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, <i>K</i>_D values were obtained that differ not more than by a factor of 4

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

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

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 <i>k</i>_on(QCM) = 7.7 × 10³ M^–1·s^–1 and <i>k</i>_off(QCM) = 4.5 × 10^–3 s^–1 (<i>K</i>_d(QCM) = 580 nM) were determined by QCM and were found to be in agreement with values for <i>k</i>_off 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

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 p<i>K</i>_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 <i>E</i>_1/2 pH 7.4 = −97 ± 3 mV and <i>E</i>_1/2 pH 7.0 = −80 ± 4 mV have been determined

Flavin Storage and Sequestration by <i>Mycobacterium tuberculosis</i> 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 <i>Pseudomonas aeruginosa</i>, <i>Streptococcus pneumonia</i>, <i>Ralstonia solanacearum</i>, and <i>Mycobacterium tuberculosis</i> (<i>M. tuberculosis</i>). In this work, we seek to understand the function of dodecins from <i>M. tuberculosis</i> 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 <i>M. tuberculosis</i> dodecin function and advance the current understanding of dodecins as flavin storage and sequestering proteins. We suggest that the dodecin in <i>M. tuberculosis</i> 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

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

Flavin Storage and Sequestration by <i>Mycobacterium tuberculosis</i> 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 <i>Pseudomonas aeruginosa</i>, <i>Streptococcus pneumonia</i>, <i>Ralstonia solanacearum</i>, and <i>Mycobacterium tuberculosis</i> (<i>M. tuberculosis</i>). In this work, we seek to understand the function of dodecins from <i>M. tuberculosis</i> 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 <i>M. tuberculosis</i> dodecin function and advance the current understanding of dodecins as flavin storage and sequestering proteins. We suggest that the dodecin in <i>M. tuberculosis</i> 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