MicroSurfaces Inc. develop surface coating technologies for microarray, microfluidic and biosensor applications.

Their proprietary core technology is derived from chemical processes which allow the control and engineering of surface properties to immobilise biomolecules in particular proteins.

MSI provide functional coatings on glass slides, covers slips, silicon wafers and other surfaces.

Their most recent development is a new class of robust PEG surface coatings that can withstand harsh experimental conditions.


Product Categories

What does MicroSurfaces, Inc. do?

MicroSurfaces, Inc (MSI) develops and markets products/services that support universities, government research institutions, pharmaceutical and biotech companies worldwide in their efforts to improve the human condition. Specifically, we supply surface coating technology to the rapidly growing microarray, microfluidics, and biosensor industries. The core of this technology is derived from MSI’s proprietary chemical processes for the control and engineering of surface properties for immobilizing biomolecules, particularly proteins. Proteins are the primary structural, functional and signaling elements in the human body, thus, a comprehensive analysis of proteins is required to obtain a complete picture of normal and disease processes in the body. MSI’s surface coating chemistry allows the immobilization of proteins and other biomolecules with exceptionally low background and excellent conformational control.

MSI sells functional coatings on glass slides, coverslips, silicon wafers, nanoparticles, and other customer surfaces for the immobilization of biomolecules. MSI provides customized coating services to MEMS and BioMEMS industries. MSI’s glass slide is more advantageous over current glass slides on the market. This advantage is reflected in its exceptionally low background, high uniformity, and high chemical reactivity. MSI has combined novel technologies from both surface chemistry and protein chemistry to create a class of high quality functional glass slides and surface modification technologies for microfluidics, biosensors, and other bio-analytical and bio-physical experiments.

MSI is also offering high throughput screening (HTS) services of compound libraries, drug candidates, & nanoparticle-based medicine. We specialize in the HTS of cell surface interactions in proteomics and glycomics analysis using our FluidArray® cell-membrane microarray platform.

Technology

Proteins tend to adsorb nonspecifically to most solid surfaces. This is because a protein molecule has various hydrophobic domains, charged sites, and hydrogen bond donor/acceptor groups and can bind strongly with hydrophobic surfaces, oppositely charged sites, and hydrogen bond acceptor/donor groups. Strong non-specific adsorption is a problem both at the probe immobilization stage and at the probe-target interaction stage: a) excessive interaction between an immobilized protein molecule and a solid surface can result in the disruption of its 3D structure and eventually denaturation; and b) non-specific adsorption of target protein molecules during assay leads to background signal. A successful strategy to circumvent the nonspecific adsorption problem is to start with an inert coating which is intrinsically repulsive to protein adsorption. The coating can be activated for the selective immobilization of probe protein molecules while the surrounding surface remains repulsive.

We have successfully developed a proprietary technology which allows us to coat glass, silicon wafer, quartz, & other substrates with a robust, high density layer of poly-ethyleneglycol (PEG) or Dextran brush. The density of PEG or Dextran functionality on the surface is orders of magnitude higher than those from the conventional silane coupling chemistry. The PEG or Dextran coating is intrinsically inert towards the adsorption of proteins, cells and other biomolecules, thus providing a zero-background starting surface in a variety of biomedical experiments. We have also developed various surface conjugation chemistry to allow user to immobilize a wide variety of biomolecues on the otherwise zero background PEG or Dextran brush. Available funcitonalities include -NHS leaving groups for covalent attachment to -NH2 functional group, biotin for conjugation with the biotin/avidin chemistry, chelated metal ions for binding to poly-histidine tags, alkyne for specific interaction with azide (i.e., Click Chemistry), and maleimide for linking to -SH groups.

These coatings are available on standard microscope slides, coverslips, silicon wafers. We also provide customer coating service for specific customer samples. Our customers have successfully applied these PEG surfaces for a range of applications, including protein sensors, protein microarrays, single molecule spectroscopy, biological atomic force microscopy and other biophysical studies.

 

Copy Right© 2015 Athena Guo. All rights reserved.

Fig. 1. Forming fluidic & air-stable supported lipid bilayers on the FluidicArray surface Fig. 2. Fluorescence recovery after photobleaching (FRAP) for an SLB fromed on the FluidArray surface after air-drying and rehydration

As drug delivery, therapy, and medical imaging are becoming increasingly cell-specific, there is a critical need for high fidelity and high-throughput screening methods for cell surface interactions. Membrane-mimicking surfaces, i.e., supported lipid bilayers (SLBs), formed from traditional methods do not possess sufficient robustness to meet this need. We have developed a groundbreaking technology to form fluidic and air-stable SLBs through tethered and dispersed cholesterol groups incorporated into the bottom leaflet (Fig. 1). The SLB remains fluidic after repeated exposure to air, drying, and rehydration (Fig. 2). Achieving air-stability allows one to easily fabricate SLB microarrays from direct robotic spotting of vesicle solutions. One can reconstitute peripheral as well as integral membrane components into the SLB to fabricate content microarrays in high throughput studies, e.g., the screening of drugs and nanomedicine targeting cell surface receptors. Read more in J. Am. Chem. Soc. 2008, 130, 6267; J. Am. Chem. Soc. 2009, 131, 13646; ACS Chem. Biol. 2014, 9, 1877.

