08.02.2015
Polysilazane in the ThreeBond 6644G transforms to glass based coating that has excellent durability.
ThreeBond 6644G is made from newly-developed polysilazane material that has Si(silicon)-N(nitrogen) chain structure. Polysilazane transfers to glass based layer (Si-O chain structure) reacting with moisture in the air. The glass based TB6644G has longer durability compare to other commercial silicone or silane based products. Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Overview of selected commercial ready-to-use cytokine antibody microarrays for laboratory use only (n.a.
When looking at the antibody microarrays for cytokine detection that are on the market (Table 1), it can be noticed that they all tend to have a similar set-up and geometry. To enable the covalent immobilization of the antibodies on the amine-modified slide, a further modification was necessary. Substrate characterization of aminosilane (A), epoxysilane (B), and NHS ester (C) modified surfaces.
It is remarkable that the resulting spot sizes are smallest on NHS-3D slides (~50 µm) compared to the BS3 activated amine slide and the epoxy slide, which both have about the same diameter of ~150 µm (Figure 4). One possibility to further increase the loading of capture antibodies on the NHS modified hydrogel would be to further reduce the spot size significantly below 50 µm, which was not possible here with the given slide and instrumentation, or to increase the spotting concentration.
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Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. IntroductionBreath analysis is an interesting and motivating field of research; most recently, it has been found that dogs can smell lung cancer with high probability if trained accordingly [1].
All suppliers offer a 3D matrix on a solid support either for covalent immobilization or physisorption of antibodies, possibly due to the option of a denser loading and a longer stability for the antibodies. The commercially available homobifunctional crosslinker Bis[sulfosuccinimidyl]suberate (Figure 2, BS3, Thermo Fisher Scientific, Rockford, IL, USA) was used at a concentration of 50 mM in phosphate buffered saline (PBS) buffer and was incubated on the slide for one hour on a shaker at room temperature. Prior to antibody immobilization the substrates showed following composition as determined by X-ray photoelectron spectroscopy (XPS) and contact angles with water. Optimized Antibody Spot Size and Signal DensityTo obtain a high density TNF? or IL-6 antibody microarray with small, homogeneous, reproducible spots and short spot to spot distance for later sensitive cytokine detection, we visualized the spotting of fluorescently labeled antibodies on different surfaces. From the contact angle measurements of virgin slides, realized with water droplets (Figure 3), one would expect a slightly different result. The second option has been investigated and neglected, as high concentrations of antibodies are spotted, but also washed off afterwards due to an already good saturation of antibodies on each spot. Optimum Opti-Lens Headlight Coating forms a covalent bond with the headlight and cross-links to blanket the lens with a durable, protective film. Optimum Opti-Glass Coating is a resin-based glass coating that forms a durable layer of protection against environmental contaminants, leaving your windshield and exterior glass clean and protected.
Optimum Opti-Glass Coating creates such an immense amount of surface tension on glass surfaces that water will simply bead up and roll off at speeds above 35 mph, rendering your windshield wipers virtually useless at highway speeds! Optimum Opti-Glass Clean & Protect Concentrate can be used as a stand-alone glass sealant or as a booster to Optimum Opti-Glass Coating. It is applied to the aluminium through a spray gun, which applies a positive electrostatic charge to the powder particles. Here we report on the development of an array comprised of different anti-cytokine antibodies on an activated solid support coupled with a fluorescence readout mechanism. Furthermore, lung diseases like asthma and chronic obstructive pulmonary disease (COPD) show similar somatic symptoms: the airways are inflamed but different medication is required. The readout strategies are based on fluorescence or chemiluminescence without a further signal amplification step. The chemical structure of the homobifunctional BS3 linker used in this study for the covalent immobilization of antibody to the amine modified substrate is given.
Afterwards, the slide was washed with water, dried with N2, and used as the other slides (see below).The epoxy and NHS-3D slides did not need further surface modification before spotting as the surfaces form directly a covalent peptide bond with the antibodies.
The three selected surfaces—amine, epoxy, and NHS-3D functionalized hydrogel substrates—were spotted with the Nanoplotter, using a picopipette and the resulting spot patterns were visualized with fluorescence microscopy. The highest contact angle implicating a small spot size is given in case of the amino slide (86°).
Another possibility to increase the antibody loading per spot would be to exchange the fluorescent label to one of a smaller effective size, or even to utilize label-free antibodies.
