Composite surface for blocking bacterial adsorption on protein biochips

The design and fabrication of protein biochips requires characterization of blocking agents that minimize nonspecific binding of proteins or organisms. Nonspecific adsorption of Escherichia coli, Listeria innocua, and Listeria monocytogenes is prevented by bovine serum albumin (BSA) or biotinylated BSA adsorbed on SiO(2) surfaces of a biochip that had been modified with a C(18) coating. Biotinylated BSA forms a protein-based surface that in turn binds streptavidin. Because streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins, including antibodies. Hence, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfacing protein, avidin or streptavidin, which in turns anchors biotinylated antibody. In our case, the antibody is C11E9, an IgG-type antibody that binds Listeria spp. Nonspecific adsorption of another bacterium, Escherichia coli, is also minimized due to the blocking action of the BSA. The blocking characteristics of BSA adsorbed on C(18)-derivatized SiO(2) surfaces for construction of a protein biochip for electronic detection of pathogenic organisms is investigated.     

Temperature control of biotin binding and release with A streptavidin-poly(N-isopropylacrylamide) site-specific conjugate.

The many laboratory and diagnostic applications utilizing streptavidin as a molecular adaptor rely on its high affinity and essentially irreversible interaction with biotin. However, there are many situations where recovery of the biotinylated molecules is desirable. We have previously shown that poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, can reversibly block biotin association as the polymer's conformation changes at its lower critical solution temperature (LCST). Here, we have constructed a streptavidin-PNIPAAm conjugate which is able to bind biotin at room temperature or lower and release bound biotin at 37 degrees C. The conjugate can repeatedly bind and release biotin as temperature is cycled through the LCST. A genetically engineered streptavidin mutant, E116C, which has only one cysteine residue, was conjugated site specifically via the sulfhydryl groups with a PNIPAAm that has pendent sulfhydryl-reactive vinyl sulfone groups. The conjugation site is near the tryptophan 120 residue, which forms a van der Waals contact with biotin that is important in generating the large binding free energy. The temperature-induced conformational change of the polymer at position 116 may lead to structural changes in the region of tryptophan 120 that are responsible for the reversible binding between biotin and the conjugated streptavidin.     

Quantification of streptavidin adsorption in microtitration wells

Streptavidin-coated microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm(2) (370 ng) and consequently the theoretical adsorption capacity of a C12-format microtitration well (200 microl liquid, coated area 1.54 cm(2)) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350-380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.     

Enzymatic surface erosion of poly(trimethylene carbonate) films studied by atomic force microscopy

In this article, the surface erosion of spin-coated poly(trimethylene carbonate) (PTMC) films by lipase solutions from Thermomyces lanuginosus was studied using atomic force microscopy (AFM). PTMC films (23-48 nm thick) were stable in water at 37 degrees C for 16 h, while after immersion in lipase solutions at 37 degrees C for 30 s and 1 min, the average thickness of the film decreased in time at a rate of 11.0 +/- 3.7 nm/min. The initially smooth films became significantly rougher during the erosion process. When the immersion time of the films in the lipase solutions was limited to less than 5 s, degradation of the surface was minimal and individual lipase molecules adsorbed on PTMC films could be discerned. By microcontact printing of the PTMC surfaces using a patterned PDMS stamp and lipase solution for 30 s, a predefined micropattern consisting of parallel, 5-microm-wide lines lying 5-nm deep and separated at a distance of 2 microm was formed. Friction images showed differences in surface properties between the recessed and protruding lines in the pattern.     

Streptavidin 2D Crystal Substrates for Visualizing Biomolecular Processes by Atomic Force Microscopy

Flat substrate surfaces are a key to successful imaging of biological macromolecules by atomic force microscopy (AFM). Although usable substrate surfaces have been prepared for still imaging of immobilized molecules, surfaces that are more suitable have recently been required for dynamic imaging to accompany the progress of the scan speed of AFM. In fact, the state-of-the-art high-speed AFM has achieved temporal resolution of 30 ms, a capacity allowing us to trace molecular processes played by biological macromolecules. Here, we characterize three types of streptavidin two-dimensional crystals as substrates, concerning their qualities of surface roughness, uniformity, stability, and resistance to nonspecific protein adsorption. These crystal surfaces are commonly resistant to nonspecific protein adsorption, but exhibit differences in other properties to some extent. These differences must be taken into consideration, but these crystal surfaces are still useful for dynamic AFM imaging, as demonstrated by observation of calcium-induced changes in calmodulin, GroES binding to GroEL, and actin polymerization on the surfaces.     