To demonstrate the application of the fluidic and air-stable SLB technology, we show in Fig. 3 the quantitative determination of multivalent binding curves for two E-coli strains on SLB microarrays with varying mannose densities. In this experiment, mannose-linked lipids are mixed into the lipid solution for robotic spotting on our surfaces to form fluidic mannose microarrays with precisely known surface mannose densities. The microarray is incubated with E-coli and the density of adhered E-coli cells on each spot is determined from microscopy. This experiment can yield a complete binding curve in 2-3 hours, as compared to days-weeks in traditional assays. Another application is the inhibition assay, as demontrated in Fig. 4 for the inhibition of E-coli adsorption by mannose-presenting nanoparticles.

Fig. 3. Multivalent binding curves of two E-coli strains on mannose density gradient microarrays (see inset). The upper shows two optical microscope images of E-coli cells adsorbed on SLB spots with 1% and 10% mannose, respectively Fig. 4. Number density of adsorbed E-coli cells on the lipid membrane surface containing 7.5% mannose as a function of solution phase nanoparticle (inhibitor) concentration.

Applications of the FluidArray® technology include a wide range of cell surface interactions, ranging from pathogen detection and characterization to the screening of nanomedicine. We supply FluidArray® series of coated surfaces (glass slides, coverslips, etc.) as well as associated reagents and protocols. We provide customer coating services for specific samples such as membrane based bio-sensors. We also provide high throughput screening (HTS) services for a wide range of cell surface interactions. Contact us with your R&D questions, problems, or ideas.

 

Copy Right © 2015 Athena Guo. All rights reserved.

Application Note:
We have turned silane coating, the “black magic”, into a reproducible and quality controlled silane monolayer on a variety of material surfaces. Our customers have successfully applied the coatings for a broad range of applications, such as DNA combing.

Since its introduction by Sagiv over three decades ago [J. Am. Chem. Soc., 1980, 102, 92-98], the formation of silane monolayers has been developed into a versatile tool for controlling surface chemical and physical properties. The physical and chemical properties of a solid surface are largely determined by those of the outmost layer of atoms or chemical groups. As a result, coating the surface with a monolayer allows us to easily tune the desired properties, such as hydrophobic, hydrophilic, specific reactivity towards a particular functional group, positive or negative charge under physiological conditions, etc. The basic principle for silane monolayer formation is illustrated in Figure 1. The starting molecule for functionalization usually consists of three parts: the reactive silane group (trichloro, trimethoxy, or triethoxy) for attachment to the solid surface, an alkyl spacer, and an end group (X) for the functionality of interest. In the presence of a trace amount of water in the solvent, the silane groups hydrolyze into silanols (Si-OH) and assemble on a hydrophilic surface. Subsequent cross-linking among the silanol groups and attachment to the surface results in a robust monolayer, with the functional group of interest, X, exposed on the top most surface.

The process of forming a silane monolayer simply involves dipping a solid surface in a solution of the silane molecule of interest in a suitable solvent (e.g., toluene) for a fixed time and then rinsing the surface with the solvent. This procedure is deceivingly simple and most users have attempted this procedure in house. However, the success rate of obtaining high quality silane coatings is not particularly high. The problem lies in the exceptional sensitivity of the silane coating chemistry to experimental conditions, such as the solvent, humidity of the ambient air, the concentration of the trace amount of water in the solvent, surface cleaning, and temperature, etc. Competitive reactions can easily lead to polymer and aggregate formation due to cross linking in the solution phase. As a result, the quality of the silane coating varies greatly from lab to lab and from person to person.

For over a decade, we have carefully studied and optimized the fabrication of silane monolayers on a variety of material surfaces. We have turned the “black magic” into a consistent, reproducible, and quality controlled technology. We now make our expertise available to you by offering a broad range of functional silane coating at customer specifications. These coatings are available on standard microscope slides, coverslips, silicon wafers. We also provide customer coating service for specific customer samples. Our customers have successfully applied various silane coatings for a broad range of applications. Talk to our scientists about your needs and we will help you achieve your R&D goals.

Copyright © 2015 Athena Guo. All rights reserved.

Application Note:
we have optimized the fabrication of polylysine coatings on glass, quartz, and other hydrophilic surfaces. Our polylysine coatings are homogeneous, stable, and reproducible, and present a high density of positive charge under physiological conditions.
Polylysine (Fig. 1) is a bio-compatible cationic polymer. It comes in both L or D type, with the former digestible by some enzymes and latter more stable. Under physiological conditions, the amine group on the lysine molecules are protonated. The cationic nature makes it an attractive molecular coating for the adhesion of negatively charged biomolecules, particularly DNAs. It is also ideally suited for the adhesion of cells, since the cell surface is known to be negatively charged. Thus, polylysine coatings are widely used for the adhesion of cells in cell biology experiments, for the fabrication of DNA or cell microarrays, and as biocompatible coatings for biomaterials, as illustrated in Fig. 2. In addition, poly-lysine coatings are also used as anchors for other biocompatible polymers, such is the case of polylysine-polyethyleneglycol (PEG) block copolymers.

The process of forming a polylysine coating in principle very simple. For a common substrate such as glass, the clean surface usually possesses net negative charge. As a result, polylysine can easily adsorbed onto the negatively charged surface. For sufficiently high polylysine molecular weight (>100 kD) and sufficient electrostatic attraction, the adsorbed polylysine layer can be robust for subsequent use in a variety of experiments. However, the seeming simple adsorption process is not easily reproducible. The amount of polylysine adsorption and the stability of the adsorbed layer can be low. As a result, one often obtains a polylysine coating with very low surface coverage (average thickness ~ 1 nm). This introduces uncertainty and irreproducibility for subsequent adsorption of DNAs or cells. In addition, it can lead to substantial wasting of the very expensive polylysine molecules that are washed off during coating and during subsequent exposure to solutions.

