Rapid determination of binding parameters of chitin binding domains using chitin-coated quartz crystal microbalance sensor chips.

Chitin present in fungal cell walls has been considered as a diagnostic polymer for the detection of fungal infections. Chitin staining can be achieved with different dyes such as Calcofluor white or Congo red, but these methods have not entered into clinical routine diagnosis due to problems with sensitivity and specificity. More accurate detection can be achieved using chitin binding domains (CBDs) from a large variety of naturally occurring proteins that specifically interact with chitin. The chitin binding properties of most of these proteins have not yet been determined, because chitin is an insoluble fibrillar material rendering accurate determination of chitin binding kinetics challenging. Here we report a quartz crystal microbalance with dissipation monitoring (QCM-D) based method to determine binding constants of CBDs on chitin-coated gold surfaces. For this purpose, chitin was trimethylsilylated and coated onto the sensor chips. After desilylation, regular fibril-like structures with a typical center-to-center spacing of 85 nm were observed by atomic force microscopy. Using different experimental conditions and data evaluation methods for QCM-D measurements, we determined kon and koff and calculated the KD values for binding of a recombinant CBD from Bacillus circulans chitinase A1. Depending on the evaluation method, the KD values ranged between 0.6 and 2.5 µM. The obtained KD values were in good agreement with those measured for other bacterial CBDs usually ranging between 1 to 10 µM. Hence, we propose that the experimental approach developed in this study can be applied to determine yet unknown binding affinities of various CBDs from different origin.

Langmuir Analysis of the Binding Affinity and Kinetics for Surface Tethered Duplex DNA and a Ligand-Apoprotein Complex.

In this work, the hybridization and dehybridization of ssDNA with 20 bases at gold coated sensor surfaces modified with complementary 20 bases capture probe ssDNA was investigated at 18 °C by quartz crystal microbalance measurements with dissipation monitoring (QCM-D). A sequence of 20 base pairs with a melting temperature of about 64 °C was chosen, since in many biosensor studies the target molecules are DNA or RNA oligomers of similar length. It turned out that at the applied experimental conditions the DNA hybridization was irreversible, and therefore the hybridization and dehybridization process could not be described by the Langmuir model of adsorption. Nevertheless, quantitative dehybridization could be achieved by rinsing the sensor surface thoroughly with pure water. When in contrast the hybridization of a target with only 10 bases complementary to the outermost 10 bases of the 20 bases capture probe was studied, binding and unbinding were reversible, and the hybridization/dehybridization process could be satisfactorily described by the Langmuir model. For the 10 base pair sequence, the melting temperature was about 36 °C. Apparently, for Langmuir behavior, it is important that the experiments are applied at a temperature sufficiently close to the melting temperature of the sequence under investigation to ensure that at least traces of the target molecules are unhybridized (i.e., there needs to be an equilibrium between hybridized and dehybridized target molecules). To validate the reliability of our experimental approach we also studied the reconstitution and disassembly of the flavoprotein dodecin at flavin-terminated DNA monolayers, as according to previous studies it is assumed that the apododecin-flavin system can be well described by the Langmuir model. As a result, this assumption could be verified. Using three different approaches, KD values were obtained that differ not more than by a factor of 4.

Flavin Storage and Sequestration by Mycobacterium tuberculosis Dodecin.

Dodecins are small flavin binding proteins occurring in archaea and bacteria. They are remarkable for binding dimers of flavins with their functional relevant aromatic isoalloxazine rings deeply covered. Bacterial dodecins are widely spread and found in a large variety of pathogens, among them Pseudomonas aeruginosa, Streptococcus pneumonia, Ralstonia solanacearum, and Mycobacterium tuberculosis ( M. tuberculosis). In this work, we seek to understand the function of dodecins from M. tuberculosis dodecin. We describe flavin binding in thermodynamic and kinetic properties and achieve mechanistic insight in dodecin function by applying spectroscopic and electrochemical methods. Intriguingly, we reveal a significant pH dependence in the affinity and specificity of flavin binding. Our data give insight in M. tuberculosis dodecin function and advance the current understanding of dodecins as flavin storage and sequestering proteins. We suggest that the dodecin in M. tuberculosis may specifically be important for flavin homeostasis during the elaborate lifestyle of this organism, which calls for the evaluation of this protein as drug target.

