Two studies in the field of protein adsorption are presented. First, we comprehensively investiga... more Two studies in the field of protein adsorption are presented. First, we comprehensively investigated the nonspecific adsorption mechanism of β-Lactoglobulin on a solid interface by monitoring adsorption kinetics under varying conditions. The experimental observations are translated into mathematical concepts resulting in a complete and accurate mechanistic model which is exploited to calculate long term kinetics and an adsorption isotherm. Second, we propose a new and very simple method to create highly porous functionalized surface coatings with specific protein adsorption characteristics. Such surfaces are considered to have a high potential in biosensor applications.
Journal of Biosensors and Bioelectronics, Jul 3, 2013
S upercritical Angle Fluorescence (SAF) Microscopy has been developed for observing fluorescence ... more S upercritical Angle Fluorescence (SAF) Microscopy has been developed for observing fluorescence signals generated at different surfaces. In particular fluorescently labeled biomolecules can be detected with very high sensitivity (even down to single molecules) and high spatial resolution (in axial direction down to a few nanometers) even in presence of many emitting fluorophors. i.e., real time measurements of binding processes are possible. Hence, even kinetic data are available from simple on-step procedures. Further, SAF can be combined with Förster Resonance Energy Transfer (FRET) in order to obtain spatial information of molecular interactions and with Fluorescence Correlation Spectroscopy (FCS). In this presentation we show several examples of application, e.g. the super resolution microscopy in axial direction, the kinetic study of adsorption of proteins at surfaces, an aggregation study of a-synuclein inducing plaque formation responsible for Parkinson's disease, even the movements of aggregates at the surface and simple and low cost method to detect antibody-antigen reactions, i.e. a simple SAF sensor.
The first high resolution infrared spectrum of the ionic complex N2..H+..N2 and its deuterated de... more The first high resolution infrared spectrum of the ionic complex N2..H+..N2 and its deuterated derivative is reported. The spectra were obtained in direct absorption in a supersonic slit nozzle plasma. The observed rovibrational transitions were assigned to the nu3 antisymmetric NN stretching vibration and the spectrum is consistent with a linear centrosymmetric equilibrium structure. The band origin is found at
$^{a}$D. Verdes, H. Linnartz, J.P. Maier, P. Botschwina, R. Oswald, P. Rosmus, and P.J. Knowles, ... more $^{a}$D. Verdes, H. Linnartz, J.P. Maier, P. Botschwina, R. Oswald, P. Rosmus, and P.J. Knowles, J. Chem. Phys. 111. 8400(1999).
Using fluorescence microscopy combined with the FRET technique we explore the formation mechanism... more Using fluorescence microscopy combined with the FRET technique we explore the formation mechanisms of protein aggregates on surfaces. We present examples for completely different scenarios including the assembly of proteins into lose, 2-dimensional protein patches, the growth of protein aggregates on the surface, and the deposition of protein clusters from the solution onto the surface.
Despite many experimental studies on cooperative effects during protein adsorption events, this p... more Despite many experimental studies on cooperative effects during protein adsorption events, this phenomenon is still poorly characterized and subject of much controversy. In this study, we address the topic of cooperativity using two distinct experimental approaches, namely, kinetic analysis and surface imaging, both based on supercritical angle fluorescence (SAF) microscopy. Several model systems comprising the two proteins BSA and fibrinogen, two different ionic strength conditions and varying pH environments were investigated. The combination of the experimental information obtained from kinetic analysis and from real-time in situ scan images unravel a clear correlation between cooperative adsorption and a heterogeneous protein layer buildup. We propose a mechanistic model of protein adsorption based on an overlap of classical Langmuir-type adsorption on unoccupied surface areas and an additional cooperative adsorption pathway near preadsorbed proteins which is consistent with the experimental observations. Moreover, the growth of two-dimensional surface clusters as an often assumed element of cooperativity could be excluded for the studied systems. The model includes the often observed phenomenon that the adsorption rate decelerates abruptly above a certain coverage limit. Furthermore, the observed evolution of the heterogeneous protein distribution on the surface is in good agreement with the proposed model.
