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Biomedical subjects

Fredrik Höök

Publications and source records attributed to Fredrik Höök.

At least 19 recordsLinked to original sources
PubMed2006

Use of PLL-g-PEG in micro-fluidic devices for localizing selective and specific protein binding.

By utilizing flow-controlled PLL-g-PEG and PLL-g-PEGbiotin modification of predefined regions of a poly(dimethylsiloxane) (PDMS) micro-fluidic device, with an intentionally chosen large (approximately 1 cm2) internal surface area, we report rapid (10 min), highly localized (6 x 10(-6) cm2), and specific surface-based protein capture from a sample volume (100 microL) containing a low amount of protein (160 attomol in pure buffer and 400 attomol in serum). The design criteria for this surface modification were achieved using QCM-D (quartz crystal microbalance with energy dissipation monitoring) of serum protein adsorption onto PLL-g-PEG-modified oxidized PDMS. Equally good, or almost as good, results were obtained for oxidized SU-8, Topas, and poly(methyl metacrylate) (PMMA), demonstrating the generic potential of PLL-g-PEG for surface modification in various micro-fluidic applications.

Adsorption↗
PubMed2006

Quantification of oligonucleotide modifications of small unilamellar lipid vesicles.

We present a new method for quantification of the coupling efficiency between amphiphilic oligonucleotides and suspended small unilamellar lipid vesicles (SUVs). The method employs a supported (phospho)lipid bilayer (SLB)-modified sensor template, which upon exposure to a mixture of SUVs and amphiphilic DNA reacts neither with free SUVs nor with DNA-modified SUVs, but with free DNA only. Using calibration curves obtained by recording the concentration dependence of the initial binding rate of free amphiphilic DNA (in the absence of SUVs), it is demonstrated how concentration determinations of both free and bound DNA in the two-component mixture (amphiphilic DNA and lipid vesicles) can be obtained. The calibration curves and the binding analysis were obtained using a quartz crystal microbalance with dissipation (QCM-D) monitoring. The binding efficiency of DNA coupled to SUVs (Ø approximately 50 nm) with two cholesterol moieties revealed that the bivalent coupling is essentially 100% in the range of approximately 1 to approximately 35 oligonucleotides per vesicle, whereas reversible coupling was confirmed in the case of monovalent coupling. Coupling of DNA via two cholesterol moieties was obtained by prehybridization of two single-stranded DNA strands modified with single cholesterol moieties in their 3' and 5' ends, respectively, and the monovalent coupling was obtained using single-stranded DNA. In the latter case, the analysis of the amount of free DNA at different DNA-SUV ratios also allowed for a determination of the maximum number of available binding sites on the SUVs, shown to be in good agreement with data obtained for DNA coupling on planar surfaces. With the only requirement that the SLB-modified sensor template react with one of the components in the two-component mixture only, as verified through fingerprint analysis of frequency, f, and energy dissipation, D, QCM-D measurements, it is emphasized that the method is generic and offers a fast and reliable method for evaluations of biomolecular modifications of any type of colloidal nanoparticles.

Base Sequence↗
PubMed2006

Improving the instrumental resolution of sensors based on localized surface plasmon resonance.

The colorimetric variations induced upon changes in interfacial refractive index of nanoscale noble metal structures exhibiting localized surface plasmon resonance (LSPR) provides a convenient means of label-free, affinity-based detection of biomolecular recognition reactions. However, despite being similar in nature to conventional SPR, LSPR has so far suffered from significantly lower data quality in terms of its signal-to-noise ratio (S/N) in typical biomolecular recognition analysis. In this work, generic data analysis algorithms and a simple experimental setup that provide a S/N upon protein binding that is comparable to that of state-of-the art SPR systems are presented. Specifically, it is demonstrated how temporal variations (rate approximately 0.5 Hz) in parameters proportional to the resonance peak position can be recorded simultaneously, yielding a peak position precision of <5 x 10(-4) nm and an extinction noise level of <5 x 10(-6) absorbance units (Abs). This, in turn, is shown to provide a S/N of approximately 2000 (equivalent to a detection limit of <0.1 ng/cm(2)) for typical protein binding reactions. Furthermore, the importance of utilizing changes in both peak position and magnitude is highlighted by comparing different LSPR active noble metal architectures that respond differently to bulk and interfacial refractive index changes.