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As with other surface care protection products manufactured by Optimum Polymer Technologies, Opti-Glass Clean & Protect Concentrate is incredibly easy to use! Through countless hours of research, development and testing Optimum Polymer Technologies, led by Dr.
You can maintain its water sheeting properties by regularly using Opti-Glass Clean & Protect Concentrate. As standard we quote in Dulux Duralloy powder coat colours (which is backed by a Dulux 10 year durability warrenty), but we can also do Colorbond colours, other Dulux powder coat colours (like pearl effects) and of course anodised metal (which is the natural metal look). This charge causes the powder to bond with the electrically grounded surface of the aluminium. Optimization of the array preparation was done in regard of spot homogeneity and spot size. To distinguish between the diseases, classical diagnostic methods like imaging, spirometry, analysis of bronchoalveolar lavage (BAL), and invasive bronchoscopy are currently applied [2,3,4]. In the case of the epoxy and NHS-3D surfaces a peptide bond is formed directly between surface and antibody. The NHS-3D slide was stored at ?20 °C, and was tempered to room temperature about one hour prior to use in order to avoid condensation on the substrate surface.
A scheme of the resulting immobilized antibodies on the surfaces together with the imaged fluorescence spots before and after washing with PBS buffer of the different slides are shown in Figure 4. At the beginning, the amine terminated surface showed low hydrophilicity; it can be assumed that during the conditioning of the surface with BS3 in PBS buffer it becomes more hydrophilic due to the protonation of the amine groups and the interaction with the ionic BS3 linker. The latest innovation from Optimum Polymer Technologies promises to deliver the convenience of a rinseless car wash with the cutting-edge protection of advanced UV inhibitors and high-grade carnauba wax. The result is a headlight-specific coating that ensures your vehicle?s headlights retain optimum clarity throughout the lifespan of the vehicle. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNF?) and Interleukin 6 (IL-6) were chosen as the first targets of interest. The vision for tomorrow, however, will be as in many medical fields the point-of-need diagnosis, a non-invasive breath analysis in order to spare patients invasive examinations, to increase the specificity of analysis, and to reduce costs. Please note that in the scheme the antibody’s orientation is drawn in the oriented form for simplicity; however this does not reflect the real situation, and the antibodies are arbitrarily immobilized. This could result in a more hydrophilic surface and hence bigger spots after antibody spotting.
The point is that the label at this stage is only used for visualization and quantification estimations before detecting the cytokine molecules.
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First, the solid support for covalent antibody immobilization and an adequate fluorescent label were selected. Critical high concentrations of nitrogen monoxide (NO) in breath, a potential marker for asthma, are already detected by chemical gas sensors, but the gaseous fraction of the breath varies strongly depending on personal habits [5,6]. Note that the fluorescence images also indicate the exposure time, showing the need to increase it from 200 ms to 600 ms for the amine and epoxy slide after washing in order to visualize the spot with the fluorescence microscope. On the other side, the epoxy slide and the NHS-3D slide show similar contact angles of 56° and 60°, respectively, but the spot on the NHS-3D slide is much smaller with the same spotting buffer and solution. This is important to consider as density calculations show an error and most probably the pure antibody is packed even more densely on the NHS-3D slide. Regular application of Opti-Glass Clean & Protect Concentrate will keep your vehicle?s exterior glass clean and clear.
Our showroom stocks these powder coat colour charts should you wish to come in, but colour cards and samples can also be requested from Dulux directly. Three differently functionalized glass substrates for spotting were compared: amine and epoxy, both having a two-dimensional structure, and the NHS functionalized hydrogel (NHS-3D). On the other hand, and similar to other body fluids, like blood, urine, and saliva, monitoring of the cytokine pattern in exhaled breath condensate (EBC) can contribute to the identification and diagnosis of diseases [7,8,9,10,11].
The aim of this work is the development of a high-density fluorescent immunoassay with the option to further amplify the signal. The antibody concentration and its influence on the spotting results were investigated and the results are also given in the Supplementary Materials. The theoretical optimum would be a dense layer of capture molecules; however, this is not possible due to steric hindrances and other protein-protein interactions, resulting in variable distances in-between antibodies.
The NHS-hydrogel functionalization of the substrate was best suited to antibody immobilization.
Depending on the kind of disease, the body reacts with a certain distinct immune response, and thus different cytokines are present in specific amounts and different cytokine patterns are characteristic, as described for COPD and asthma in Barnes et al.