Cooperative effects play a vital role in protein adsorption events on biological interfaces. Desp... more Cooperative effects play a vital role in protein adsorption events on biological interfaces. Despite a number of studies in this field molecular adsorption mechanisms that include cooperativity are still under debate. In this work we use a Monte Carlo-type simulation to explore the microscopic details behind cooperative protein adsorption. The simulation was designed to implement our previously proposed mechanism through which proteins are not necessarily rejected if they approach the surface to an occupied region. Instead, we suggest that proteins can be tracked laterally for a certain distance due to the influence of preadsorbed proteins in order to reach the nearest available binding site. The simulation results were compared with experimental data obtained by using the supercritical angle fluorescence (SAF) microscopy technique. It was found that the tracking distance may be up to 2.5 times the protein's diameter depending on the investigated system. The general validity of this tracking mechanism is supported by a number of linear or upward concave adsorption kinetics reported in the literature which are consistent with our simulation results. Furthermore, the self-organization of proteins adsorbing under cooperative conditions on the surface is shown to necessarily cause density inhomogeneities in the surface distribution of proteins which is also in agreement with experimental observations.
In this study we investigate the behavior of protein clusters on solid surfaces. It is shown that... more In this study we investigate the behavior of protein clusters on solid surfaces. It is shown that clusters consisting of up to several hundreds of protein monomers can form in solution even at low monomer concentrations and subsequently adsorb to the surface. Using FRET ...
Difficulties in preparing cluster ions for spectroscopic studies have limited our understanding o... more Difficulties in preparing cluster ions for spectroscopic studies have limited our understanding of intermolecular forces in charged complexes that are typical of many reactive intermediates. Here, the infrared spectrum of the charge transfer complex [Ar-N2]+, recorded in a supersonic planar plasma with a tunable diode laser spectrometer, is presented. More than 70 adjacent rovibrational transitions were measured near 2272 wave numbers and assigned to the molecular nitrogen stretching fundamental in the 2Sigma+ ground state of [Ar-N2]+. The accurate structural parameters that were determined confirm a linear structure and show that the major part of the charge is located at the argon atom. The latter result is surprising and implies a charge switch of the cationic center upon complexation.
A sensitive and generally applicable technique for high resolution infrared spectroscopy of weakl... more A sensitive and generally applicable technique for high resolution infrared spectroscopy of weakly bound ionic complexes is presented. The method is based on the direct absorption of tunable diode laser radiation in an expansion cooled planar plasma. The plasma is generated by electron impact ionization of gas that is expanded supersonically through a long and narrow slit. This technique allows a fast and effective production modulation. Online monitoring by a quadrupole mass spectrometer yields direct information on the plasma characteristics. Rotational temperatures as low as 15 K and densities of the order of 10 9 -10 10 complexes/cm 3 are routinely obtained. The technique is discussed on the example of N 4 ϩ and demonstrated with results on the proton bound complexes N 2 -H ϩ -N 2 , Ar-HN 2 ϩ , and Ar-HCO ϩ .
We report a noninvasive fluorescence microscopy method and demonstrate nanometer resolution along... more We report a noninvasive fluorescence microscopy method and demonstrate nanometer resolution along the optical axis. The technique is based on the influence of the microscope slide on the angular intensity distribution of fluorescence. Axial positions are determined by measuring the proportion of light emitted below the critical angle of total internal reflection, which behaves in a classical way, and light emitted above the critical angle, which is exponentially dependent on the distance of the fluorophore from the microscope slide.