A thermoresponsive chitosan-NIPAAm/vinyl laurate copolymer vector for gene transfection

A carboxyl-terminated N-isopropylacrylamide/vinyl laurate (VL) copolymer was prepared and coupled with chitosan (molecular weight = 2000) to produce a chitosan-NIPAAm/VL copolymer (PNVLCS) vector. The aqueous solution of PNVLCS displayed an obvious thermoresponsive behavior with a lower critical solution temperature (LCST) about 26 degrees C. The transmission electron microscopy (TEM) showed that the size of PNVLCS/DNA complexes varied with charge ratios (+/-), and the smaller nanoparticles were formed at higher charge ratios. DLS revealed that the size of complex particles was dependent on temperature. The results of temperature-variable circular dichroism (CD), UV, and electrophoresis retardation indicated that at lower charge ratios, DNA in the complexes assume a B conformation, whereas increasing charge ratios caused B --> C type conformation transformation; the dissociation-formation of PNVLCS/DNA complexes could be tuned by varying temperature: at 37 degrees C, the collapse of PNIPAAm in PNVLCS was favorable for the formation of compact complexes, shielding more DNA from exposure; at 20 degrees C, the hydrated and extended PNIPAAm chains facilitated the unpacking of DNA from PNVLCS, increasing the exposure of DNA. PNVLCS was used to transfer plasmid-encoding beta-galactosidase into C2C12 cells. The level of gene expression could be controlled by varying incubation temperature. The transfection efficiency of PNVLCS was well improved by temporarily reducing culture temperature to 20 degrees C, whereas naked DNA and Lipofectamine 2000 did not demonstrate the characteristics of thermoresponsive gene transfection.     

Argon-plasma-induced ultrathin thermal grafting of thermoresponsive pNIPAm coating for contractile patterned human SMC sheet engineering

A new method for ultrathin grafting of pNIPAm on PDMS surfaces is introduced that employs plasma activation of the surface followed by thermal polymerization. This method is optimized for human primary SMC attachment and subsequent intact cell sheet detachment by lowering the temperature. The contractile gene expression of the cells showed that the contractile phenotype of the SMCs which is induced by aligning the cells through micropatterning is more preserved after thermoresponsive cell sheet detachment in contrast with enzymatic detachment. Given its simplicity and low cost, this thermoresponsive grafting method can be utilized for engineering patterned cell sheets for future bottom-up tissue engineering techniques.     

The synthesis, turnover, and artificial restoration of a major cell surface glycoprotein.

A large glycoprotein (molecular weight equals 220,000 daltons) can be isolated from the surfaces of chick embryo fibroblasts. Such a protein had been shown by others to be present on normal cells in vitro, but missing from transformed variants of these cells, suggesting a possible role in growth control. We have estimated the turnover rate of this cell surface protein (CSP) and show that it is not more rapid than that of other cell proteins. We have also examined the resynthesis of CSP after removal from cells by trypsinization. This restoration is gradual, requiring more than 24 hr, and is affected by cell density. Restoration can be blocked by cycloheximide. Using cycloheximide to maintain cells depleted of CSP, we have demonstrated that isolated CSP can be progressively reabsorbed at 37 degrees C onto these denuded cell surfaces, and that this adsorption is not observed at 4 degrees C. This information on the turnover, depletion, and restoration of CSP may provide a means of directly testing its function.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus) : II. Effects of pretreatment with a small number of HVJ

Ehrlich ascites tumor cell membranes were completely modified after incubation at 37 °C for 30 min with a small dose of HVJ (about 0.7% of the maximum number of the virus particles that could be adsorbed onto the cells). After this treatment, the cells could adsorb further added HVJ onto their surfaces at 0 °C. But the cell agglutination which was induced by viral adsorption at 0 °C was very weak, and the interaction of the adsorbed virus with the lipid layer of the cell membrane at 37 °C preceding fusion or lysis of the cells was not strong. A discrepancy was observed between acquisition of the modification and liberation of sialic acid (destruction of viral receptors) by viral neuraminidase. The modification proceeded well on incubation at 37 °C but not at lower temperatures. The possibility that the modification is induced by fusion of viral envelopes with cell membranes is discussed.     