Nanostructures↗
PubMed2006

Investigation of binding event perturbations caused by elevated QCM-D oscillation amplitude.

We report measurements with the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, with focus on how the shear oscillation amplitude of the sensor surface influences biorecognition binding events. Technically, this is made as reported recently (M. Edvardsson, M. Rodahl, B. Kasemo, F. Höök, Anal. Chem., 2005, 77(15), 4918-4926) by operating the QCM in dual frequency mode; one harmonic (n = n1) is utilized for continuous excitation of the QCM-D sensor at resonance at variable driving amplitudes (1-10 V), while the second harmonic (n not equaln(1)) is used for combined f and D measurements. By using one harmonic as a "probe" and the other one as an "actuator", elevated amplitudes can be used to perturb - or activate - binding reactions in a controlled way, while simultaneously maintaining the possibility of probing the adsorption and/or desorption events in a non-perturbative manner using combined f and D measurements. In this work we investigate the influence of oscillation amplitude variations on the binding of NeutrAvidin-modified polystyrene beads (slashed circle approximately 200 nm) to a planar biotin-modified lipid bilayer supported on an SiO2-modified QCM-D sensor. These results are further compared with data on an identical system, except that the NeutrAvidin-biotin recognition was replaced by fully complementary DNA hybridization. Supported by micrographs of the binding pattern, the results demonstrate that there exists, for both systems, a unique critical oscillation amplitude, A(c), below which binding is unaffected by the oscillation, and above which binding is efficiently prevented. Associated with A(c), there is a critical crystal radius, r(c), defining the central part of the crystal where binding is prevented. From QCM-D data, A(c) for the present system was estimated to be approximately 6.5 nm, yielding a value of r(c) of approximately 3 mm--the latter number was nicely confirmed by fluorescent- and dark-field micrographs of the crystal. Furthermore, the fact that A(c) is observed to be identical for the two types of biorecognition reactions suggests that it is neither the strength, nor the number of contact points, that determine the amplitude at which binding is prevented. Rather, particle size seems to be the determining parameter.

Journal Article↗
PubMed2005

Gravimetric antigen detection utilizing antibody-modified lipid bilayers.

Lipid bilayers containing 5% nitrilotriacetic acid (NTA) lipids supported on SiO2 have been used as a template for immobilization of oligohistidine-tagged single-chained antibody fragments (scFvs) directed against cholera toxin. It was demonstrated that histidine-tagged scFvs could be equally efficiently coupled to an NTA-Ni2+-containing lipid bilayer from a purified sample as from an expression supernatant, thereby providing a coupling method that eliminates time-consuming protein prepurification steps. Irrespective of whether the coupling was made from the unpurified or purified antibody preparation, the template proved to be efficient for antigen (cholera toxin) detection, verified using quartz crystal microbalance with dissipation monitoring. In addition, via a secondary amplification step using lipid vesicles containing GM1 (the natural membrane receptor for cholera toxin), the detection limit of cholera toxin was less than 750 pM. To further strengthen the coupling of scFvs to the lipid bilayer, scFvs containing two histidine tags, instead of just one tag, were also evaluated. The increased coupling strength provided via the bivalent anchoring significantly reduced scFv displacement in complex solutions containing large amounts of histidine-containing proteins, verified via cholera toxin detection in serum.

Biological Assay↗
PubMed2005

Hydrogels from a water-soluble zwitterionic polythiophene: dynamics under pH change and biomolecular interactions observed using quartz crystal microbalance with dissipation monitoring.

The water-soluble zwitterionic polythiophene, poly(3-((S)-5-amino-5-carboxyl-3-oxapentyl)-2,5-thiophene) hydrochloride (POWT), is a conjugated polyelectrolyte (CPE) with properties well suited for biochip applications. CPEs readily form hydrogels when exposed to water-based buffer solutions or biomolecule solutions. In this work, we used in situ quartz crystal microbalance with dissipation (QCM-D) monitoring to collect information on the interaction between POWT films exposed to buffers with different pH and POWT/DNA chains. Our data show that POWT swells significantly when exposed to low-pH buffers, such as pH 4 acetate, this is seen as an increase in thickness and decrease in viscosity obtained via a Voight-based modeling of combined f and D QCM-D measurements. The magnitude of thickness and viscosity change upon changing from a pH 10 carbonate buffer to pH 4 acetate is 100% increase in thickness and 50% decrease in viscosity. The response of the hydrogel under pH change is well correlated with fluorescence data from POWT films on glass. The state of the hydrogel is important during interaction with biomolecules; illustrated by the observation that a swollen CPE hydrogel adsorbs a higher amount of DNA than a compacted one. In agreement with previous results, the QCM-D data confirmed that the POWT/DNA hydrogel sense complementary DNA specifically and with negligible binding of noncomplementary DNA. These results are important for efficient constructions of biochips in water environments using this class of materials.