We explore a new confocal microscope for the detection of surface-generated fluorescence. The ins... more We explore a new confocal microscope for the detection of surface-generated fluorescence. The instrument is designed for high resolution imaging as well as for the readout of large biochips. Special feature is the separated collection of two different fluorescence emission modes. One optical path covers the emission into the glass at low surface angles, the other captures high angles, exceeding the critical angle of the water/glass interface. Due to the collection of the supercritical angle fluorescence (SAF) the confocal detection volume is strictly confined to the interface, whereas the low angles collect much deeper from the aqueous analyte solution. Hence the system can deliver information about surfacebound and unbound fraction of fluorescent analyte simultaneously.
We present a simple and versatile technique of tailoring functionalized surface structures for pr... more We present a simple and versatile technique of tailoring functionalized surface structures for protein enrichment and purification applications based on a superhydrophobic silicone nanofilament coating. Using amino and carboxyl group containing silanes, silicone nanofilament templates were chemically modified to mimic anionic and cationic exchange resins. Investigations on the selectivity of the functionalized surfaces toward adsorption of charged model proteins were carried out by means of fluorescence techniques. Due to a high contact area resulting from the nanoroughness of the coating, excellent protein retention characteristics under various conditions were found. The surfaces were shown to be highly stable and reusable over several retention-elution cycles. Especially the full optical transparency and the possibility to use glass substrates as support material open new opportunities for the development of optical biosensors, open geometry microfluidics, or lab-on-a-chip devices.
A high resolution infrared absorption spectrum of the ionic complex N 2 •••Ar ϩ •••N 2 has been o... more A high resolution infrared absorption spectrum of the ionic complex N 2 •••Ar ϩ •••N 2 has been observed by tunable diode laser spectroscopy through a supersonic planar plasma. Seventy-six rovibrational transitions were measured involving excitation of the antisymmetric NN ( 3 ) stretching fundamental. The band origin is at 2288.7272͑2͒ cm Ϫ1 and the ground state rotational constant is determined as B 0 ϭ0.034 296͑7͒ cm Ϫ1 . The spectrum shows a clear spin-statistical intensity alternation and is consistent with a linear and centrosymmetric equilibrium geometry and a ground state with 2 ⌺ u ϩ symmetry. The charge is predominantly located on the argon atom. The assignment is supported by high level ab initio calculations carried out. The best estimate for the equilibrium geometry is R e ͑NN͒ϭ1.1014 Å and r e ͑Ar•••N͒ϭ2.3602 Å, yielding B e ϭ0.034 297 cm Ϫ1 . The equilibrium dissociation energy D e for fragmentation into N 2 and ͓ArN 2 ] ϩ is calculated to be ϳ1.7 eV.
A combined theoretical and experimental study of the 1 and 2 stretching vibrations of Ar¯HN 2 ϩ i... more A combined theoretical and experimental study of the 1 and 2 stretching vibrations of Ar¯HN 2 ϩ is presented. These correlate asymptotically with the almost local NH and NN stretching vibrations of free HN 2 ϩ , but undergo mode mixing and are strongly influenced by vibrational anharmonicity in the complex. The first observation of the strong 2 band by means of diode laser absorption spectroscopy in a supersonic planar plasma is reported. Its band origin is found at 2041.1802͑3͒ cm Ϫ1 . New results are presented for the 1 band with origin at 2505.5000͑2͒ cm Ϫ1 . The ground-state rotational constant is determined from both band systems as 0.080 868͑6͒ cm Ϫ1 .
ABSTRACT The origin band of the A 2&Pgr;←X 2&Pgr; electronic transition of cyanot... more ABSTRACT The origin band of the A 2&Pgr;←X 2&Pgr; electronic transition of cyanotriacetylene cation has been detected in the gas phase. The ions were generated in a liquid-nitrogen cooled hollow cathode discharge incorporated in a multiple reflection cell. The technique of frequency modulation absorption spectroscopy is used to observe the band at Doppler-limited resolution. Analysis of the rotational structure yields accurate values of the rotational constants in the two electronic states, confirming a linear geometry. Comparison with astronomical measurements of diffuse interstellar bands is made.