Taking advantage of our extensive knowledge and expertise in surface coating chemistry, as well as quantitative surface characterization techniques, e.g., X-ray photoelectron spectroscopy (XPS), ellipsometry, atomic force microscopy (AFM), and attenuated total internal reflection Fourier transform infrared (ATR-FTIR) spectroscopy, we have optimized the fabrication of polylysine coatings on glass, quartz, and other hydrophilic surfaces. Our polylysine coatings are homogeneous, stable, and reproducible, and present a high density of positive charge under physiological conditions. You can have peace of mind when you apply our high-density polylysine coating products in your research and development projects. Talk to our scientists about your needs and we will help you achieve your goals.

Fig. 1. Molecule structure of polylysine.

Fig. 2. Schematic illustration of the adsorption of cell or DNA molecule (with negative charges) on the positively charged polylysine coated glass surface.

 

Copy Right© 2015 Athena Guo. All rights reserved.

Products

Application Note:
A zero background surface is ideal for biophysical experiments, such as single molecule spectroscopy, biological atomic force microscopy, and as starting surfaces for bioconjugation .

It is well known that oligo- or poly(ethyleneglycol) (PEG) is the most “inert” chemical group toward protein adsorption. The inertness or non-fouling property of PEG is attributed to its hydrophilic nature. The PEG backbone is extensively hydrogen-bonded to water molecules, resulting in the formation of a partially structured hydration layer. Adsorption of a protein molecule requires the disruption of this structured water layer and is enthalpically inhibited. Protein adsorption also leads to the compression of the PEG layer towards the solid surface and is entropically unfavorable, Fig. 1. Indeed, PEG and other oligo- or poly-ethers have been successfully used as inert surfaces for the immobilization of protein molecules. Examples include oligoethyleneglycol (OEG) terminated alkanethiol self-assembled monolayers (SAMs) on Au, as demonstrated by Whitesides’ group and others, as well as PEG films anchored to glass or other oxide terminated surface by silane coupling chemistry. The OEG-thiol SAM approach is restricted mainly to noble metal surfaces, particularly gold. The “graft-onto” strategy based on silane chemistry suffers from low PEG density and instability due to hydrolysis. To overcome the above limitations, we has successfully developed a proprietary high density brush of poly-ethyleneglycol (PEG). The density of grafted PEG groups is 1-2 orders of magnitude higher than those from the conventional silane coupling chemistry. Such high PEG density is the reason for the exceptional repulsiveness of the surface towards protein adsorption, as demonstrated in Fig. 2, which compares the adsorption of two protein molecules on the PEG brush surface and on a clean glass surface without PEG coating. The low background PEG surface can be directly used in a variety of experiments and applications demanding repulsiveness towards proteins. It is also the starting point to create functional surfaces for biomolecular immobilization, as detailed in other application notes.

 

Fig, 1. How PEG resists protein adsorption. Protein adsorption on the PEG surface Fig. 2. Fluorescence microscope images of the adsorption of fibrinogen (FBN 1mg/ml) and membrane-type matrix metalloproteinase (MMP5, 1mg/ml) on clean and PEG coated glass surfaces. Detection was achieved by primary antibody and CY3-tagged secondary antibody.

 

Copy Right © 2015 Athena Guo. All rights reserved.

Application Note:
Covalent linking between a maleimide group and a sulfhydryl group is one of the most selective, facile, and convenient reactions in bioconjugation chemistry. We have now made this favorite reaction available on the ZeroBkg® surface.

Development of a solid-phase environment that provides optimum bioactivity without biomolecule loss, displacement, or surface migration is a common goal of research scientists, clinical laboratories, and diagnostic kit manufacturers. One likely approach is the facile and covalent immobilization of protein molecules without the use of any special tag or chemical modification. This can be achieved conveniently via chemical reactive group, maleimide, towards the available sulfhydryl (–SH) groups on the surface of protein molecules. We have developed a maleimide surface based on the zero background PEG coating, as shown in Figure 1. The Maleimide derivatized surface covalently binds sulphydryl groups that become available in biomolecules after the reduction of disulphide bonds or after the chemical modification of primary amines through the introduction of SH groups with specific reagents. The PEG functionality ensures that binding of particular molecules to the surface is only through the specific interaction with the immobilized protein molecule during the assay and the commonly seen background problem is solved.

Fig. 1. The zero background maleimide surface consists of the reactive double bound, tethered to the high-density PEG coating. A protein molecule is attached via surface –SH group(s) but is otherwise repelled from the PEG coating. Excess -SH groups are easily de-activated in a simple washing step. The same immobilization reaction applies to peptides, antibodies, and oligonucleotides. Fig. 2. Fluorescence images of dye-conjugated oligo (-SH) and antibody microarrays fabricated (hand spotting) on maleimide-PEG/glass slides. The diameter of each spot is about 500 um.