Pristine DNA Hydrogels from Biotechnologically Derived Plasmid DNA.

DNA hydrogels are of great interest for a variety of biomedical applications owing to their biocompatibility and biodegradability but the advantages of DNA hydrogels have not been exploited yet because of their limited availability. Thus far, DNA hydrogels have been prepared from synthetically derived building blocks, and their production on large scale would be far too expensive. As an alternative, here the generation of DNA hydrogels from plasmid DNA is reported. Plasmid DNA can be prepared on large scale at reasonable costs by a fermentation process. The desired linear DNA building blocks are then obtained from the plasmid DNA by enzymatic digestion. Gel formation is carried out by covalent bond formation between individual building blocks via enzymatic ligation. The generation of pristine DNA hydrogels from plasmid DNA is thus presented for the first time. The viscoelastic properties of the hydrogels were studied by rheology, which confirmed that the gels have storage moduli G' of >100 Pa.

Influence of the Thiol Anchor on the Orientation of Surface-Grafted dsDNA Assemblies.

The orientation of surface-grafted dsDNA assemblies relative to the surface depends strongly on the nature of the employed thiol anchor. This was shown by ssDNA capture probe strands of 20 bases grafted to a gold surface by three dithiane rings or a single mercaptohexyl group. The capture probe strands were hybridized to one end of complementary ssDNA strands (target) comprising 40, 60, or 80 bases (T40 , T60 , and T80 ). At the other end of the targets a fluorophore-labeled reporter probe ssDNA strand of 20 bases was hybridized. To stiffen the DNA assemblies, the targets T60 and T80 were further hybridized to ssDNA patches of 20 or 40 bases. Whether the fluorescence intensity, and thus the distance between surface and fluorophore, increases or decreases with increasing target length depends on the thiol anchor. Attempts were made to heal the nicks that are present in the formed dsDNA assemblies by ligation. For enzymatic ligation, the presence of a phosphate at the 5'-end of the reporter probe and a patch is required, which may also influence the fluorescence intensity.

Thickness Dependence of Bovine Serum Albumin Adsorption on Thin Thermoresponsive Poly(diethylene glycol) Methyl Ether Methacrylate Brushes by Surface Plasmon Resonance Measurements.

This study reports on the dependence of the temperature-induced changes in the properties of thin thermoresponsive poly(diethylene glycol) methyl ether methacrylate (PDEGMA) layers of end-tethered chains on polymer thickness and grafting density. PDEGMA layers with a dry ellipsometric thickness of 5-40 nm were synthesized by surface-initiated atom transfer radical polymerization on gold. To assess the temperature-induced changes, the adsorption of bovine serum albumin (BSA) was investigated systematically as a function of film thickness, temperature, and grafting density by surface plasmon resonance (SPR), complemented by wettability and quartz crystal microbalance with dissipation monitoring (QCM-D) measurements. BSA adsorption on PDEGMA brushes is shown to differ significantly above and below an apparent transition temperature. This surface transition temperature was found to depend linearly on the PDEGMA thickness and changed from 35 °C at 5 nm thickness to 48 °C at 23 nm. Similarly, a change of the grafting density enables the adjustment of this transition temperature presumably via a transition from the mushroom to the brush regime. Finally, BSA that adsorbed irreversibly on polymer brushes at temperatures above the transition temperature can be desorbed by reducing the temperature to 25 °C, underlining the reversibly switchable properties of PDEGMA brushes in response to temperature changes.

A sandwich-like strategy for the label-free detection of oligonucleotides by surface plasmon fluorescence spectroscopy (SPFS).