In situ harvesting of adhered target cells using thermoresponsive substrate under a microscope: principle and instrumentation

A novel technique and instrumented device were developed to harvest target cells from multicellular mixture of different cell types under a microscope. The principle of the technique is that cells cultured on a thermoresponsive-substance-coated dish were detached by a region-specific cooling device and simultaneously harvested using a micropipette, both of which were assembled in an inverted microscope. Thermoresponsive coating consists of the mixture of poly(N-isopropylacrylamide) (PNIPAAm) and PNIPAAm-grafted gelatin. The former non-cell-adhesive polymer dissolves below at 32.1 degrees C in water and precipitates over that temperature (called lower critical solution temperature, LCST), and the latter cell-adhesive polymer has LCST of 34.1 degrees C. The appropriate mixing ratio of these thermoresponsive polymers exhibited high cell adhesion at physiological temperature and complete cell detachment at room temperature. A device developed as to cool at only a tiny area of the bottom of the dish, beneath which a cell that was targeted under a microscope, was assembled in a microscope. It was demonstrated that single cell or two cells that adhered to each other was detached from the surface and harvested by a micropipette within approximately 30s.     

Protein-reactive, thermoresponsive copolymers with high flexibility and biodegradability

A family of injectable, biodegradable, and thermosensitive copolymers based on N-isopropylacrylamide, acrylic acid, N-acryloxysuccinimide, and a macromer polylactide-hydroxyethyl methacrylate were synthesized by free radical polymerization. Copolymers were injectable at or below room temperature and formed robust hydrogels at 37 degrees C. The effects of monomer ratio, polylactide length, and AAc content on the chemical and physical properties of the hydrogel were investigated. Copolymers exhibited lower critical solution temperatures (LCSTs) from 18 to 26 degrees C. After complete hydrolysis, hydrogels were soluble in phosphate buffered saline at 37 degrees C with LCSTs above 40.8 degrees C. Incorporation of type I collagen at varying mass fractions by covalent reaction with the copolymer backbone slightly increased LCSTs. Water content was 32-80% without collagen and increased to 230% with collagen at 37 degrees C. Hydrogels were highly flexible and relatively strong at 37 degrees C, with tensile strengths from 0.3 to 1.1 MPa and elongations at break from 344 to 1841% depending on NIPAAm/HEMAPLA ratio, AAc content, and polylactide length. Increasing the collagen content decreased both elongation at break and tensile strength. Hydrogel weight loss at 37 degrees C was 85-96% over 21 days and varied with polylactide content. Hydrogel weight loss at 37 degrees C was 85-96% over 21 days and varied with polylactide content. Degradation products were shown to be noncytotoxic. Cell adhesion on the hydrogels was 30% of that for tissue culture polystyrene but increased to statistically approximate this control surface after collagen incorporation. These newly described thermoresponsive copolymers demonstrated attractive properties to serve as cell or pharmaceutical delivery vehicles for a variety of tissue engineering applications.     

Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability

The influence of protein stability on the adsorption and desorption behavior to surfaces with fundamentally different properties (negatively charged, positively charged, hydrophilic, and hydrophobic) was examined by surface plasmon resonance measurements. Three engineered variants of human carbonic anhydrase II were used that have unchanged surface properties but large differences in stability. The orientation and conformational state of the adsorbed protein could be elucidated by taking all of the following properties of the protein variants into account: stability, unfolding, adsorption, and desorption behavior. Regardless of the nature of the surface, there were correlation between (i) the protein stability and kinetics of adsorption, with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability. All of the above correlations could be explained by the rate of denaturation and the conformational state of the adsorbed protein. In conclusion, protein engineering for increased stability can be used as a strategy to decrease irreversible adsorption on surfaces at a liquid-solid interface.     

Chitosan-mediated synthesis of gold nanoparticles on patterned poly(dimethylsiloxane) surfaces

Synthesis of gold nanoparticles on surfaces has been accomplished by the incubation of poly(dimethylsiloxane) (PDMS) films in tetrachloroauric(III) acid and chitosan solution at room temperature and 4 degrees C. One important point in the present study is that the synthesis selectively occurred on the PDMS surface. These observations are substantially different from the reaction in solution, in which no particles can be formed at room temperature. Computation of surface plasmon bands (SPBs) based on Mie theory suggests that the particles are partially coated by chitosan molecules, and the experimental results confirm the theoretical calculations. The proposed mechanism is that chitosan molecules adsorbed or printed on the PDMS surfaces act as reducing/stabilizing agents. Furthermore, PDMS films patterned with chitosan could induce localized synthesis of gold nanoparticles in regions capped with chitosan only. In this way, colloidal patterns were fabricated on the surfaces with high spatial selectivity simultaneously with the synthesis of the particles. Surface-induced fluorescence quenching was observed in the regions capped with gold nanoparticles as well.     