To demonstrate the performance, we show in Figure 2 thiol-conjugated oligo microarrays (upper panel) and antibody microarrays (lower panel) fabricated on Mal/PEG/glass slides. Briefly, nanoliter droplets of dye-conjugated oligo (30 mers) or antibody solution containing 10% glycerol are deposited on the glass slide and incubated for 30 minutes. SH-groups in remaining area are removed by a deactivating buffer for 30 minutes at room temperature. The immobilized antibody on the surface is detected by incubation with cy3-conjugated secondary antibody. The glass slides are imaged on a laser scanner. The most important result is the exceptionally low background due to the PEG coating. The Mal/PEG coated glass slides are ideal for biomolecules containing free –SH group (s). Disulphide bonds two cysteine residues in proteins are very stable, but can be reduced with reducing agents (R-SH), such as Dithiothreitol, 2-Mercaptoethanol, etc.. The non-fouling property of the high density PEG coating is critically important for maintaining protein activities at the surface and for minimizing non-specific adsorption of other abundant biomolecules in crude samples, like serum.

These coatings are available on standard microscope slides, coverslips, silicon wafers. We also provide customer coating service for specific customer samples. Our customers have successfully applied the Mal/PEG surfaces for a range of applications, including protein sensors, protein microarrays, single molecule spectroscopy, biological atomic force microscopy and other biophysical studies.

 

Copy Right © 2015 Athena Guo. All rights reserved.

Application Note:
All functionalities are available for gold surfaces used in surface plasmon resonance (SPR) sensing. Either PEG brush or dextran base.

Surface Plasmon Resonance (SPR) is the most popular technique in bio-sensing. The chief advantage of SPR is its capability of quantitative detection in real time, with sensitivity as high as a fraction of a monolayer of small molecules and, most of all, with no need for labeling. This label-free nature has enabled SPR to be used in a wide range of bio- and chemical analysis applications, ranging from establishing the specificity of biomolecular interactions to the measurement of binding/dissociation kinetics and binding affinity. Recent development of SPR imaging in array formats has made this technique a high throughput tool. SPR is now a standard tool in drug discovery/development and in biomedical research. One critical issue in the reliable application of SPR is the need to eliminate or minimize non-specific adsorption. The SPR signal essentially measures changes of mass (more precisely refractive index) near a sensor surface (typically gold metal film). As a result, any non-specific adsorption will contribute to a false-positive signal. There is a clear requirement for a surface which minimizes non-specific interaction, maintains probe activity, and optimizes probe-target interaction. MicroSurfaces’ ZeroBkg® line of functional surfaces is ideal for meeting this requirement. Each coating consists of a high-density poly-ethylene-glycol (PEG) brush to prevent non-specific adsorption, robust bonds to the solid substrate for long term coating stability, specific functional groups for the facile immobilization of protein and other bio or organic molecules of interest, and a hydrophilic surface chemical environment for optimal target-probe interaction. We now offer the complete line of ZeroBkg® functional surfaces to SPR users. To illustrate the performance of the surfaces, we show specific adsorption on our functional SPR surfaces. Fig. 1 shows the immobilization of fibrinogen on the in-situ activated NHS surface (red). Fig. 2 shows the specific adsorption of streptavidin on the biotin-activated surface. Fig. 3 shows the specific immobilization of 6xHis-tagged green fluorescent protein (GFP) on the chelated Cu2+/PEG/Au surface. The blue curves in all three figures are controls showing the absence of nonspecific fibrinogen (Fbn) adsorption.

Fig.1 Fibrinogen binding to ZeroBkg® SPR surface with in situ NHS activation (red). The blue curve shows SPR response on the surface without NHS activation. Fig. 2 Streptavidin binding to biotin-activated ZeroBkg® SPR surface (red). The blue curve shows the resistance of the surface to nonspecific adsorption of fibrinogen. Fig. 3. SPR response (red) showing the specific adsorption of 6xHis-tagged GFP on the chelated Cu2+/PEG/Au surface. The blue curve is control showing the absence of nonspecific adsorption.

In addition to SPR sensors, our customers are also applying the ZeroBkg® surfaces on a variety of other biosensors, microfluidics, biochips, and bioMEMS devices. Tell us what you need. We will find the surface coating technology to meet your demand.

 

Copy Right © 2015 Athena Guo. All rights reserved.

Application Note:
This product comes either in the form of a biotin surface for activation with streptavidin or in the form of pre-adsorbed streptavidin. The PEG environment ensures exceptionally low background. Control streptavidin amount at the surface is important.

Biotin-streptavidin interaction is one of the most widely used in bioconjugation chemistry, owing to the strong affinity and high specificity of the interaction. The binding strength between biotin and streptavidin is about four orders of magnitude stronger than that typical of antigen-antibody interaction and is the strongest among all noncovalent interactions. The presence of four binding sites on each streptavidin molecule makes it possible to link together biotin-tagged molecules or biotin-tagged molecules to a biotin functionalized surface. The exceptionally strong interaction ensures the integrity of the linker under a wide range of experimental conditions. When this interaction is used for the immobilization of a biotin tagged protein molecule to a solid surface, the interaction is through the tag, thus minimizing disturbance to protein structure.

To take advantage of the biotin-streptavidin interaction for protein immobilization, we have developed a surface of high density biotin groups on an otherwise zero background poly-ethyleneglycol (PEG) brush, Figure 1. As a demonstration of the specificity and exceptional low background of this surface, we show in Figure 2 the adsorption of Cy3-labeled streptavidin on the biotin/PEG suface. The surface remains resistant to the adsorption of fibrinogen.