For the detection of oligonucleotides a sandwich-like detection strategy has been developed by which the background fluorescence is significantly lowered in comparison with surface-bound molecular beacons. Surface bound optical molecular beacons are DNA hairpin structures comprising a stem and a loop. The end of the stem is modified with a fluorophore and a thiol anchor for chemisorption on gold surfaces. In the closed state the fluorophore is in close proximity to the gold surface, and most of the fluorescence is quenched. After hybridization with a target the hairpin opens, the fluorophore and surface become separated, and the fluorescence drastically increases. Using this detection method the sensitivity is limited by the difference in the fluorescence intensity in the closed and open state. As the background fluorescence is mainly caused by non-quenched fluorophores, a strategy to reduce the background fluorescence is to cut the beacon in two halves. First a thiolated ssDNA capture probe strand (first half) is chemisorbed to a gold surface together with relatively short thiol spacers. Next the target is hybridized by one end to the surface-anchored capture probe and by the other to a fluorophore-labeled reporter probe DNA (second half). The signal readout is done by surface plasmon fluorescence spectroscopy (SPFS). Using this detection strategy the background fluorescence can be significantly lowered, and the detection limit is lowered by more than one order of magnitude. The detection of a target takes only a few minutes and the sensor chips can be used for multiple detection steps without a significant decrease in performance.

Critical View on Electrochemical Impedance Spectroscopy Using the Ferri/Ferrocyanide Redox Couple at Gold Electrodes.

Electrochemical or faradaic impedance spectroscopy (EIS) using the ferri/ferrocyanide couple as a redox probe at gold working electrodes was evaluated with respect to its ability to monitor consecutive surface modification steps. As a model reaction, the reversible hybridization and dehybridization of DNA was studied. Thiol-modified single stranded DNA (ssDNA, 20 bases, capture probe) was chemisorbed to a gold electrode and treated with a solution of short thiols to release nonspecifically adsorbed DNA before hybridization with complementary ssDNA (20 bases, target) was carried out. Reversible dehybridization was achieved by intense rinsing with pure water. The experimental procedures were optimized by kinetic surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation (QCM-D) measurements to maximize the increase in reflectivity or decrease in frequency upon hybridization before hybridization/dehybridization was also monitored by EIS. In contrast to SPR and QCM-D, repeatable EIS measurements were not possible at first. Combined SPR/EIS and QCM-D/EIS measurements revealed that during EIS the gold surface is seriously damaged due to the presence of CN(-) ions, which are released from the ferri/ferrocyanide redox probe. Even at optimized experimental conditions, etching the gold electrodes could not be completely suppressed and the repeatability of the EIS measurements was limited. In three out of four experimental runs, only two hybridization/dehybridization steps could be monitored reversibly by EIS. Thereafter etching the gold electrode significantly contributed to the EIS spectra whereas the QCM-D response was still repeatable. Hence great care has to be taken when this technique is used to monitor surface modification at gold electrodes.

Nanomechanical properties of protein-DNA layers with different oligonucleotide tethers.

The multi-ligand binding flavoprotein dodecin is reconstituted on top of flavin-terminated oligonucleotide monolayers. A detailed quartz crystal microbalance with a dissipation monitoring (QCM-D) study showing how the length and flexibility of the oligonucleotide tethers influence the stability and the viscoelastic properties of the resulting DNA-protein layers is presented. Relatively dense protein layers can be obtained, if the length of the tethers is in the same range as the diameter of dodecin. When significantly longer tethers are used, less dense layers are formed. When rather short tethers are used, the reaching area of individual tethers is too low to capture single apododecin molecules cooperatively, and the formation of stable and dense protein layers is not possible. On top of the DNA-dodecin layers additional flavin-DNA ligands may be captured to form sandwich-type DNA-protein-DNA layers. Differences in the binding and unbinding behavior of flavin-dsDNA and flavin-ssDNA ligands are measured by QCM-D and surface plasmon fluorescence spectroscopy (SPFS). Both type of ligands show relatively low kon values, which might be explained by the structural rigidity of the binding pockets allowing a ligand to enter only when it approaches precisely in the right orientation. Apparently apododecin-flavin binding follows Fischer's classic lock-and-key binding model.

