Dorothee Wasserberg

Directed Supramolecular Surface Assembly of SNAP‐tag Fusion Proteins

Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized SNAP-fusion proteins on cyclodextrin-coated surfaces yielded stable monolayers. The binding of the fusion proteins is specific and occurs with an affinity in the order of 10(6) M(-1) as determined by surface plasmon resonance. Reversible micropatterns of the fusion proteins on micropatterned cyclodextrin surfaces were visualized by using fluorescence microscopy. Furthermore, the guest-functionalized proteins could be assembled out of solution specifically onto the surface of cyclodextrin vesicles. The SNAP-tag labeling of proteins thus allows for assembly of modified proteins through a host-guest interaction on different surfaces. This provides a new strategy in fabricating protein patterns on surfaces and takes advantage of the high labeling efficiency of the SNAP-tag with designed supramolecular elements.

Oriented protein immobilization using covalent and noncovalent chemistry on a thiol-reactive self-reporting surface.

We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin.

Patterning of peptide nucleic acids using reactive microcontact printing.

PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.

Dendritic ruthenium(II)-based dyes tuneable for diagnostic or therapeutic applications.

The development of novel highly fluorescent agents is of paramount importance for biological and medical diagnostic applications. In the last decade, a number of nanosized materials, for example, semiconductor quantum dots and silica nanoparticles, have been developed, but the lack of control over the functionalisation stoichiometry, together with potential toxicity effects of such nanoparticles, are restraining their further implementation. Dendrimers are a class of polymers with well-defined hyperbranched structures, which, depending on the type and generation, have a size of 1–10 nm. Control over the functionalisation stoichiometry and stepwise introduction of multiple label, targeting, or therapeutic functionalities make dendrimers an ideal platform for in vitro and in vivo biomedical applications. Ironically, increasing the number of fluorescent moieties on a dendrimer does not necessarily increase their fluorescence intensity, due to propensity for p-stacking and self-quenching typical of most organic dyes. Octahedral six-coordinated transition-metal-based luminophores generally show a large Stokes shift and give negligible stacking, with a reduced self-quenching. Here we present the use of transition-metal complexes that do not suffer from selfquenching and yield highly fluorescent dendrimers. Ruthenium(II) luminophores have been used both for non-biological, for example, solar cells, and biological applications, for example, electro-chemiluminescence, protein labelling, in vivo imaging and photodynamic therapy. A few examples of ruthenium(II)-based luminescent dendriACHTUNGTRENNUNGmers have been reported but their potential as bright labels for biomedicinal applications has not yet been explored. We present here the synthesis, characterisation and photophysical properties of two luminescent dendrimers based on ruthenium(II)-dyes, carrying 32 positive (1) or negative (2) charges (Scheme 1). We report on the uptake in tumour cell lines, the fluorescent imaging characteristics, and the remarkable differences in phototoxicity of 1 and 2, which were synthesised according to Scheme 1 (see also Supporting Information). A second-generation PAMAM (polyamidoamine) dendrimer, with cystamine core and sixteen surface amino groups was decorated with a bipyridine or phenanthroline derivative by means of DCC/HOBt coupling (DCC= N,N’-dicyclohexylcarbodiimide, HOBt=hydroxylbenzotriazole), and further functionalised with [Ru ACHTUNGTRENNUNG(bpy)2Cl2] or [RuACHTUNGTRENNUNG(pheS)2Cl2] (bpy= 2,2’-bipyridine and pheS=4,4’-(1,10-phenanthroline4,7-diyl)dibenzene sulfonate) to obtain a positively or negatively charged dendrimer, respectively. The bpyand phenACHTUNGTRENNUNGantroline-functionalised dendrimers were characterised by NMR spectroscopy and mass spectrometry, proving the complete functionalisation of all 16 end groups of the second-generation PAMAM dendrimer. Also the Ru-decorated dendrimers were characterised by NMR spectroscopy, although for dendrimers this is notoriously difficult, but particularly 1 shows surprisingly high-resolution data, allowing full assignment of peaks through 1D and 2D homoand heteronuclear experiments. Comparison of the integration values of the dendrimer core peak signals with the aromatic and bpy–ethyl peak signals indicate derivatisation of all 16 endgroups with [Ru ACHTUNGTRENNUNG(bpy)3]2+ units. In particular pulse-field gradient spin echo (PFGSE) DOSY experiments proved to be a powerful tool in the [a] A. Ruggi, Prof. Dr. D. N. Reinhoudt, Dr. A. H. Velders Supramolecular Chemistry and Technology MESA + Institute for Nanotechnology University of Twente, PO Box 217 7500 AE, Enschede (The Netherlands) Fax: (+31) 534894645 E-mail : a.h.velders@utwente.nl [b] Dr. C. Beekman, Dr. F. W. B. van Leeuwen Division of Diagnostic Oncology The Netherlands Cancer Institute, Plesmanlaan 121 1066CX, Amsterdam (The Netherlands) [c] Dr. D. Wasserberg, Prof. Dr. V. Subramaniam Nanobiophysics, MESA + Institute for Nanotechnology University of Twente, PO Box 217 7500 AE, Enschede (The Netherlands) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201002514.

