Poly(N-isopropyl acrylamide) (pNIPAM) undergoes a phase change in a physiologically relevant temperature range that leads to cell release. Above its lower critical solution temperature (LCST, ~32^oC), pNIPAM is relatively hydrophobic, and when grafted to a surface, it takes on a packed conformation. There have been numerous studies on the conformation change of pNIPAM across its LCST. Although it is known how pNIPAM chains tethered to a substrate behave when the temperature is changed, no study has probed the influence of a temperature gradient on the behavior of cells attached to the polymer. In this work, we present the investigation of cell detachment from pNIPAM-grafted surfaces resting on a temperature-gradient device. The polymer was deposited on the surface using plasma polymerization. This deposition technique creates a conformal, sterile film that is compatible with any surface chemistry, including the transparent well plates required for these experiments. Prior to their use for cell culture, it is imperative to characterize the pNIPAM films for film thickness, surface chemistry, and thermoresponse, as the surface characteristics determine cell attachment and detachment. The characterization was performed via interferometry, X-ray photoelectron spectroscopy (XPS), and contact angle measurements. Using a device fabricated in our laboratory, we studied whether there is a gradual progression of cellular detachment from the polymer along the temperature gradient, or if there is an abrupt step-change in the detachment. This work will have valuable insights regarding the optimal temperature for cell detachment from pNIPAM.

Protein resistant oligo(ethylene glycol) terminated (OEG) self-assembled monolayers (SAMs) of thiols on gold are commonly used for suppression of nonspecific protein adsorption in biology and biotechnology. The standard preparation for these SAMs is the solution method (SM) that involves immersion of the gold surface in an OEG solution. Here we present the preparation of 11-(mercaptoundecyl)-triethylene glycol (HS(CH₂)₁₁(OCH₂CH₂)₃OH) SAMs on gold surface by vapor deposition (VD) in vacuum. We compare the properties of SAMs prepared by VD and SM using X-ray photoelectron spectroscopy (XPS), polarisation modulation infrared reflection absorption spectroscopy (PM-IRRAS) and surface plasmon resonance (SPR) measurements. VD and SM SAMs exhibit similar packing density and show a similar resistance to the nonspecific adsorption of various proteins (bovine serum albumin, trypsin, and myoglobin) under physiological conditions. A very high sensitivity of the OEG SAMs to X-ray radiation is found, which allows tuning their protein resistance. These results show a new path to in situ engineering, analysis and patterning of protein resistant OEG SAMs by high vacuum and ultra high vacuum techniques.

[1] L. Kankate, H. Großmann, U. Werner, R. Tampé, A. Turchanin, A. Gölzhäuser, Biointerphases 5, 30 (2010)

The printable biological ink on gelatin is a biopolymer that could be use in a handheld, portable device, such as a ink jet of printer and become a substrate for advanced wound care. Skin is very affected by when burned, and also by diabetic venous ulcers. Currently, some skin substitutes exist that for treatment of diabetic foot ulcers, but these products are really expensive. Based on this we want to create a low cost wound care material that helps not only people with diabetic foot ulcers and venous leg ulcers, but also with burned skin. We selected sodium alginate because it is a FDA approved carbohydrate that has many applications for tissue regeneration and cell therapy, and it is very compatible with human body. Oxidation of sodium alginate has been investigated to obtained alginate dialdehyde (ADA). Alginate dialdehyde is a fast acting self-crosslinking degradable polymer, when applied over collagen or gelatin. We investigated, the degree of oxidation, the degree of cross-linking and mechanical properties of the materials. The degree of crosslinking was determined by trinitrobenzene sulphonic acid assay maintaining constant ADA concentration in borax or PBS buffer and varying concentration of gelatin in borax or PBS. An increase of the degree of oxidation of sodium alginate, an increase in the cross-linking and a decrease of the gelling time was observed with increasing ADA concentration. We investigated the effect of ADA concentration on viscosity and found that at concentrations of about 10% ADA, the solutions are viscous. We will present data on the use of ADA solution as an ink in a printing device and the effect of printed ADA to cross-link gelatin. In general, control over the concentrations of ADA as well as the spatial dispensing via printing should allow us to generate wound dressings of tunable properties. The use of a portable device makes this solution attractive to low resource settings. Future work will include testing the wound dressing in a small animal model and investigating the effect of adding keratinocytes as well as endothelial cells to the material on healing and wound contraction.

An electrochemical impedance spectroscopic (EIS) instrument with indium-tin oxide (ITO) culture chip module and lock-in impedance readout module was developed. The lock-in impedance readout module achieves impedance measurements with a small, bio-harmless AC signal. Moreover, transparent ITO culture-chip module is experience friendly; other optical inspections can be applied on the chip. The impedance readout module is designed to perform in a range from 1 to 10 kHz; the phase measuring errors are within 5.1% in that range. Typical examples of PBS solutions, BSA proteins and cell culturing tests are discussed in experiments. Higher concentration levels of PBS produced lower impedance. Higher concentration level of BSA solution also produced lower impedance. Furthermore, experiments of 0.25% BSA dissolved in sDDW and 1X PBS show that the mediums influence system impedance. Moreover, the log phase (period of cell proliferation) of B16F10 cell culture tests ended at 9, 16, 23, and 65 h for seeded cell densities of 5×10⁴, 1×10⁵, 2×10⁵, and 4×10⁵/mL, respectively. Finally, cultures are incubated and doped to demonstrate the monitoring of cell proliferation.