Smart thermoresponsive coatings and surfaces for tissue engineering: switching cell-material boundaries

The smart thermoresponsive coatings and surfaces that have been explicitly designed for cell culture are mostly based on poly(N-isopropylacrylamide) (PNIPAAm). This polymer is characterized by a sudden precipitation on heating, switching from a hydrophilic to a hydrophobic state. Mammalian cells cultured on such thermoresponsive substrates can be recovered as confluent cell sheets, while keeping the newly deposited extracellular matrix intact, simply by lowering the temperature and thereby avoiding the use of deleterious proteases. Thermoresponsive materials and surfaces are powerful tools for creating tissue-like constructs that imitate native tissue geometry and mimic its spatial cellular organization. Here we review and compare the most representative methods of producing thermoresponsive substrates for cell sheet engineering.     

Comparison of the adsorbed conformation of barley lipid transfer protein at the decane-water and vacuum-water interface: a molecular dynamics simulation

Molecular dynamics simulation is used to model the adsorption of the barley lipid transfer protein (LTP) at the decane-water and vacuum-water interfaces. Adsorption at both surfaces is driven by displacement of water molecules from the interfacial region. LTP adsorbed at the decane surface exhibits significant changes in its tertiary structure, and penetrates a considerable distance into the decane phase. At the vacuum-water interface LTP shows small conformational changes away from its native structure and does not penetrate into the vacuum space. Modification of the conformational stability of LTP by reduction of its four disulphide bonds leads to an increase in conformational entropy of the molecules, which reduces the driving force for adsorption. Evidence for changes in the secondary structure are also observed for native LTP at the decane-water interface and reduced LTP at the vacuum-water interface. In particular, intermittent formation of short (six-residue) regions of beta-sheet is found in these two systems. Formation of interfacial beta-sheet in adsorbed proteins has been observed experimentally, notably in the globular milk protein beta-lactoglobulin and lysozyme.     

Adsorption of zein on surfaces with controlled wettability and thermal stability of adsorbed zein films

Adsorption characteristics of zein protein on hydrophobic and hydrophilic surfaces have been investigated to understand the orientation changes associated with the protein structure on a surface. The protein is adsorbed by a self-assembly procedure on a monolayer-modified gold surface. It is observed that zein shows higher affinity toward hydrophilic than hydrophobic surfaces on the basis of the initial adsorption rate followed by quartz crystal microbalance studies. Reflection absorption infrared (RAIR) spectroscopic studies reveal the orientation changes associated with the adsorbed zein films. Upon adsorption, the protein is found to be denatured and the transformation of alpha-helix to beta-sheet form is inferred. This transformation is pronounced when the protein is adsorbed on hydrophobic surfaces as compared to hydrophilic surfaces. Electrochemical techniques (cyclic voltammetry and impedance techniques) are very useful in assessing the permeability of zein film. It is observed that the zein moieties adsorbed on hydrophilic surfaces are highly impermeable in nature and act as a barrier for small molecules. The topographical features of the deposits before and after adsorption are analyzed by atomic force microscopy. The protein adsorbed on hydrophilic surface shows rod- and disclike features that are likely to be the base units for the growth of cylindrical structures of zein. The thermal stability of the adsorbed zein film has been followed by variable-temperature RAIR measurements.     

Strategy for allosteric analysis based on protein-patterned stationary phase in microfluidic chip

An effective method is presented for the on-chip analysis of chiral interactions with a successful depression of nonspecific adsorption. The alumina gel-derived protein network on poly(methyl methacrylate) (PMMA) microchannel was explored to form a protein-stationary phase and then used to carry out electrophoresis for fast enantioseparation coupled with electrochemical detection. On the basis of the chemical modification of a synthesized copolymer containing silane-functionalized scaffold, alumina sol-gel could react readily with the silane groups and form steady microstructure on the chip surface achieving the encapsulation of functional biomolecules. Compared with the native PMMA microchannels, the modified surfaces exhibited much better wettability, more stable and enhanced electroosmotic mobility, and less nonspecific adsorption. The water contact angle and EOF of alumina-gel-derived PMMA substrate were 22 degrees and 4.3 x 10(-4) cm(2) V(-1) s(-1), compared to those of 73 degrees and 1.9 x 10(-4) cm(2) V(-1) s(-1) from the untreated one, respectively. Bovine serum albumin, acting as a target protein, could be stably and homogeneously immobilized in the modified PMMA microchannel to fabricate a protein-stationary phase. Under a mild condition, D- and L-tryptophan were efficiently separated with a resolution of 1.57. The as-prepared microchip can perform chiral separations within short time, indicating that the general protocol has the potential to provide a platform for high throughput screening of enantiomer candidates such as those biochemical drugs with protein targets and the research of receptor interactions.     