Adsorption↗
PubMed2005

A dual-frequency QCM-D setup operating at elevated oscillation amplitudes.

An often raised, but rarely addressed, question with respect to applications of the quartz crystal microbalance technique is whether the shear oscillation of the sensor surface influences the adsorption kinetics or binding events being studied. Motivated by this uncertainty, as well as by the possibility of using elevated amplitudes to influence and steer specific biomolecular interactions, we have further developed the quartz crystal microbalance with dissipation monitoring (QCM-D) technique to operate in dual-frequency mode. One mode (one harmonic) is utilized for continuous excitation of the QCM-D sensor at resonance, at variable driving amplitudes, while the other mode (another harmonic) is used for combined frequency and energy dissipation (damping) measurements. To evaluate this experimental approach, we investigated the following: (i) the well-established process by which intact lipid vesicles adsorb and decompose into a planar supported lipid bilayer on SiO2, recently shown to be very sensitive to external perturbations, and (ii) specific streptavidin binding to biotin-modified surfaces. In the former case, we observed a clear influence of elevated oscillation amplitudes on the bilayer formation kinetics, while in the latter case, no influence was observed for protein monomers. However, binding was inhibited when the biotin-binding protein was coupled to colloidal particles (o.d. approximately 200 nm).

Journal Article↗
PubMed2005

Viscoelastic modeling of template-directed DNA synthesis.

In the present study, we have used the QCM-D technology to study the replication of surface attached oligonucleotide template strands using Escherichia coli DNA polymerase I (Klenow fragment, KF). Changes in resonance frequency (F) and energy dissipation (D) for DNA hybridization and polymerization were recorded at multiple harmonics. Formation of the polymerase/DNA complex led to a significant decrease in energy dissipation, which is consistent with a conformational change induced upon enzyme binding. This interpretation was further strengthened by a data analysis using a Voigt-based viscoelastic model. The analysis revealed a significant increase in shear viscosity and shear modulus during KF binding, whereas the viscoelastic properties of single- and double-stranded templates were almost identical. During the actual DNA synthesis, an initial increase in rigidity (shear viscosity) was followed by a gradual decrease that has two components corresponding to the release of enzyme and to the presence of the catalytically active enzyme/substrate complex. The corresponding decrease in surface concentration was found to underestimate the rate of enzyme release due to viscously coupled water that compensates for the loss in enzyme mass. Furthermore, the modeling elucidates that significant changes in both F and D originate from variations in the viscoelastic properties, which means that changes in F alone should be used with care for estimations of coupled mass and kinetics. Therefore, the modeled temporal variation in effective thickness, being proportional to coupled mass and, thus, independent of structural changes, was used to estimate the catalytic constants of the polymerization reaction. The reported work is the first example providing this type of structural information for the catalytic action of an enzyme, thereby demonstrating the potential of the technique for advanced analysis of complex biological reactions, including proper analysis of enzyme kinetics.

DNA, Bacterial↗
PubMed2005

Localized surface plasmon resonance sensing of lipid-membrane-mediated biorecognition events.

Supported phospholipid bilayers (SPBs) have emerged as important model systems for studies of the natural cell membrane and its components, which are essential for the integrity and function of cells in all living organisms, and also constitute common targets for therapeutic drugs and in disease diagnosis. However, the preferential occurrence of spontaneous SPB formation on silicon-based substrates, but not on bare noble-metal surfaces, has so far excluded the use of the localized surface plasmon resonance (LSPR) sensing principle for studies of lipid-membrane-mediated biorecognition reactions. This is because the LSPR phenomenon is associated with, and strongly confined to, the interfacial region of nanometric noble-metal particles. This problem has been overcome in this study by a self-assembly process utilizing localized rupture of phospholipid vesicles on silicon dioxide in the bottom of nanometric holes in a thin gold film. The hole-induced localization of the LSPR field to the voids of the holes is demonstrated to provide an extension of the LSPR sensing concept to studies of reactions confined exclusively to SPB-patches supported on SiO2. In particular, we emphasize the possibility of performing label-free studies of lipid-membrane-mediated reaction kinetics, including the compatibility of the assay with array-based reading (approximately 7 x 7 microm2) and detection of signals originating from bound protein in the zeptomole regime.