Two studies in the field of protein adsorption are presented. First, we comprehensively investiga... more Two studies in the field of protein adsorption are presented. First, we comprehensively investigated the nonspecific adsorption mechanism of β-Lactoglobulin on a solid interface by monitoring adsorption kinetics under varying conditions. The experimental observations are translated into mathematical concepts resulting in a complete and accurate mechanistic model which is exploited to calculate long term kinetics and an adsorption isotherm. Second, we propose a new and very simple method to create highly porous functionalized surface coatings with specific protein adsorption characteristics. Such surfaces are considered to have a high potential in biosensor applications.
Journal of Biosensors and Bioelectronics, Jul 3, 2013
S upercritical Angle Fluorescence (SAF) Microscopy has been developed for observing fluorescence ... more S upercritical Angle Fluorescence (SAF) Microscopy has been developed for observing fluorescence signals generated at different surfaces. In particular fluorescently labeled biomolecules can be detected with very high sensitivity (even down to single molecules) and high spatial resolution (in axial direction down to a few nanometers) even in presence of many emitting fluorophors. i.e., real time measurements of binding processes are possible. Hence, even kinetic data are available from simple on-step procedures. Further, SAF can be combined with Förster Resonance Energy Transfer (FRET) in order to obtain spatial information of molecular interactions and with Fluorescence Correlation Spectroscopy (FCS). In this presentation we show several examples of application, e.g. the super resolution microscopy in axial direction, the kinetic study of adsorption of proteins at surfaces, an aggregation study of a-synuclein inducing plaque formation responsible for Parkinson's disease, even the movements of aggregates at the surface and simple and low cost method to detect antibody-antigen reactions, i.e. a simple SAF sensor.
The first high resolution infrared spectrum of the ionic complex N2..H+..N2 and its deuterated de... more The first high resolution infrared spectrum of the ionic complex N2..H+..N2 and its deuterated derivative is reported. The spectra were obtained in direct absorption in a supersonic slit nozzle plasma. The observed rovibrational transitions were assigned to the nu3 antisymmetric NN stretching vibration and the spectrum is consistent with a linear centrosymmetric equilibrium structure. The band origin is found at
$^{a}$D. Verdes, H. Linnartz, J.P. Maier, P. Botschwina, R. Oswald, P. Rosmus, and P.J. Knowles, ... more $^{a}$D. Verdes, H. Linnartz, J.P. Maier, P. Botschwina, R. Oswald, P. Rosmus, and P.J. Knowles, J. Chem. Phys. 111. 8400(1999).
Using fluorescence microscopy combined with the FRET technique we explore the formation mechanism... more Using fluorescence microscopy combined with the FRET technique we explore the formation mechanisms of protein aggregates on surfaces. We present examples for completely different scenarios including the assembly of proteins into lose, 2-dimensional protein patches, the growth of protein aggregates on the surface, and the deposition of protein clusters from the solution onto the surface.
Despite many experimental studies on cooperative effects during protein adsorption events, this p... more Despite many experimental studies on cooperative effects during protein adsorption events, this phenomenon is still poorly characterized and subject of much controversy. In this study, we address the topic of cooperativity using two distinct experimental approaches, namely, kinetic analysis and surface imaging, both based on supercritical angle fluorescence (SAF) microscopy. Several model systems comprising the two proteins BSA and fibrinogen, two different ionic strength conditions and varying pH environments were investigated. The combination of the experimental information obtained from kinetic analysis and from real-time in situ scan images unravel a clear correlation between cooperative adsorption and a heterogeneous protein layer buildup. We propose a mechanistic model of protein adsorption based on an overlap of classical Langmuir-type adsorption on unoccupied surface areas and an additional cooperative adsorption pathway near preadsorbed proteins which is consistent with the experimental observations. Moreover, the growth of two-dimensional surface clusters as an often assumed element of cooperativity could be excluded for the studied systems. The model includes the often observed phenomenon that the adsorption rate decelerates abruptly above a certain coverage limit. Furthermore, the observed evolution of the heterogeneous protein distribution on the surface is in good agreement with the proposed model.