With the adsorption of streptavidin on the biotin/PEG surface, the unoccupied binding sites on the tetravalent streptavidin molecule can be used for the immobilization of biotin-tagged proteins, as illustrated in Figure 3 for the adsorption of biotinlated BSA. The streptavidin surface can be freshly prepared on the biotin/PEG surface to ensure optimal interaction. The surface remains resistant to the adsorption of a “sticky” protein, fibrinogen.

The biotin/streptavdin surface is available on standard microscope slides, cover slips, and silicon wafers. We also provide customer coating service for specific samples and devices. Our customers have successfully applied the biotin/PEG surfaces for a range of applications, including protein sensors, protein microarrays, single molecule spectroscopy, biological atomic force microscopy and other biophysical experiments.

Fig. 1. Left: biotin groups tethered to the high-density PEG coating. Right: the immobilization of a biotinlated protein molecule to the biotin/PEG surface via the intermediate streptavidin. biotin-streptavidin
Fig. 2. Left: the specific adsorption of Cy3-labeledto the biotin-PEG surface. Note that the biotin surface remains resistant to the non-specific adsorption of fibrinogen (middle). Right: an array Cy3-streptavidin. protein immobilization on biotin surface
Fig. 3. Left: adsorption of biotin-conjugated BSA on the streptavidin/biotin/PEG surface. The immobilized BSA is detected by immonostaining; Right: the streptavidin / biotin / PEG surface resists the nonspecific adsorption of a sticky protein, fibrinogen

 

Copy Right © 2015 Athena Guo. All rights reserved.

Application Note:
A protein molecule is linked to the surface only through the poly-His tag with controlled orientation but otherwise stays away from the surface as much as possible. This ensures protein activity of the native state. No sample pre-purification is needed.
Figure 1. The zero background Cu2+ surface consists of chelated Cu2+ ion tethered to the high-density PEG coating. A protein molecule is attached specifically via a poly-His tag.

Recombinant proteins are often produced with poly-histidine (His) tags to facilitate purification. These proteins are now used in the fabrication of protein microarrays. To take advantage of the availability of the poly-His tag, we have developed a chelated Cu2+ surface based on the zero background PEG coating, as shown in Figure 1. The use of this surface essentially eliminates the purification step and incorporates it directly in the protein immobilization process, i.e., one can directly use the crude lysate in microarray fabrication, as illustrated in Figure 2 for green fluorescent protein (GFP). Except for the poly-His-tag on the N- or C-terminus, each immobilized protein molecule stays away from and minimizes its interaction with the surface due to the repulsive nature of the PEG environment. As a result, there is minimal disturbance to the native conformation of the protein. Both the inertness of the chemical surrounding and the controlled orientation contribute to an ideal environment for the immobilized protein molecule to retain its native conformation and activity, as illustrated in Figure 3. For 6xHis tagged sulfotransferase and alkaline phosphatase immobilized with controlled orientation on the Cu2+/PEG surface, their activities are nearly identical to those in the solution phase. For comparison, enzymes immobilized with random orientation on the 3-aminopropyltriethoxysilane (gamma-APS) coated surface show only ~10% of the activities.

Figure 3. A comparison of enzyme activities in the solution phase, with those of random orientation on gamma-APS surface or controlled orientation on the Cu2+/PEG surface. Figure 2. The left shows the intrinsic fluorescence of 6xHis tagged GFP adsorbed on the chelated Cu2+/PEG surface. The surface resists the non-specific adsorption of GFP without His tag (right). Spot diameter ~0.2 mm.

The repulsive 2D chemical environment on the Cu2+/PEG surface is of critical importance not only to the activity of immobilized protein molecules, but also to the maintenance and revival of this activity. The latter is demonstrated in fluorescence microscope images (Figure 4) obtained after repeated cycles of denaturing and refolding of 6xHis-GFP immobilized on three different surfaces: (a) the Cu2+/PEG surface; (b) Cu2+ ions chelated to surface iminodiacetic acid groups on a 3-aminopropyltriethoxysilane (gama-APS) functionalized surface; and (c) the gamma-APS surface for nonspecific protein adsorption. The intrinsic fluorescence intensities are very different on the three surfaces, although immunostaining reveals similar amounts of GFP. The fluorescence intensity on the Cu2+ /gamma-APS or the gamma-APS surface is 70% or < 20% of that on the Cu2+/ PEG surface. While no fluorescence is detected after the denaturing step on all three surfaces, result of refolding is a strong function of the chemical nature of the surface. On the Cu2+/PEG surface, most of the fluorescence intensity is recovered after the refolding step. In contrast, little fluorescence intensity is left on the Cu2+/gamma-APS or gamma-APS surface. On the Cu2+/PEG surface, each immobilized GFP molecule is linked only by the 6xHis tag but otherwise prefers to stay away from the surface due to the repulsive nature of the PEG functionality. This repulsive or non-fouling nature of the surface ensures that the weak protein-surface interaction does not introduce additional barriers on the energy landscape for protein refolding. On the gamma-APS surface, there is attractive and non-specific interaction between GFP and the “sticky” or fouling –NH2 functional groups in the immediate surrounding. Upon denaturing, such non-specific interaction with the sticky environment is expected to increase, thus effectively introduces insurmountable barriers on the energy landscape for protein refolding. The capability of the Cu2+/PEG surface for on-chip refolding of protein molecules opens the door to many potential applications, e.g., direct on-chip generation of protein microarrays, the removal of inclusion body from recombinant proteins, etc. These coatings are available on standard microscope slides, cover slips, & silicon wafers. Our customers have successfully applied the Cu2+/PEG surfaces for a range of applications, including protein sensors, protein microarrays, single molecule spectroscopy, biological atomic force microscopy and other biophysical studies. Coming soon: a three-dimensional (3D) version of the Cu2+/PEG surface is under development. The large surface area afforded by the microporous 3D coating allows high loading of poly-His tagged probe molecules. This is ideal for the detection of very low concentrations of biomarkers.