Adsorption↗
PubMed2005

Plasmonic sensing characteristics of single nanometric holes.

The optical response of isolated holes in 20 nm thin gold is probed as a function of alkanethiol CH(3)(CH2)x SH (x epsilon in 1-15) and protein adsorption using dark-field spectroscopy. We establish that the plasmon excitations of single and short-range ordered 60 nm holes exhibit similar E-field decay lengths delta approximately 10-20 nm and that a single hole can be used to resolve the successive adsorption of a protein (biotin-BSA) and its interaction partner (neutravidin). The data confirm the localized character of the hole plasmon and demonstrate that its applicability for bio/chemosensing is similar to that of particle plasmons.

Journal Article↗
PubMed2004

Micropatterning of DNA-tagged vesicles.

We present a novel concept for the creation of lipid vesicle microarrays based on a patterning approach termed Molecular Assembly Patterning by Lift-off (MAPL). A homogeneous MAPL-based single-stranded DNA microarray was converted into a vesicle array by the use of vesicles tagged with complementary DNAs, permitting sequence-specific coupling of vesicles to predefined surface regions through complementary DNA hybridization. In the multistep process utilized to fulfill this achievement, active spots consisting of PLL-g-PEGbiotin with a resistant PLL-g-PEG background, as provided by the MAPL process, was converted into a DNA array by addition of complexes of biotin-terminated DNA and NeutrAvidin. This was then followed by addition of POPC vesicles tagged with complementary cholesterol-terminated DNA, thus providing specific coupling of vesicles to the surface through complementary DNA hybridization. Quartz crystal microbalance with dissipation (QCM-D) and optical waveguide lightmode spectroscopy monitoring were used to optimize the multistep surface modification process. It was found that the amount of adsorbed biotinDNA-NeutrAvidin complexes decreases with increasing molar ratio of biotinDNA to NeutrAvidin and decreasing ionic strength of the buffer solution. Modeling of the QCM-D data showed that the shape of the immobilized vesicles depends on the amount of available anchoring groups between the vesicles and the surface. Fluorescent microscopy images confirmed the possibility to create well-defined patterns of DNA-tagged, fluorescently labeled vesicles in the micrometer range.

Biotin↗
PubMed2004

Simultaneous surface plasmon resonance and quartz crystal microbalance with dissipation monitoring measurements of biomolecular adsorption events involving structural transformations and variations in coupled water.

Simultaneous quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) measurements are used to analyze the surface kinetics of two biomacromolecular systems, one lipid and one protein based, undergoing surface-induced conformational changes. First we establish a theoretical platform, which allows quantitative analysis of the combined SPR and QCM-D data. With this theoretical base, new information can be extracted, not obtainable with either technique alone. As an example we demonstrate how time-resolved measurements with these two techniques in combination--yielding three independent measured quantities--add new information about (i) kinetics, i.e. number of adsorbed molecules per unit area versus time, and (ii) temporal variation in the mass fraction of coupled water versus coverage. In particular, it is demonstrated for the first time, how the kinetics of the process during which adsorbed vesicles are spontaneously transformed into a supported phospholipid bilayer (SPB) on SiO(2) can be quantitatively separated into its two dominating states: adsorbed vesicles and supported planar bilayer patches. In addition, the relevance of dynamically coupled water for interpretation and modeling of the QCM-D response during bilayer formation is discussed and further illustrated with a second model system: streptavidin adsorption on a biotin-modified SPB. A very strong coverage dependence in the number of water molecules per protein sensed by the QCM is demonstrated, with strong implications for the use of QCM as a tool for quantitative determination of protein mass uptake kinetics.

Adsorption↗
PubMed2004

Utilizing adsorbed proteoliposomes trapped in a non-ruptured state on SiO2 for amplified detection of membrane proteins.