To make cytokine detection in potential low concentrations, such as later in exhaled breath condensate, possible, we chose to fabricate a planar 2D or 3D support, non-bead based, antibody microarray. Additionally, it has also been reported that a highly dense antibody layer does not automatically mean that more analyte can be detected, again due to steric hindrances and interactions [14]. Then, the optimization of plotting parameters and geometry as well as buffer media were investigated, considering the ambient analyte theory of Roger Ekins. As interesting targets and starting point for experiments we selected Tumor Necrosis Factor alpha (TNF?, MW = 17 kDa) and Interleucin-6 (IL-6, MW = 20 kDa) as they are, among other cytokines, involved in asthma and COPD development [8]. Finally, for a highly sensitive cytokine array the first step is the selection of an appropriate antibody-antigen pair with specific and high association constant K.
As a first step towards real sample studies, a proof of principle of cytokine detection has been established. Thus, the detection mechanism of the antibody microarray has to be very sensitive in case a pre-concentration of the sample needs to be avoided. To improve the sensitivity of the antibody microarray, small spot sizes were targeted as already postulated by Roger Ekins [16,17]. It is possible that the epoxy surface is more glycerol-philic compared to the NHS-3D slide with unknown polymer composition and the spotting buffer on the NHS modified slide results in a higher contact angle than with water.
This required high sensitivity of antibody microarrays is a general problem in non-invasive diagnostics, and a solution would be of advantage for many different applications.
Ekins’ ambient analyte theory concludes that the reduction of spot sizes improves signal-to-noise ratios as signal density and not the total sum of signal is relevant to define the lower LoD. The incubation time of one hour after antibody spotting and before washing was found to be enough for all three different surfaces. Several groups are working on this challenge applying different strategies to improve the limit of detection (LoD) of antibody microarrays.
To print smallest possible spots of antibodies on a solid support, automated nanoplotting can be used. This could be observed by varying the incubation time from 1–8 hours on all solid surfaces.
All spots on one kind of slide time-independently showed the same intensities after washing, meaning the same grey values, regardless of whether they were incubated for one or 8 hours.


As illustrated and calculated in Figure 5, the estimated remaining antibody density after washing within one spot differs strongly in spot size at a constant antibody concentration spotting. The first results will be shown to demonstrate the proof of the principle and to draw a first conclusion.
Assuming an antibody size of 150 kDa, about 6 ? 106 TNF? antibody molecules would be deposited within one spot before washing.
While DNA microarrays are already used in several fields of research and diagnostics, protein microarrays are not yet well established in diagnostics.
On the NHS-3D slide the spots are smallest and hence show a more intense fluorescent signal compared to the epoxy and amine slide, as the 6 ? 106 available antibodies are more densely packed.
To achieve this detection in the small spots with the calculated low analyte (antigen) concentration, the diffusion coefficient plays an important role to transport the analyte from the sample solution to the spot. One reason is that in contrast to DNA, it is not trivial to maintain the antibody’s structure and function when it is immobilized on a solid surface [14]. However, different functionalized surfaces have been found to be suitable for protein immobilization. For the densest monolayer, there is theoretically space for ~4 ? 106 labeled antibodies in the case of 50 µm spots as we have achieved them on the NHS-3D hydrogel surface and ~33 ? 106 spaces in case of 150 µm spots of the amino and epoxy functionalized supports. Admittedly, the label size plays an important role and the results will be different if no label is used for later cytokine detection. However, spotting smaller spots will result in an excess of capture antibodies for the potential immobilization of also the third dimension on the solid support, and thus a more effective binding is possible. On all three surfaces the spots lost intensity after the PBS washing procedure because non-immobilized labeled antibodies were washed away. In a biochemical laboratory, however, protein microarrays for diagnostic purposes are still not well accepted in the medical field, and the Enzyme Linked Immunosorbent Assay (ELISA) is still the golden standard also for the measurement of cytokine concentrations. Residues of antibodies from the washing process in form of non-specific binding were not observed, leading to the conclusion of an accordingly slow kinetic for the immobilization process under given parameters [19].The ~150 µm spot on the amine surface lost its fluorescence intensity after the washing process and could only be observed with the fluorescent microscope by increasing the exposure time from 200 ms to 600 ms.
Nevertheless, only a few types of antibody and cytokine microarrays are available on the market for research purposes and only few are, for example, used in the field of biomarker discovery and validation [15]. Antigen DetectionWhen cytokines were labeled for detection, the LYNX Rapid RPE Antibody Conjugation Kit (AbD Serotec, Puchheim, Germany) was used.