Multi-Ligand-Binding Flavoprotein Dodecin as a Key Element for Reversible Surface Modification in Nano-biotechnology.

In this paper the multiple (re)programming of protein-DNA nanostructures comprising generation, deletion, and reprogramming on the same flavin-DNA-modified surface is introduced. This work is based on a systematic study of the binding affinity of the multi-ligand-binding flavoprotein dodecin on flavin-terminated DNA monolayers by surface plasmon resonance and quartz crystal microbalance with dissipation (QCM-D) measurements, surface plasmon fluorescence spectroscopy (SPFS), and dynamic AFM force spectroscopy. Depending on the flavin surface coverage, a single apododecin is captured by one or more surface-immobilized flavins. The corresponding complex binding and unbinding rate constants kon(QCM) = 7.7 × 10(3) M(-1)·s(-1) and koff(QCM) = 4.5 × 10(-3) s(-1) (Kd(QCM) = 580 nM) were determined by QCM and were found to be in agreement with values for koff determined by SPFS and force spectroscopy. Even though a single apododecin-flavin bond is relatively weak, stable dodecin monolayers were formed on flavin-DNA-modified surfaces at high flavin surface coverage due to multivalent interactions between apododecin bearing six binding pockets and the surface-bound flavin-DNA ligands. If bi- or multivalent flavin ligands are adsorbed on dodecin monolayers, stable sandwich-type surface-DNA-flavin-apododecin-flavin ligand arrays are obtained. Nevertheless, the apododecin flavin complex is easily and quantitatively disassembled by flavin reduction. Binding and release of apododecin are reversible processes, which can be carried out alternatingly several times to release one type of ligand by an external redox trigger and subsequently replace it with a different ligand. Hence the versatile concept of reprogrammable functional biointerfaces with the multi-ligand-binding flavoprotein dodecin is demonstrated.

Molecular beacon modified sensor chips for oligonucleotide detection with optical readout.

Three different surface bound molecular beacons (MBs) were investigated using surface plasmon fluorescence spectroscopy (SPFS) as an optical readout technique. While MB1 and MB2, both consisting of 36 bases, differed only in the length of the linker for surface attachment, the significantly longer MB3, consisting of 56 bases, comprised an entirely different sequence. For sensor chip preparation, the MBs were chemisorbed on gold via thiol anchors together with different thiol spacers. The influence of important parameters, such as the length of the MBs, the length of the linker between the MBs and the gold surface, the length and nature of the thiol spacers, and the ratio between the MBs and the thiol spacers was studied. After hybridization with the target, the fluorophore of the longer MB3 was oriented close to the surface, and the shorter MBs were standing more or less upright, leading to a larger increase in fluorescence intensity. Fluorescence microscopy revealed a homogeneous distribution of the MBs on the surface. The sensor chips could be used for simple and fast detection of target molecules with a limit of detection in the larger picomolar range. The response time was between 5 and 20 min. Furthermore, it was possible to distinguish between fully complementary and singly mismatched targets. While rinsing with buffer solution after hybridization with target did not result in any signal decrease, complete dehybridization could be carried out by intense rinsing with pure water. The MB modified sensor chips could be prepared in a repeatable manner and reused many times without significant decrease in performance.

Determination of the pH dependent redox potential of glucose oxidase by spectroelectrochemistry.

The pH dependent redox potential of the oxidoreductase glucose oxidase (GOx) from Aspergillus niger, which is the most frequently applied enzyme in electrochemical glucose biosensors and biofuel cells, was measured between pH 4.5 and 8.5 using UV/vis spectroelectrochemistry. In the entire pH range under investigation, the flavin adenine dinucleotide cofactor of GOx changed directly from the oxidized quinone to the doubly reduced hydroquinone. No stable semiquinoid species could be detected if electrochemical equilibrium was reached. From the pH dependency of the GOx redox potential, a pK(a) of 7.2 has been determined for the GOx flavohydroquinone. At pH values ≤6.0, a dependency of the reduction mechanism and the GOx redox potential on the presence of halides, especially on Cl(-), was observed. For the development of glucose biosensors and glucose biofuel cell anodes working at physiological or neutral pH, the GOx redox potentials at pH 7.4 and pH 7.0 are of main interest. Here values of E(1/2 pH 7.4) = -97 ± 3 mV and E(1/2 pH 7.0) = -80 ± 4 mV have been determined.