An EIS using microelectrode arrays has gained much attention as a promising, label free, fast, and real-time method for cellular analysis. The electric cell substrate impedance sensing (ECIS; Applied BioPhysics) and real time cell electronic sensing (RT-CES; Roche Applied Sciences) systems are widely applied in measuring cell proliferation, attachment and spreading, motility, toxicology, barrier function, wounding, and migration. The sensing chips are opaque and the influence of solutions is generally disregarded. However, transparent chips for optically related detection are required in many applications. And, in fact, chemical additives for biochemical treatments induce impedance variations.

This study develops an EIS instrument with an ITO culture chip module and lock-in impedance readout module. The instrument can apply and extract bio-harmless small signals by lock-in technology. Experimental results prove impedance readout module performance. The ITO culture chip module electrodes are connected via the electrolyte medium and the route of cell/tissue. Typical examples of the effects of solutions and cell culturing tests are discussed. A higher level of PBS causes lower impedance. Further study of solution ingredients will improve the measurement accuracy and results analysis. The ESI instrument with an ITO culture chip module and lock-in impedance readout module can be applied in other conductive liquid sample investigations.

Although many studies have tried to elucidate the lubrication mechanisms that occur in articular cartilage, the molecular details of how constituents of the synovial fluid interact with cartilage surfaces and mediate cartilage to cartilage interaction still remain poorly understood. One of the major constituents of the synovial fluid that is thought to be responsible for boundary lubrication is the glycoprotein lubricin; however, details of the molecular mechanisms by which lubricin carries out its vital functions still remain largely unknown. Here, we examine i) the molecular details in which lubricin interacts with type II collagen, the main component of cartilage that provides structural integrity and tensile strength, ii) whether collagen structure can affect lubricin binding and change the adhesive interactions and boundary lubrication between collagen surfaces. We found that lubricin adsorbed strongly onto denatured, amorphous and fibrillar collagens surfaces. Furthermore, we found large repulsive interactions, between the collagen surfaces in presence of lubricin, increase with increasing lubricin concentration. Lubricin attenuated the large friction and also the long-ranged adhesion between the fibrillar collagen surfaces. Interestingly, lubricin mediated the frictional response between the denatured and native amorphous collagen surfaces equally, and showed no preference on the supramolecular architecture of collagen. We speculate that in mediating interactions at the cartilage surface, an important role of lubricin is to attach to the cartilage surface and provide a protective coating that maintains the contacting surfaces in a sterically repulsive state.

Poly(N-isopropyl acrylamide) (pNIPAM) is a thermoresponsive polymer that is widely used in bioengineering applications, including tissue culture engineering, single cell adhesion/detachment, and biofouling prevention. Although there are many ways to treat surfaces with pNIPAM, plasma polymerization is one of the more adaptable ways for coating surfaces for the use in tissue culture experiments. While plasma polymerization creates a sterile environment useful for tissue culture, occasionally, additional sterilization techniques must be used. Some sterilization methods include using UV light to sterilize the surfaces, and the use of different solvents, such as ethanol. To date, there have been no studies on the effect of these sterilization techniques on the reversible adhesion of biological cells on pNIPAM treated surfaces. In this work, we investigate the effect of different sterilization techniques (e.g., ethanol and UV light) on the thermoresponsive nature of pNIPAM. Substrates were coated using plasma polymerization (ppNIPAM), after which they were sterilized using solvents, and characterized to determine if the solvents changed the thermoresponsive nature of the polymer. X-ray photoelectron spectroscopy (XPS) and interferometry were used to determine the surface chemistry and thickness of our ppNIPAM surfaces. Goniometry was used to confirm the thermoresponsive nature of our surfaces. Finally, we tested the adhesion and detachment of cells on the surfaces using bovine aortic endothelial cells (BAECs). We found that the use solvents as sterilizing agents does have an effect on the thermoresponsive nature of pNIPAM (as demonstrated by the decreased detachment of cells from the surfaces), even when the pNIPAM film’s chemistry appears unaffected.

Our recent work has made extensive use of X-ray photoelectron spectroscopy (XPS) in order to identify molecular interactions at a series of bio-inorganic and bio-organic interfaces.

In the first project, biologically-synthesized silica-enzyme and silica-peptide nanocomposite materials were analyzed by means of high resolution XPS. Biosilicification, i.e., rapid precipitation of silica mediated by a biological catalyst at ambient conditions, provides an efficient method for controlled synthesis of complex nanoscale structures. The process mimics reactions that organisms use to form rigid structures such as diatom exoskeletons and sponge spicules. The molecular interactions that allow these peptides to induce silica mineralization have not been elucidated. The focus of our study used the antimicrobial decapeptide KSL (KKVVFKVKFK), which induces rapid biosilica formation from pre-hydrolyzed tetramethyl orthosilicate in phosphate buffer. XPS was used to probe elemental composition and coordination chemistry of hybrid peptide-silica nanoparticle surfaces and ultimately identify the molecular interactions at the bio-inorganic interface.