Figure 4. Fluorescence microscope images of 6xHis-GFP before and after cycles of denaturing (De) and refolding (Re) on three surfaces: (a) the Cu2+/PEG; (b) Cu2/ /gamma-APS; (c) gamma-APS. Denaturing involves immersion of the 6xHis-GFP coated surface in a buffer solution at pH = 3.5, while refolding corresponds to incubating the sample with a buffer solution containing 1xPBS, 20% sucrose and 10% glycerol at pH = 8.1.

READ Publications using our Cu2+ surfaces: Proteomics, 2005, 5, 416;  Proteomics, 2007, 7, 1771;  Nature Method, 2008, 5, 507;  BMC Biotech., 2009, 1. etc..

 

Copy Right © 2015 Athena Guo. All rights reserved.

Application Note:
The advantage of this surface is that it allows for very fast immobilization and the remaining chemical groups are easily washed off to expose the zero background PEG brush. Ideal for ultra-small quantities and crude tissue samples.

In many proteomics applications, one is interested in the facile and covalent immobilization of protein molecules without the use of any special tag or chemical modification. This is most conveniently achieved via chemical reactivity towards the commonly available –NH2 groups on the surface of protein molecules. One of the most efficient leaving groups towards –NH2 is N-hydroxysuccinimide (NHS) attached via an ester bond. We have developed an NHS surface based on the zero background PEG coating, as shown in Figure 1. It allows for fast immobilization reactions with the remaining NHS groups easily washed off to expose the zero background PEG coating. In subsequent assays, the PEG functionality ensures that binding of particular molecules to the surface is only through the specific interaction with the immobilized protein molecule and the commonly seen background problem is solved without the need of a blocking step. To demonstrate the performance, we show in Figure 2 peptide and protein microarrays fabricated on NHS/PEG/glass slides. Briefly, nanoliter droplets of peptide (21 amino-acids) or protein (fibrinogen) solution containing 10% glycerol are deposited on the glass slide with a robotic arrayer and incubated for 10 minutes. NHS-groups in remaining area are removed by a deactivating buffer for 30 minutes at room temperature. The immobilized peptide or protein on the surface is detected by incubation with the primary antibody specifically against the peptide or fibrinogen, followed by wash and incubation with cy3-conjugated secondary antibody. The glass slides are imaged on a laser scanner. The most important result is the exceptionally low background due to the PEG coating.

Fig. 1. ZeroBkg® NHS activated surfaces for the immobilization of proteins, peptides, & antibodies Fig. 2. Fluorescence images of peptide (left) and protein (Fibrinogen, right) microarrays fabricated on NHS/PEG/glass slides and detected by immunostaining. The diameter of each spot is ~100 μm.

While the NHS/PEG coated glass slides are ideal for protein, peptide, and antibody arrays, they are also useful as low background surfaces for other microarrays, such as oligonucleotides, carbohydrates, and other small molecules. The non-fouling property of the high density PEG coating becomes critically important when one uses such an array for the study of complex biological samples, such as plasma or serum. In order to detect molecules of low abundance, such as cancer biomarkers, one needs to minimize non-specific adsorption of other abundant biomolecules.

These coatings are available on standard microscope slides, coverslips, silicon wafers. We also provide customer coating service for specific customer samples. Our customers have successfully applied the NHS/PEG surfaces for a range of applications, including protein sensors, protein microarrays, single molecule spectroscopy, biological atomic force microscopy and other biophysical studies.

 

Copy Right © 2015 Athena Guo. All rights reserved.

Application Note:
The so-called “Click chemistry” is becoming one of the most reliable conjugation strategies in organic and biochemistry. One can easily link azide (-N3) functionalized molecules to the alkyne groups on the ZeroBkg® surface. The reaction is fast.

Since its introduction by Sharpless almost 10 years ago [Angew. Chem. Intern. Ed. 2001, 40, 2004], the so-called “Click Chemistry” has been developed into a versatile tool for the conjugation of a wide variety of organic, polymeric, and biological molecules. This process requires a highly selective and facile conjugation reaction between two small chemical groups under benign reaction conditions, such as the most popular Huisgen cycloaddition reaction between azide and alkyne in aqueous solution catalyzed by Cu(I). This powerful method has also been demonstrated for the conjugation of organic and biomolecules to solid surfaces. In response to popular demand, we have recently extended our ZeroBkg® line of surfaces to include the powerful Click chemistry, Fig. 1. We start with our high density PEG coating and covalently attach alkyne groups at controlled density to the PEG surface. Users of this surface coating product can then easily attach azide-conjugated molecules, such as proteins or glycans, to the PEG brush surface. The hydrophilic PEG brush ensures exceptionally low background and high activity of the immobilized probe molecule.