The quartz crystal microbalance with dissipation (QCM-D) technique was used to monitor the formation of supported phospholipid bilayers (SPBs) on SiO2 using proteoliposomes with reconstituted proton translocating nicotinamide nucleotide transhydrogenase (TH). Exposure of the surface to such proteoliposomes creates a lipid film composed of a mixture of proteolipid bilayers and adsorbed non-ruptured proteoliposomes, where the fraction of the latter is reduced if the TH-liposomes are pretreated with trypsin to remove the water soluble domains of TH [Langmuir 19 (2003) 842]. In the present work, the latter study is complemented by investigating the influence of trypsin treatment of the mixed adlayer (proteolipid bilayer + non-ruptured proteoliposomes) after adsorption on the surface. This demonstrates how trypsin-cleavage induced rupture of adsorbed TH-liposomes can be utilized to detect the presence of less than 0.04 pmol/cm2 of immobilized TH.

Adsorption↗
PubMed2004

Bivalent cholesterol-based coupling of oligonucletides to lipid membrane assemblies.

By mimicking Nature's way of utilizing multivalent interactions, we introduce in the present work a novel method to improve the strength of cholesterol-based DNA coupling to lipid membranes. The bivalent coupling of DNA was accomplished by hybridization between a 15-mer DNA and a 30-mer DNA, being modified with cholesterol in the 3' and 5' end, respectively. Compared with DNA modified with one cholesterol moiety only, the binding strength to lipid membranes appears to be significantly stronger and even irreversible over the time scale investigated ( approximately 1 hr). First, this means that the bivalent coupling can be used to precisely control the number of DNA per lipid-membrane area. Second, the strong coupling is demonstrated to facilitate DNA-hybridization kinetics studies. Third, exchange of DNA between differently DNA-modified vesicles was demonstrated to be significantly reduced. The latter condition was verified via site-selective and sequence-specific sorting of differently DNA-modified lipid vesicles on a low-density cDNA array. This means of spatially control the location of different types of lipid vesicles is likely to find important applications in relation to the rapid progress currently made in the protein chip technology and the emerging need for efficient ways to develop membrane protein arrays.

Cholesterol↗
PubMed2003

Characterization of DNA immobilization and subsequent hybridization on a 2D arrangement of streptavidin on a biotin-modified lipid bilayer supported on SiO2.

We show how the water content (and effective density) of thin adsorbed films composed of biomolecules can be determined using combined quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) analysis. In particular, these techniques, combined with theoretical treatment using a Voigt-based viscoelastic model, were applied to analyze the state of surface immobilized single stranded biotin-modified probe DNA (b-DNA) coupled via streptavidin to a biotin-doped supported phospholipid bilayer (b-SPB)). From a proper analysis, it is demonstrated how changes in effective thickness, delta(f), and the viscoelastic components (shear viscosity, eta(f), and shear elasticity, mu(f))) can be obtained during both DNA immobilization and hybridization with single stranded fully complementary target DNA. In particular, it is demonstrated how this type of analysis can be used to control the state of streptavidin arrangement for improved measurements of DNA hybridization kinetics. The latter is demonstrated by identifying a surface-coverage dependent viscoelastic behavior of immobilized b-DNA, which is shown to influence the hybridization efficiency.

Biotin↗
PubMed2002

Protein adsorption on supported phospholipid bilayers.

Quartz crystal microbalance with dissipation (QCM-D) measurements were used to investigate the adsorption of human fibrinogen, human serum albumin, bovine hemoglobin, horse heart cytochrome c, human immunoglobulin (hIgG), and 10% fetal bovine serum on supported bilayers of egg-phosphatidylcholine (eggPC) lipids. For comparison the adsorption of fibrinogen and hIgG to eggPC bilayers was also studied with surface plasmon resonance (SPR). The supported bilayers were formed in situ by vesicle adhesion and spontaneous fusion onto a SiO(2) surface. The supported lipid bilayer is highly protein resistant: The irreversible adsorption measured with the QCM-D technique was below the detection level, while reversible protein adsorption was detected for all the proteins in the range 0.3-4% of the saturation coverage on a hydrophobic thiol monolayer on gold. The adsorbed amounts were slightly higher for the SPR measurements. Possible mechanisms for the protein resistance of eggPC bilayers are briefly discussed.

Adsorption↗