Immobilization of Ferrocene-Modified SNAP-Fusion Proteins

The supramolecular assembly of proteins on surfaces has been investigated via the site-selective incorporation of a supramolecular moiety on proteins. To this end, fluorescent proteins have been site-selectively labeled with ferrocenes, as supramolecular guest moieties, via SNAP-tag technology. The assembly of guest-functionalized SNAP-fusion proteins on cyclodextrin- and cucurbit [7]uril-coated surfaces yielded stable monolayers. The binding of all ferrocene fusion proteins is specific as determined by surface plasmon resonance. Micropatterns of the fusion proteins, on patterned cyclodextrin and cucurbituril surfaces, have been visualized using fluorescence microscopy. The SNAP-fusion proteins were also immobilized on cyclodextrin vesicles. The supramolecular SNAP-tag labeling of proteins, thus, allows for the assembly of modified proteins via supramolecular host-guest interaction on different surfaces in a controlled manner. These findings extend the toolbox of fabricating supramolecular protein patterns on surfaces taking advantage of the high labeling efficiency of the SNAP-tag with versatile supramolecular moieties.

Patterning perylenes on surfaces using thiol–ene chemistry

This article describes the patterning of alkene-terminated perylenes on thiolated surfaces. A photochemical thiol–ene click reaction was locally induced (i) in the contact area between the stamp and the substrate, (ii) on surfaces, which were modified with thiolated nanolithographic patterns, immersed in solution and (iii) using a laser scanning confocal microscope over a thiolated substrate immersed in solution. The chemoselective attachment of the alkene-terminated perylenes was demonstrated. All patterning methods yielded homogeneous fluorescent patterns; while, the solution-based methods limit the presence of aggregates of perylenes. The patterns were characterized using atomic force microscopy, fluorescence spectroscopy and fluorescence lifetime measurements revealing monolayers of oriented perylenes in high coverages.

Controlling protein surface orientation by strategic placement of oligo-histidine tags

We report oriented immobilization of proteins using the standard hexahistidine (His6)-Ni2+:NTA (nitrilotriacetic acid) methodology, which we systematically tuned to give control of surface coverage. Fluorescence microscopy and surface plasmon resonance measurements of self-assembled monolayers (SAMs) of red fluorescent proteins (TagRFP) showed that binding strength increased by 1 order of magnitude for each additional His6-tag on the TagRFP proteins. All TagRFP variants with His6-tags located on only one side of the barrel-shaped protein yielded a 1.5 times higher surface coverage compared to variants with His6-tags on opposite sides of the so-called β-barrel. Time-resolved fluorescence anisotropy measurements supported by polarized infrared spectroscopy verified that the orientation (and thus coverage and functionality) of proteins on surfaces can be controlled by strategic placement of a His6-tag on the protein. Molecular dynamics simulations show how the differently tagged proteins reside at the surface in “end-on” and “side-on” orientations with each His6-tag contributing to binding. Also, not every dihistidine subunit in a given His6-tag forms a full coordination bond with the Ni2+:NTA SAMs, which varied with the position of the His6-tag on the protein. At equal valency but different tag positions on the protein, differences in binding were caused by probing for Ni2+:NTA moieties and by additional electrostatic interactions between different fractions of the β-barrel structure and charged NTA moieties. Potential of mean force calculations indicate there is no specific single-protein interaction mode that provides a clear preferential surface orientation, suggesting that the experimentally measured preference for the end-on orientation is a supra-protein, not a single-protein, effect.