Construction of three-dimensional DNA hydrogels from linear building blocks.

A three-dimensional DNA hydrogel was generated by self-assembly of short linear double-stranded DNA (dsDNA) building blocks equipped with sticky ends. The resulting DNA hydrogel is thermoresponsive and the length of the supramolecular dsDNA structures varies with temperature. The average diffusion coefficients of the supramolecular dsDNA structures formed by self-assembly were determined by diffusion-ordered NMR spectroscopy (DOSY NMR) for temperatures higher than 60 °C. Temperature-dependent rheological measurements revealed a gel point of 42±1 °C. Below this temperature, the resulting material behaved as a true gel of high viscosity with values for the storage modulus G' being significantly larger than that for the loss modulus G''. Frequency-dependent rheological measurements at 20 °C revealed a mesh size (ξ) of 15 nm. AFM analysis of the diluted hydrogel in the dry state showed densely packed structures of entangled chains, which are also expected to contain multiple interlocked rings and catenanes.

A reusable sensor for the label-free detection of specific oligonucleotides by surface plasmon fluorescence spectroscopy.

The development of a reusable molecular beacon (MB)-based sensor for the label-free detection of specific oligonucleotides using surface plasmon fluorescence spectroscopy (SPFS) as the readout method is described. The MBs are chemisorbed at planar gold surfaces serving as fluorescence quenching units. Target oligonucleotides of 24 bases can be detected within a few minutes at high single-mismatch discrimination rates.

Strategies for "wiring" redox-active proteins to electrodes and applications in biosensors, biofuel cells, and nanotechnology.

In this tutorial review the basic approaches to establish electrochemical communication between redox-active proteins and electrodes are elucidated and examples for applications in electrochemical biosensors, biofuel cells and nanotechnology are presented. The early stage of protein electrochemistry is described giving a short overview over electron transfer (ET) between electrodes and proteins, followed by a brief introduction into experimental procedures for studying proteins at electrodes and possible applications arising thereof. The article starts with discussing the electrochemistry of cytochrome c, the first redox-active protein, for which direct reversible ET was obtained, under diffusion controlled conditions and after adsorption to electrodes. Next, examples for the electrochemical study of redox enzymes adsorbed on electrodes and modes of immobilization are discussed. Shortly the experimental approach for investigating redox-active proteins adsorbed on electrodes is outlined. Possible applications of redox enzymes in electrochemical biosensors and biofuel cells working by direct ET (DET) and mediated ET (MET) are presented. Furthermore, the reconstitution of redox active proteins at electrodes using molecular wire-like units in order to "wire" the proteins to the electrode surface and possible applications in nanotechnology are discussed.

Synthesis, spectroscopic properties, and electropolymerization of azulene dyads.

Four azulene dyads have been synthesized and studied by spectroscopic and electrochemical methods. A triarylamine, a boron-dipyrromethene (BDP or BODIPY), a porphyrin, and an isoalloxazine moiety have been linked to an extended π electron system at the 2-position of azulene, leading to the dyads 1-4, respectively. For the synthesis of 1-4, first 2-(4-ethynyl-phenyl)azulene (EPA) was prepared, which was further reacted with the halogenated chromophores by Pd-catalyzed cross-coupling reactions. The dyads 1-4 exhibit strong absorption bands in the visible range, which are dominated by the absorption spectra of the individual subchromophores. Fluorometric studies of 2-4 revealed that after excitation of the subchromophoric unit attached to the parent azulene moiety, quenching mainly through energy transfer to azulene is effective, whereas possible charge-transfer interactions play only a minor role. Potentiodynamic oxidation of the dyads 1-4 leads to the formation of polymer films, which are deposited at the electrode. The polymer film derived from 1 was further characterized by spectroelectrochemistry. During positive doping of poly-1, a strong absorption band appears at 13,200 cm(-1), which is typical for triarylamine radical cations. This band is overlapping with a broad absorption band in the low-energy region that might be caused by charge-transfer interactions within the polymer.