Fluorine-free mixed amphiphilic polymers based on PDMS and PEG side chains for fouling release applications

Fluorine-free mixed amphiphilic block copolymers with mixtures of short side groups of polydimethyl siloxane (PDMS) and polyethylene glycol (PEG) were synthesized and studied for their ability to influence the surface properties and control the adhesion of marine organisms to coated surfaces. The settlement (attachment) and strength of adhesion of two different marine algae, the green seaweed Ulva and the diatom Navicula, were evaluated against the surfaces. It is known that hydrophobic coatings based on polydimethyl siloxane elastomers (PDMSe) are prone to protein adsorption and accumulation of strongly adherent diatom slimes, in contrast to PEG-based hydrophilic surfaces that inhibit protein adsorption and moderate only weak adhesion of diatoms. By incorporating both PDMS and PEG side chains into the polymers, the effect of incorporating both polar and non-polar groups on fouling-release could be studied. The dry surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The ability of these mixed amphiphilic polymers to reconstruct in water was examined using underwater bubble contact angle and dynamic water contact angle experiments. To understand more about surface reconstruction behavior, protein adsorption experiments were carried out with fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) on both dry and pre-soaked surfaces.     

Chemiluminescence-based detection and comparison of protein amounts adsorbed on differently modified silica surfaces

The biological consequences of protein adsorption on biomaterial surfaces are considered to be of utmost importance for their biocompatibility. A new method based on amino group-labeling coupled to a chemiluminescence reaction for direct determination of proteins adsorbed on material surfaces was employed. This method was used to explore the effects of surface chemistry and surface roughness on protein adsorption in a silicon oxide model system. Corundum sandblasting was applied to silicon wafers to create roughened surfaces while immobilization of fluorocarbon-, hydrocarbon-, and poly(ethylene glycol)-containing silanes produced surfaces of varying wettability. The adsorption behavior of two complex body fluids, human serum and saliva, and of two purified components, human serum albumin and fibronectin, was strongly influenced by the surface parameters. A general tendency to higher amounts of adsorbed protein was found on roughened surfaces and modification with poly(ethylene glycol) or with fluorocarbon moieties reduced protein adsorption. The values obtained with the new method could be confirmed by a colorimetric determination of protein amounts adsorbed on identically modified silica beads and were in accordance with those previously reported utilizing established methods for protein quantification. The presented method, which was methodically simple to perform and allowed the simultaneous measurement of a large number of samples, may be of future value for high-throughput surveying of the protein adsorption characteristics of biomaterials.     

Salivary protein adsorption onto hydroxyapatite and sds‐mediated elution studied by in situ ellipsometry

Whole unstimulated saliva from two donors was investigated both with respect to adsorption characteristics and SDS‐induced elutability. Salivary protein adsorption onto hydroxyapatite (HA) discs was studied by means of in situ ellipsometry in the concentration range 0.1–20% saliva. The adsorbed amounts on HA were found to be similar to those on silica, but the rates of adsorption were lower. Protein adsorption was virtually unaffected by the presence of Na⁺, whereas Ca²⁺ induced nucleation of calcium phosphate at the surface, the deposition rate being influenced by the pellicle age but not by the presence of saliva in bulk solution. The SDS elutability of adsorbed pellicles was determined on HA as well as on silica surfaces. Desorption from both surfaces was found to occur in the same SDS concentration range, although a residual layer was observed on HA. The slight net positive charge and lower charge density of HA as compared to the strongly negatively charged silica, may, at least partly, account for this observation by causing a reduction in the repulsive force between protein‐surfactant complexes and the surface. Inter‐individual differences, observed in the adsorption as well as elution experiments, are thought to relate to the compositional differences observed by SDS‐PAGE.