To demonstrate the performance, we show in Fig. 2 the specific immobilization of an azide-conjugated protein, fibrinogen. As negative control, we show the absence of adsorption for same protein without the azide-tag. Here detection of adsorbed protein was achieved by primary antibody, followed by CY3-labeled secondary antibody. We draw two important conclusions from these results: 1) the conjugation of the azide-tagged protein to the alkyne presenting surface is highly efficient; 2) the PEG coating remains repulsive to protein molecules without the azide tag, thus ensuring high selectivity and low background. No blocking is involved! Combining the Click Chemistry with the low background PEG brush allows us to offer a powerful product. Users of these surfaces can easily immobilize a variety of biomolecules, such as proteins, glycans, and DNA.

Figure 1. The zero background alkyne surface consists of the reactive terminal alkyne groups tethered to the high-density PEG coating. A protein molecule is attached via azide (-N3) group(s) but is otherwise repelled from the PEG coating. The same immobilization reaction applies to peptides, antibodies, glycans, small molecules, and oligonucleotides. Figure 2. Fluorescence images of azide-conjugated fibrinogen (four spots on the left) covalently attached to the alkyne/PEG surface via Click Chemistry. The left part shows four spots from negative control, i.e., the adsorption of fibrinogen without azide-label. The spots of immobilized fibrinogen were obtained from hand spotting (pippeting) on the alkyne/PEG/glass slides. The diameter of each spot was about 1 mm. Detection was achieved by primary antibody followed by CY3-labeled secondary antibody.

These coatings are available on standard microscope slides, coverslips, silicon wafers. We also provide customer coating service for specific customer samples. Our customers have successfully applied the alkyne/PEG surfaces for a range of applications, including protein sensors, protein microarrays, single molecule spectroscopy, biological atomic force microscopy and other biophysical studies.

 

Copy Right © 2015 Athena Guo. All rights reserved.

Application Note:
We have turned silane coating, the “black magic”, into a reproducible and quality controlled silane monolayer on a variety of material surfaces. Our customers have successfully applied the coatings for a broad range of applications, such as DNA combing.

Since its introduction by Sagiv over three decades ago [J. Am. Chem. Soc., 1980, 102, 92-98], the formation of silane monolayers has been developed into a versatile tool for controlling surface chemical and physical properties. The physical and chemical properties of a solid surface are largely determined by those of the outmost layer of atoms or chemical groups. As a result, coating the surface with a monolayer allows us to easily tune the desired properties, such as hydrophobic, hydrophilic, specific reactivity towards a particular functional group, positive or negative charge under physiological conditions, etc. The basic principle for silane monolayer formation is illustrated in Figure 1. The starting molecule for functionalization usually consists of three parts: the reactive silane group (trichloro, trimethoxy, or triethoxy) for attachment to the solid surface, an alkyl spacer, and an end group (X) for the functionality of interest. In the presence of a trace amount of water in the solvent, the silane groups hydrolyze into silanols (Si-OH) and assemble on a hydrophilic surface. Subsequent cross-linking among the silanol groups and attachment to the surface results in a robust monolayer, with the functional group of interest, X, exposed on the top most surface.

The process of forming a silane monolayer simply involves dipping a solid surface in a solution of the silane molecule of interest in a suitable solvent (e.g., toluene) for a fixed time and then rinsing the surface with the solvent. This procedure is deceivingly simple and most users have attempted this procedure in house. However, the success rate of obtaining high quality silane coatings is not particularly high. The problem lies in the exceptional sensitivity of the silane coating chemistry to experimental conditions, such as the solvent, humidity of the ambient air, the concentration of the trace amount of water in the solvent, surface cleaning, and temperature, etc. Competitive reactions can easily lead to polymer and aggregate formation due to cross linking in the solution phase. As a result, the quality of the silane coating varies greatly from lab to lab and from person to person.

For over a decade, we have carefully studied and optimized the fabrication of silane monolayers on a variety of material surfaces. We have turned the “black magic” into a consistent, reproducible, and quality controlled technology. We now make our expertise available to you by offering a broad range of functional silane coating at customer specifications. These coatings are available on standard microscope slides, coverslips, silicon wafers. We also provide customer coating service for specific customer samples. Our customers have successfully applied various silane coatings for a broad range of applications. Talk to our scientists about your needs and we will help you achieve your R&D goals.

Copyright © 2015 Athena Guo. All rights reserved.

Application Note:
we have optimized the fabrication of polylysine coatings on glass, quartz, and other hydrophilic surfaces. Our polylysine coatings are homogeneous, stable, and reproducible, and present a high density of positive charge under physiological conditions.
Polylysine (Fig. 1) is a bio-compatible cationic polymer. It comes in both L or D type, with the former digestible by some enzymes and latter more stable. Under physiological conditions, the amine group on the lysine molecules are protonated. The cationic nature makes it an attractive molecular coating for the adhesion of negatively charged biomolecules, particularly DNAs. It is also ideally suited for the adhesion of cells, since the cell surface is known to be negatively charged. Thus, polylysine coatings are widely used for the adhesion of cells in cell biology experiments, for the fabrication of DNA or cell microarrays, and as biocompatible coatings for biomaterials, as illustrated in Fig. 2. In addition, poly-lysine coatings are also used as anchors for other biocompatible polymers, such is the case of polylysine-polyethyleneglycol (PEG) block copolymers.