A third generation glucose biosensor based on cellobiose dehydrogenase from Corynascus thermophilus and single-walled carbon nanotubes.

A third generation glucose biosensor working under physiological conditions with a linear range of 0.1-30 mM, a detection limit of 0.05 mM, and a sensitivity of 222 nA µM(-1) cm(-2) has been developed by co-adsorption of cellobiose dehydrogenase (CDH) from the ascomycete Corynascus thermophilus (CtCDH) and oxidatively shortened single-walled carbon nanotubes (SWCNTs).

Increasing the coulombic efficiency of glucose biofuel cell anodes by combination of redox enzymes.

A highly efficient anode for glucose biofuel cells has been developed by a combination of pyranose dehydrogenase from Agaricus meleagris (AmPDH) and cellobiose dehydrogenase from Myriococcum thermophilum (MtCDH). These two enzymes differ in how they oxidize glucose. AmPDH oxidizes glucose at the C(2) and C(3) carbon, whereas MtCDH at the C(1) carbon. Both enzymes oxidize efficiently a number of other mono- and disaccharides. They do not react directly with oxygen and produce no H(2)O(2). Electrodes were prepared by embedding (i) only AmPDH (in order to study this enzyme separately) and (ii) a mixture of AmPDH and MtCDH in an Os redox polymer hydrogel. Single-walled carbon nanotubes (SWCNTs) were added in order to enhance the current density. The electrodes were investigated with linear sweep and cyclic voltammetry in the presence of different substrates at physiological conditions. The electrochemical measurements revealed that the product of one enzyme can serve as a substrate for the other. In addition, a kinetic pathway analysis was performed by spectrophotometric measurements leading to the conclusion that up to six electrons can be gained from one glucose molecule through a combination of AmPDH and MtCDH. Hence, the combination of redox enzymes can lead to an enzymatic biofuel cell anode with an increased coulombic efficiency far beyond the usual yields of two electrons per substrate molecule.

Tryptophan repressor-binding proteins from Escherichia coli and Archaeoglobus fulgidus as new catalysts for 1,4-dihydronicotinamide adenine dinucleotide-dependent amperometric biosensors and biofuel cells.

The tryptophan (W) repressor-binding proteins (WrbA) from Escherichia coli (EcWrbA) and Archaeoglobus fulgidus (AfWrbA) were investigated for possible use in 1,4-dihydronicotinamide adenine dinucleotide (NADH) dependent amperometric biosensors and biofuel cells. EcWrbA and AfWrbA are oligomeric flavoproteins binding one flavin mononucleotide (FMN) per monomer and belonging to a new family of NAD(P)H:quinone oxidoreductases (NQOs). The enzymes were covalently linked to a low potential Os redox polymer onto graphite in the presence of single-walled carbon nanotube (SWCNT) preparations of varying average lengths. The performance of the enzyme modified electrodes for NADH oxidation was strongly depending on the average length of the applied SWCNTs. By blending the Os redox polymer with SWCNTs, the electrocatalytic current could be increased up to a factor of 5. Results obtained for AfWrbA modified electrodes were better than those for EcWrbA. For NADH detection, a linear range between 5 microM and 1 mM, a lower limit of detection of 3 microM, and a sensitivity of 56.5 nA microM(-1) cm(-2) could be reached. Additionally spectroelectrochemical measurements were carried out in order to determine the midpoint potentials of the enzymes (-115 mV vs NHE for EcWrbA and -100 mV vs NHE for AfWrbA pH 7.0). Furthermore, an AfWrbA modified electrode was used as an anode in combination with a Pt black cathode as a biofuel cell prototype.