Temperature-Switch Cytometry—Releasing Antibody on Demand from Inkjet-Printed Gelatin for On-Chip Immunostaining

Complete integration of all sample preparation steps in a microfluidic device greatly benefits point-of-care diagnostics. In the most simplistic approach, reagents are integrated in a microfluidic chip and dissolved upon filling with a sample fluid by capillary force. This will generally result in at least partial reagent wash-off during sample inflow. However, many applications, such as immunostaining-based cytometry, strongly rely on a homogeneous reagent distribution across the chip. The concept of initially preventing release (during inflow), followed by a triggered instantaneous and complete release on demand (after filling is completed) represents an elegant and simple solution to this problem. Here, we realize this controlled release by embedding antibodies in a gelatin layer integrated in a microfluidic chamber. The gelatin/antibody layer is deposited by inkjet printing. Maturation of this layer during the course of several weeks, due to the ongoing physical cross-linking of gelatin, slows down the antibody release, thereby reducing antibody wash-off during inflow, and consequently helping to meet the requirement for a homogeneous antibody distribution in the filled chamber. After inflow, complete antibody release is obtained by heating the gelatin layer above its sol-gel transition temperature, which causes the rapid dissolution of the entire gelatin/antibody layer at moderate temperatures. We demonstrate uniform and complete on-chip immunostaining of CD4 positive (CD4+) T-lymphocytes in whole blood samples, which is critical for accurate cell counts. The sample preparation is realized entirely on-chip, by applying temperature-switched antibody release from matured gelatin/antibody layers.

Orthogonal supramolecular protein assembly on patterned bifunctional surfaces

We report successful and selective dual protein assembly on patterned bifunctional βCD-Ni(ii)NTA surfaces, using red fluorescent protein variants with hexahistidine-tags and teal fluorescent protein variants conjugated with a peptide containing three adamantyl groups. We show that dual protein patterns can only be assembled, when opposing supramolecular interactions have been optimized and nonspecific interactions have been sufficiently suppressed.

All-printed cell counting chambers with on-chip sample preparation for point-of-care CD4 counting

We demonstrate the fabrication of fully printed microfluidic CD4 counting chips with complete on-chip sample preparation and their applicability as a CD4 counting assay using samples from healthy donors and HIV-infected patients. CD4 counting in low-income and resource-limited point-of-care settings is only practical and affordable, if disposable tests can be fabricated at very low cost and all manual sample preparation is avoided, while operation as well as quantification is fully automated and independent of the skills of the operator. Here, we show the successful use of (inkjet) printing methods both to fabricate microfluidic cell counting chambers with controlled heights, and to deposit hydrogel layers with embedded fluorophore-labeled antibodies for on-chip sample preparation and reagent storage. The maturation process of gelatin after deposition prevents antibody wash-off during blood inflow very well, while temperature-controlled dissolution of the matrix ensures complete antibody release for immunostaining after the inflow has stopped. The prevention of antibody wash-off together with the subsequent complete antibody release guarantees a homogeneous fluorescence background, making rapid and accurate CD4 counting possible. We show the successful application of our fully printed CD4 counting chips on samples from healthy donors as well as from HIV-infected patients and find an excellent agreement between results from our method and from the gold standard, flow cytometry, in both cases.