The process of forming a polylysine coating in principle very simple. For a common substrate such as glass, the clean surface usually possesses net negative charge. As a result, polylysine can easily adsorbed onto the negatively charged surface. For sufficiently high polylysine molecular weight (>100 kD) and sufficient electrostatic attraction, the adsorbed polylysine layer can be robust for subsequent use in a variety of experiments. However, the seeming simple adsorption process is not easily reproducible. The amount of polylysine adsorption and the stability of the adsorbed layer can be low. As a result, one often obtains a polylysine coating with very low surface coverage (average thickness ~ 1 nm). This introduces uncertainty and irreproducibility for subsequent adsorption of DNAs or cells. In addition, it can lead to substantial wasting of the very expensive polylysine molecules that are washed off during coating and during subsequent exposure to solutions.

Taking advantage of our extensive knowledge and expertise in surface coating chemistry, as well as quantitative surface characterization techniques, e.g., X-ray photoelectron spectroscopy (XPS), ellipsometry, atomic force microscopy (AFM), and attenuated total internal reflection Fourier transform infrared (ATR-FTIR) spectroscopy, we have optimized the fabrication of polylysine coatings on glass, quartz, and other hydrophilic surfaces. Our polylysine coatings are homogeneous, stable, and reproducible, and present a high density of positive charge under physiological conditions. You can have peace of mind when you apply our high-density polylysine coating products in your research and development projects. Talk to our scientists about your needs and we will help you achieve your goals.

Fig. 1. Molecule structure of polylysine.

Fig. 2. Schematic illustration of the adsorption of cell or DNA molecule (with negative charges) on the positively charged polylysine coated glass surface.

 

Copy Right© 2015 Athena Guo. All rights reserved.

Application Note:
The latest ground breaking technology from MicroSurfaces: supported lipid bilayer, which remains fluidic after repeated exposure to air, drying, and rehydration. This allows easy fabrication of a variety of cell membrane mimicking microarrays from simple

 

Fig. 1. Forming fluidic & air-stable supported lipid bilayers on the FluidicArray surface Fig. 2. Fluorescence recovery after photobleaching (FRAP) for an SLB fromed on the FluidArray surface after air-drying and rehydration

As drug delivery, therapy, and medical imaging are becoming increasingly cell-specific, there is a critical need for high fidelity and high-throughput screening methods for cell surface interactions. Membrane-mimicking surfaces, i.e., supported lipid bilayers (SLBs), formed from traditional methods do not possess sufficient robustness to meet this need. We have developed a groundbreaking technology to form fluidic and air-stable SLBs through tethered and dispersed cholesterol groups incorporated into the bottom leaflet (Fig. 1). The SLB remains fluidic after repeated exposure to air, drying, and rehydration (Fig. 2). Achieving air-stability allows one to easily fabricate SLB microarrays from direct robotic spotting of vesicle solutions. One can reconstitute peripheral as well as integral membrane components into the SLB to fabricate content microarrays in high throughput studies, e.g., the screening of drugs and nanomedicine targeting cell surface receptors. Read more in J. Am. Chem. Soc. 2008, 130, 6267; J. Am. Chem. Soc. 2009, 131, 13646; ACS Chem. Biol. 2014, 9, 1877.

To demonstrate the application of the fluidic and air-stable SLB technology, we show in Fig. 3 the quantitative determination of multivalent binding curves for two E-coli strains on SLB microarrays with varying mannose densities. In this experiment, mannose-linked lipids are mixed into the lipid solution for robotic spotting on our surfaces to form fluidic mannose microarrays with precisely known surface mannose densities. The microarray is incubated with E-coli and the density of adhered E-coli cells on each spot is determined from microscopy. This experiment can yield a complete binding curve in 2-3 hours, as compared to days-weeks in traditional assays. Another application is the inhibition assay, as demontrated in Fig. 4 for the inhibition of E-coli adsorption by mannose-presenting nanoparticles.

Fig. 3. Multivalent binding curves of two E-coli strains on mannose density gradient microarrays (see inset). The upper shows two optical microscope images of E-coli cells adsorbed on SLB spots with 1% and 10% mannose, respectively Fig. 4. Number density of adsorbed E-coli cells on the lipid membrane surface containing 7.5% mannose as a function of solution phase nanoparticle (inhibitor) concentration.

Applications of the FluidArray® technology include a wide range of cell surface interactions, ranging from pathogen detection and characterization to the screening of nanomedicine. We supply FluidArray® series of coated surfaces (glass slides, coverslips, etc.) as well as associated reagents and protocols. We provide customer coating services for specific samples such as membrane based bio-sensors. We also provide high throughput screening (HTS) services for a wide range of cell surface interactions. Contact us with your R&D questions, problems, or ideas.

 

Copy Right © 2014 Athena Guo. All rights reserved.


Air-Stable Lipid Bilayers

  Fig. 1. Forming fluidic & air-stable supported lipid bilayers on the FluidicArray surface Fig. 2. Fluorescence recovery after photobleaching (FRAP) Read More…

ZeroBkg® Functional Surfaces

Proteins tend to adsorb nonspecifically to most solid surfaces. This is because a protein molecule has various hydrophobic domains, charged sites, Read More…

Silane surfaces

Application Note: We have turned silane coating, the “black magic”, into a reproducible and quality controlled silane monolayer on a Read More…

Polylysine surfaces

Application Note: We have optimized the fabrication of polylysine coatings on glass, quartz, and other hydrophilic surfaces. Our polylysine coatings Read More…