, catalytic, and inhibition properties, yields novel hybrid nanobiomaterials of synergetic properties and functions. This review describes recent advances in the synthesis of biomolecule-nanoparticle/nanorod hybrid systems and the application of such assemblies in the generation of 2D and 3D ordered structures in solutions and on surfaces. Particular emphasis is directed to the use of biomolecule-nanoparticle (metallic or semiconductive) assemblies for bioanalytical applications and for the fabrication of bioelectronic devices.     

Structure-switching signaling aptamers: transducing molecular recognition into fluorescence signaling

The development of aptamer technology considerably broadens the utility of nucleic acids as molecular recognition elements, because it allows the creation of DNA or RNA molecules for binding a wide variety of analytes (targets) with high affinity and specificity. Several recent studies have focused on developing rational design strategies for transducing aptamer-target recognition events into easily detectable signals, so that aptamers can be widely exploited for detection directed applications. We have devised a generalizable strategy for designing nonfluorescent aptamers that can be turned into fluorescence-signaling reporters. The resultant signaling probes are denoted "structure-switching signaling aptamers" as they report target binding by switching structures from DNA/DNA duplex to DNA/target complex. The duplex is formed between a fluorophore-labeled DNA aptamer and an antisense DNA oligonucleotide modified with a quencher (denoted QDNA). In the absence of the target, the aptamer hybridizes with QDNA, bringing the fluorophore into close proximity of the quencher for efficient fluorescence quenching. When this system is exposed to the target, the aptamer switches its binding partner from QDNA to the target. This structure-switching event is coupled to the generation of a fluorescent signal through the departure of QDNA, permitting the real-time monitoring of the aptamer-target recognition. In this article, we discuss the conceptual framework of the structure-switching approach, the essential features of structure-switching signaling aptamers as well as remaining challenges and possible solutions associated with this new methodology.     

Analyte-receptor binding kinetics for different types of biosensors

A fractal analysis is presented for analyte-receptor binding kinetics for different types of biosensor applications. Data taken from the literature may be modeled using a single-fractal analysis, a single- and a dual-fractal analysis, or a dual-fractal analysis. The latter two methods represent a change in the binding mechanism as the reaction progresses on the surface. Predictive relationships developed for the binding rate coefficient as a function of the analyte concentration are of particular value since they provide a means by which the binding rate coefficients may be manipulated. Relationships are presented for the binding rate coefficients as a function of the fractal dimension D _f or the degree of heterogeneity that exists on the surface. When analyte-receptor binding is involved, an increase in the heterogeneity on the surface (increase in D _f ) leads to an increase in the binding rate coefficient. It is suggested that an increase in the degree of heterogeneity on the surface leads to an increase in the turbulence on the surface owing to the irregularities on the surface. This turbulence promotes mixing, minimizes diffusional limitations, and leads subsequently to an increase in the binding rate coefficient. The binding rate coefficient is rather sensitive to the degree of heterogeneity, D _f , that exists on the biosensor surface. For example, the order of dependence on D _f1 is 7.25 for the binding rate coefficient k ₁ for the binding of a Fab fragment of an antiparaquat monoclonal antibody in solution to an antigen in the form of a paraquat analog immobilized on a sensor surface. The predictive relationships presented for the binding rate coefficient and the fractal dimension as a function of the analyte concentration in solution provide further physical insights into the binding reactions on the surface, and should assist in enhancing biosensor performance. In general, the technique is applicable to other reactions occurring on different types of surfaces, such as cell-surface reactions.     

Design optimization of highly sensitive LSPR enhanced surface plasmon resonance biosensors with nanoholes

For breaking through the sensitivity limitation of conventional surface plasmon resonance (SPR) biosensors, novel highly sensitive SPR biosensors with Au nanoparticles and nanogratings enhancement have been proposed recently.But in practice, these structures have obvious disadvantages.In this study, a nanohole based sensitivity enhancement SPR biosensor is proposed and the influence of different structural parameters on the performance is investigated by using rigorous coupled wave analysis (RCWA).Electromagnetic field distributions around the nanohole are also given out to directly explain the performance difference for various structural parameters.The results indicate that significant sensitivity increase is associated with localized surface plasmons (LSPs) excitation mediated by nanoholes.Except to outcome the weakness of other LSP based biosensors, larger resonance angle shift, reflectance amplitude, and sharper SPR curves' width are obtained simultaneously under optimized structural parameters.