Cooperative effects play a vital role in protein adsorption events on biological interfaces. Desp... more Cooperative effects play a vital role in protein adsorption events on biological interfaces. Despite a number of studies in this field molecular adsorption mechanisms that include cooperativity are still under debate. In this work we use a Monte Carlo-type simulation to explore the microscopic details behind cooperative protein adsorption. The simulation was designed to implement our previously proposed mechanism through which proteins are not necessarily rejected if they approach the surface to an occupied region. Instead, we suggest that proteins can be tracked laterally for a certain distance due to the influence of preadsorbed proteins in order to reach the nearest available binding site. The simulation results were compared with experimental data obtained by using the supercritical angle fluorescence (SAF) microscopy technique. It was found that the tracking distance may be up to 2.5 times the protein's diameter depending on the investigated system. The general validity of this tracking mechanism is supported by a number of linear or upward concave adsorption kinetics reported in the literature which are consistent with our simulation results. Furthermore, the self-organization of proteins adsorbing under cooperative conditions on the surface is shown to necessarily cause density inhomogeneities in the surface distribution of proteins which is also in agreement with experimental observations.
In this study we investigate the behavior of protein clusters on solid surfaces. It is shown that... more In this study we investigate the behavior of protein clusters on solid surfaces. It is shown that clusters consisting of up to several hundreds of protein monomers can form in solution even at low monomer concentrations and subsequently adsorb to the surface. Using FRET ...
Difficulties in preparing cluster ions for spectroscopic studies have limited our understanding o... more Difficulties in preparing cluster ions for spectroscopic studies have limited our understanding of intermolecular forces in charged complexes that are typical of many reactive intermediates. Here, the infrared spectrum of the charge transfer complex [Ar-N2]+, recorded in a supersonic planar plasma with a tunable diode laser spectrometer, is presented. More than 70 adjacent rovibrational transitions were measured near 2272 wave numbers and assigned to the molecular nitrogen stretching fundamental in the 2Sigma+ ground state of [Ar-N2]+. The accurate structural parameters that were determined confirm a linear structure and show that the major part of the charge is located at the argon atom. The latter result is surprising and implies a charge switch of the cationic center upon complexation.
A sensitive and generally applicable technique for high resolution infrared spectroscopy of weakl... more A sensitive and generally applicable technique for high resolution infrared spectroscopy of weakly bound ionic complexes is presented. The method is based on the direct absorption of tunable diode laser radiation in an expansion cooled planar plasma. The plasma is generated by electron impact ionization of gas that is expanded supersonically through a long and narrow slit. This technique allows a fast and effective production modulation. Online monitoring by a quadrupole mass spectrometer yields direct information on the plasma characteristics. Rotational temperatures as low as 15 K and densities of the order of 10 9 -10 10 complexes/cm 3 are routinely obtained. The technique is discussed on the example of N 4 ϩ and demonstrated with results on the proton bound complexes N 2 -H ϩ -N 2 , Ar-HN 2 ϩ , and Ar-HCO ϩ .
We report a noninvasive fluorescence microscopy method and demonstrate nanometer resolution along... more We report a noninvasive fluorescence microscopy method and demonstrate nanometer resolution along the optical axis. The technique is based on the influence of the microscope slide on the angular intensity distribution of fluorescence. Axial positions are determined by measuring the proportion of light emitted below the critical angle of total internal reflection, which behaves in a classical way, and light emitted above the critical angle, which is exponentially dependent on the distance of the fluorophore from the microscope slide.
