Paper-based plasmonic substrates as surface-enhanced Raman scattering spectroscopy platforms for cell culture applications.

The engineering of advanced materials capable of mimicking the cellular micro-environment while providing cells with physicochemical cues is central for cell culture applications. In this regard, paper meets key requirements in terms of biocompatibility, hydrophilicity, porosity, mechanical strength, ease of physicochemical modifications, cost, and ease of large-scale production, to be used as a scaffold material for biomedical applications. Most notably, paper has demonstrated the potential to become an attractive alternative to conventional biomaterials for creating two-dimensional (2D) and three-dimensional (3D) biomimetic cell culture models that mimic the features of in vivo tissue environments for improving our understanding of cell behavior (e.g. growth, cell migration, proliferation, differentiation and tumor metastasis) in their natural state. On the other hand, integration of plasmonic nanomaterials (e.g. gold nanoparticles) within the fibrous structure of paper opens the possibility to generate multifunctional scaffolds equipped with biosensing tools for monitoring different cell cues through physicochemical signals. Among different plasmonic based detection techniques, surface-enhanced Raman scattering (SERS) spectroscopy emerged as a highly specific and sensitive optical tool for its extraordinary sensitivity and the ability for multidimensional and accurate molecular identification. Thus, paper-based plasmonic substrates in combination with SERS optical detection represent a powerful future platform for monitoring cell cues during cell culture processes. To this end, in this review, we will describe the different methods for fabricating hybrid paper-plasmonic nanoparticle substrates and their use in combination with SERS spectroscopy for biosensing and, more specifically, in cell culture applications.

Connecting quantum dots with enzymes: mediator-based approaches for the light-directed read-out of glucose and fructose oxidation.

The combination of the biocatalytic features of enzymes with the unique physical properties of nanoparticles in a biohybrid system provides a promising approach for the development of advanced bioelectrocatalytic devices. This study describes the construction of photoelectrochemical signal chains based on CdSe/ZnS quantum dot (QD) modified gold electrodes as light switchable elements, and low molecular weight redox molecules for the combination with different biocatalysts. Photoelectrochemical and photoluminescence experiments verify that electron transfer can be achieved between the redox molecules hexacyanoferrate and ferrocene, and the QDs under illumination. Since for both redox mediators a concentration dependent photocurrent change has been found, light switchable enzymatic signal chains are built up with fructose dehydrogenase (FDH) and pyrroloquinoline quinone-dependent glucose dehydrogenase ((PQQ)GDH) for the detection of sugars. After immobilization of the enzymes at the QD electrode the biocatalytic oxidation of the substrates can be followed by conversion of the redox mediator in solution and subsequent detection at the QD electrode. Furthermore, (PQQ)GDH has been assembled together with ferrocenecarboxylic acid on top of the QD electrode for the construction of a funtional biohybrid architecture, showing that electron transfer can be realized from the enzyme over the redox mediator to the QDs and subsequently to the electrode in a completely immobilized fashion. The results obtained here do not only provide the basis for light-switchable biosensing and bioelectrocatalytic applications, but may also open the way for self-driven point-of-care systems by combination with solar cell approaches (power generation at the QD electrode by enzymatic substrate consumption).

Ecotoxicity and uptake of polymer coated gold nanoparticles.

Bioconjugated gold nanoparticles (Au NPs) are a promising tool for pharmaceutical applications. However, the ecotoxicity of these types of NPs has hardly been studied. We investigated the ecotoxicity and uptake of 4-5 nm Au NPs to which two types of polymer coatings were attached. One coating was an amphiphilic polymer only and the other an amphiphilic coating to which 10 kDa polyethylene glycol chains were attached. In both 72 h algal growth inhibition tests with the alga Pseudokirchneriella subcapitata and in 24 h resazurin cytotoxicity tests with the rainbow trout gill cell line RTGill-W1, the pegylated Au NPs were found less toxic compared to the amphiphilic coated particles. No uptake or direct interaction between particles and algal cells was observed. However, uptake/adsorption in fish gill cells reached up to >10(6) particles/cell after 1 h and particles were eliminated for ≥96% after 24 h depuration. Both particle types were found within membrane enclosed vesicles in the cytoplasm of RTgill-W1 cells.

A general synthetic approach for obtaining cationic and anionic inorganic nanoparticles via encapsulation in amphiphilic copolymers.