In separate work, XPS was used to define interactions between biomolecules and various materials molecules used for bioelectrochemical architectures. One complex used the in vitro biosilicification process to help associate glucose oxidase (GOx) and carbon nanotubes (CNT) to yield a conductive composite matrix. Silica encapsulation of GOx provided an immobilization efficiency of ~25% in the presence of CNT. XPS analysis confirmed that the formation of a heterogeneous silica matrix had incorporated lysozyme, GOx and CNT. Another materials approach used layer-by layer assembly (LbL) to immobilize a redox enzyme. The LbL approach fixed bilirubin oxidase (BOD) on an electrode surface by using a glutaraldehyde as a “cross-linker” in combination with electrostatic interactions between the negatively charged enzyme and the positively charged polymer. The composite matrix was characterized using angle resolved XPS and multivariate analysis. The methods defined: the physical architecture of BOD layers immobilized on the electrode, the relative thickness of each assembled layer, along with their elemental and chemical composition.

In the third project we have studied poly-azines, which have been popular electrocatalysts of choice for NADH oxidation for sensors and biofuel cell applications using NAD-dependent enzymes. Little is known about their structure and mechanism of polymerization. Through detailed XPS analysis of monomers and polymers we were able to propose mechanism of electropolymerization and elucidate polyazine structure.

The cellular machinery dedicated to the synthesis of the polypeptide chain translated from the mRNA template is the ribosome. Clusters of translating ribosomes held together by mRNA form the so-called polysome. The study of such supramolecular assemblies has interests both in structural and functional aspects as addressed in this study. As consequence the analyses of polysomal mRNA are emerging as good estimators in directly representing cell phenotypes. Since traditional methods of polysome purification are still a time consuming and laborious procedures, high throughput polysomal purifications are attracting growing interest. Recently, more modern approaches have been developed, based on the specific affinity between genetically modified ribosomes and functional surfaces. However, native ribosomes are required for several analytical applications. This work is a first step in developing a convenient and fast strategy to purify native polysomes thanks to their adhesion ability to appropriate substrates. We studied the interaction between functionalized gold surfaces and polysome fractions purified from human cultured cells or ribosomes derived from rabbit reticulocyte lysate. Surfaces endowed with different chemical properties were obtained using functional thiols able to form self assembled monolayers on gold substrates. Substrate surface properties were studied with XPS, ToF-SIMS and AFM. The interaction of rabbit ribosomes with the surfaces was assessed with AFM and confocal microscopy.

Despite different imaging approaches have been used to visualize bacterial and eukaryotic polysomes, human native polysomes have never been observed in physiological conditions. Moreover nothing is known about the formation of the mRNA-ribosomes macroassembly. Here we present the very first AFM images of native polysomes from mammalian cell lines adherent to surfaces. For the first time to our knowledge, we could observe ribosome aggregates associated to RNA, providing the imaging of polysome assemblies. Our imaging approach suggests the existence of a complex pattern of conformations and reveals novel levels of polysomal organization.

This poster will describe surface adsorption of beta-helical scaffolds derived from peptides composed of alternating D- and L-amino acids. The monomeric and stable conformation of beta-helical peptides in solution provides a well-defined starting point for discriminating the changes in secondary structure of peptides induced by surface adsorption. NMR, CD, VCD, and XPS results will be presented.

The development of new transducing substrate materials, such as polymers, has spurred a growing interest in assessing the influences of surface topography and chemistry of the abiotic layer on the bio-functionality of the biotic layer. However, it is a challenge to differentiate the contributions of surface roughness and surface chemistry to biointerface functionality, as most surface modification methods tend to alter both at the same time. In this work, we discuss a dry and wet chemistry approach to biointerface development on polymeric substrates aimed at minimizing changes in the surface morphology. Specifically, an NRL developed electron beam-generated plasma processing system was used to functionalize the surface of polystyrene microtitre plates, which were then silanized to covalently immobilize biomolecules. Electron beam plasmas are unique in that they deliver high flux of low energy (< 5 eV) ions to substrate surfaces enabling selective, chemical alteration of the top few nanometers of any material without significant morphological modification. Thus, reactive hydroxyl groups, that make the characteristically inert material amenable to silane chemistry, can be introduced while maintaining substrate morphology. Silanization is an easy, controllable, and conformal covalent immobilization method that supports a wide variety of bio-immobilization schemes. Biomolecules covalently immobilized using this combined dry-wet chemistry approach retained functionality and demonstrated attachment efficiency comparable (and in some cases superior) to specialized commercial microtitre plates. We have used a combination of complementary surface analytical techniques to evaluate the relationships between the physical and chemical properties of the treated polymer surfaces, with particular emphasis on attachment of a functional silane layer, which is a critical parameter for efficient covalent bio-immobilization. Based on our results, we conclude that the development of novel interface materials with superior transducing capabilities is contingent on the deeper understanding of the complex physicochemical interactions between substrate polymer and the chemical/biological components attached to it.