Therefore, the cytokine was mixed according to the manual of the supplier with a modifier reagent and incubated in the dark and at room temperature overnight with the lyophilized LYNX RPE dye. It can be assumed that analyte detection in this case would lead to even weaker signals, due to a low signal to noise ratio. After incubation, a quencher reagent was added and the solution was ready for use on the microarray. The NHS groups of the BS3 linker, for example, leave, and the linker forms a stable peptide bond with the amine groups of the biomolecule and surface. In case of detection by a sandwich assay, the PE-labeled antibody from eBioscience was pre-mixed in a vial with the cytokine and shaken in the dark and at room temperature.
The advantage of this relatively easy way of preparation of amine functional groups on glass or glass-like surfaces by silanization is canceled out by the disadvantage of an intermediate step with a crosslinker. They evaluated different surfaces for antibody microarrays and found amine modified surfaces well suited regarding spot size, morphology, signal background, sensitivity and reproducibility, also in comparison with other different epoxy modified surfaces and NHS modified hydrogels [20]. The cytokine solution was left to react for at least 2 hours up to overnight in a humid, dark chamber at room temperature. Afterwards the slides were washed three times with PBS buffer, once with water, and dried with nitrogen. For signal detection, here also the exposure time had to be increased from 200 ms to 600 ms. Epoxy groups can be attached to glass or glass-like surfaces by silanization similar to amine groups. The advantage of a time efficient direct antibody binding via an amine group of the biomolecule on the epoxy surface did not lead to better results in signal intensity.In contrast to the amine and epoxy surface, the NHS functionalized hydrogel on a glass slide offers a 3D geometry of polymer with NHS ester groups and reacts directly with the amine group of an antibody. The main advantage of the 3D hydrogel is that it is claimed to be beneficial for protein stability, as the antibody can keep its native form and hence its activity, making it an interesting surface for antibody microarrays [14].
Additionally, the 3D setup theoretically offers more active binding sites per area, meaning a higher analyte binding capacity per spot of the same size and an increased signal. This means the antibody loading is maximized for smaller spots even after washing, the final microarray can be spotted more densely, and more tests per slide can be realized. Therefore we decided to fabricate our high-density protein arrays on NHS-modified hydrogel slides.
As it is still unclear how many unbound antibodies are washed away during the PBS washing procedure, the binding efficiency of immobilized antibody was estimated to be about 12%. Substrate CharacterizationPrior to the antibody spotting, the three different substrates amine, epoxy, and NHS-3D were characterized chemically by X-ray photoelectron spectroscopy (XPS) and physically by contact angle measurements using water in order to get a tendency for spotting behavior.
The aminosilane-functionalized slide showed the expected nitrogen due to the presence of amine groups and a high content of carbon, which was hard to judge as the structure of the aminosilane was unknown; however, suggesting the presence of alkyl chains longer than C3.
A quantitative evaluation of the spotting results is given below and visualized in Figure 5.
The theoretically possible antibody loading in a 50 µm spot, considering the densest monolayer, was calculated to be 4 ? 106 PE-labeled anti-TNF? molecules. Additionally, the crosslinker’s length might also influence the antibodies’ binding activity under consideration of steric hindrances and mobility.
From the XPS data it can be concluded that the alkyl chains on amine-modified slides are longer than on the epoxy-modified slides, as shown in the C1s Spectra in Figure 3.
Additionally, the epoxy slide shows the expected high content of oxygen (O1s) in the substrate due to a thinner coating and a signal, which can be attributed to the epoxy group (green in Figure 3). Taking the PE dye of 240 kDa into consideration, ~47% of a theoretical dense antibody-PE monolayer is achieved.
The exact composition of the NHS-3D modified hydrogel polymer is not known, as no information from the supplier is available. Please note that the term “monolayer” in case of a 3D surface is not appropriate but is taken for comparison and an easy imagination. The surface analysis by XPS showed a similar content in nitrogen compared to the amine slide, assuming a high modification of polymer with NHS groups. The analysis of carbon and oxygen does not give any further conclusion regarding the unknown polymer. Contact angle measurements with water indicate that the amine slide was more hydrophobic compared to epoxy and NHS-3D slides, resulting in a higher contact angle (86°) and hence showing the highest surface energy. Epoxy and NHS modified slides did not differ much in contact angle, showing 56° and 60°, respectively.



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