We explore a new confocal microscope for the detection of surface-generated fluorescence. The ins... more We explore a new confocal microscope for the detection of surface-generated fluorescence. The instrument is designed for high resolution imaging as well as for the readout of large biochips. Special feature is the separated collection of two different fluorescence emission modes. One optical path covers the emission into the glass at low surface angles, the other captures high angles, exceeding the critical angle of the water/glass interface. Due to the collection of the supercritical angle fluorescence (SAF) the confocal detection volume is strictly confined to the interface, whereas the low angles collect much deeper from the aqueous analyte solution. Hence the system can deliver information about surfacebound and unbound fraction of fluorescent analyte simultaneously.
We present a simple and versatile technique of tailoring functionalized surface structures for pr... more We present a simple and versatile technique of tailoring functionalized surface structures for protein enrichment and purification applications based on a superhydrophobic silicone nanofilament coating. Using amino and carboxyl group containing silanes, silicone nanofilament templates were chemically modified to mimic anionic and cationic exchange resins. Investigations on the selectivity of the functionalized surfaces toward adsorption of charged model proteins were carried out by means of fluorescence techniques. Due to a high contact area resulting from the nanoroughness of the coating, excellent protein retention characteristics under various conditions were found. The surfaces were shown to be highly stable and reusable over several retention-elution cycles. Especially the full optical transparency and the possibility to use glass substrates as support material open new opportunities for the development of optical biosensors, open geometry microfluidics, or lab-on-a-chip devices.
A high resolution infrared absorption spectrum of the ionic complex N 2 •••Ar ϩ •••N 2 has been o... more A high resolution infrared absorption spectrum of the ionic complex N 2 •••Ar ϩ •••N 2 has been observed by tunable diode laser spectroscopy through a supersonic planar plasma. Seventy-six rovibrational transitions were measured involving excitation of the antisymmetric NN ( 3 ) stretching fundamental. The band origin is at 2288.7272͑2͒ cm Ϫ1 and the ground state rotational constant is determined as B 0 ϭ0.034 296͑7͒ cm Ϫ1 . The spectrum shows a clear spin-statistical intensity alternation and is consistent with a linear and centrosymmetric equilibrium geometry and a ground state with 2 ⌺ u ϩ symmetry. The charge is predominantly located on the argon atom. The assignment is supported by high level ab initio calculations carried out. The best estimate for the equilibrium geometry is R e ͑NN͒ϭ1.1014 Å and r e ͑Ar•••N͒ϭ2.3602 Å, yielding B e ϭ0.034 297 cm Ϫ1 . The equilibrium dissociation energy D e for fragmentation into N 2 and ͓ArN 2 ] ϩ is calculated to be ϳ1.7 eV.
A combined theoretical and experimental study of the 1 and 2 stretching vibrations of Ar¯HN 2 ϩ i... more A combined theoretical and experimental study of the 1 and 2 stretching vibrations of Ar¯HN 2 ϩ is presented. These correlate asymptotically with the almost local NH and NN stretching vibrations of free HN 2 ϩ , but undergo mode mixing and are strongly influenced by vibrational anharmonicity in the complex. The first observation of the strong 2 band by means of diode laser absorption spectroscopy in a supersonic planar plasma is reported. Its band origin is found at 2041.1802͑3͒ cm Ϫ1 . New results are presented for the 1 band with origin at 2505.5000͑2͒ cm Ϫ1 . The ground-state rotational constant is determined from both band systems as 0.080 868͑6͒ cm Ϫ1 .
ABSTRACT The origin band of the A 2&Pgr;←X 2&Pgr; electronic transition of cyanot... more ABSTRACT The origin band of the A 2&Pgr;←X 2&Pgr; electronic transition of cyanotriacetylene cation has been detected in the gas phase. The ions were generated in a liquid-nitrogen cooled hollow cathode discharge incorporated in a multiple reflection cell. The technique of frequency modulation absorption spectroscopy is used to observe the band at Doppler-limited resolution. Analysis of the rotational structure yields accurate values of the rotational constants in the two electronic states, confirming a linear geometry. Comparison with astronomical measurements of diffuse interstellar bands is made.
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Papers by Dorinel Verdes