A series of amphiphilic copolymers with variable charge densities on their backbone is synthesized. Positively charged N,N,N-trimethylammonium-2-ethyl methacrylate iodide or negatively charged 2-(methacryloyloxy)ethylphosphonic acid and lauryl methacrylate are used as building blocks. When wrapped around hydrophobically capped inorganic nanoparticles (NPs), the latter are able to disperse in aqueous solutions. Using this method, positively as well as negatively charged colloidal NPs can be synthesized in a reliable way. The method presented herein allows the charge on the NPs to be adjusted to different negative and positive values by using polymers with a variable ratio of charged monomers and lauryl methacrylate. Virtually all kinds of hydrophobic inorganic NPs could be coated with these amphiphilic polymers. The coating procedure is demonstrated for Au particles as well as for CdSe/ZnS quantum dots. To date, wrapping amphiphilic polymers around NPs has led only to anionic NPs. The polymers synthesized in this work allow for positively charged NPs with a high colloidal stability.

Light-controlled bioelectrochemical sensor based on CdSe/ZnS quantum dots.

This study reports on the oxygen sensitivity of quantum dot electrodes modified with CdSe/ZnS nanocrystals. The photocurrent behavior is analyzed for dependence on pH and applied potential by potentiostatic and potentiodynamic measurements. On the basis of the influence of the oxygen content in solution on the photocurrent generation, the enzymatic activity of glucose oxidase is evaluated in solution. In order to construct a photobioelectrochemical sensor which can be read out by illuminating the respective electrode area, two different immobilization methods for the fixation of the biocatalyst have been investigated. Both covalent cross-linking and layer-by-layer deposition of GOD by means of the polyelectrolyte polyallylamine hydrochloride show that a sensor construction is possible. The sensing properties of this type of electrode are drastically influenced by the amount and density of the enzyme on top of the quantum dot layer, which can be advantageously adjusted by the layer-by-layer technique. By depositing four bilayers [GOD/PAH](4) on the CdSe/ZnS electrode, a fast-responding sensor for the concentration range of 0.1-5 mM glucose can be prepared. This study opens the door to multianalyte detection with a nonstructured sensing electrode, localized enzymes, and spatial read-out by light.

Multifunctional nanoparticles for dual imaging.

For imaging with different modalities, labels, which provide contrast for all modalities, are required. Colloidal nanoparticles composed out of an inorganic core and a polymer shell offer progress in this direction. Both, the core and the polymer shell, can be synthesized to be fluorescent, magnetic, or radioactive. When different cores are combined with different polymer shells, different types of particles for dual imaging can be obtained, as for example, fluorescent cores with radioactive polymer shells. Properties and perspectives of such nanoparticles for multimodal imaging are discussed.

Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles.

Inorganic colloidal nanoparticles are very small, nanoscale objects with inorganic cores that are dispersed in a solvent. Depending on the material they consist of, nanoparticles can possess a number of different properties such as high electron density and strong optical absorption (e.g. metal particles, in particular Au), photoluminescence in the form of fluorescence (semiconductor quantum dots, e.g. CdSe or CdTe) or phosphorescence (doped oxide materials, e.g. Y(2)O(3)), or magnetic moment (e.g. iron oxide or cobalt nanoparticles). Prerequisite for every possible application is the proper surface functionalization of such nanoparticles, which determines their interaction with the environment. These interactions ultimately affect the colloidal stability of the particles, and may yield to a controlled assembly or to the delivery of nanoparticles to a target, e.g. by appropriate functional molecules on the particle surface. This work aims to review different strategies of surface modification and functionalization of inorganic colloidal nanoparticles with a special focus on the material systems gold and semiconductor nanoparticles, such as CdSe/ZnS. However, the discussed strategies are often of general nature and apply in the same way to nanoparticles of other materials.

Photoactivated release of cargo from the cavity of polyelectrolyte capsules to the cytosol of cells.

Polyelectrolyte capsules with metal nanoparticles in their walls and fluorescently labeled polymers as cargo inside their cavity were prepared. Capsules were ingested by living cells with no uncontrolled release of the cargo upon the incorporation process. Photoinduced heating of the metal nanoparticles in the capsule walls lead to rupture of the capsule walls, and the polymeric cargo was released to the whole cytosol. Viability tests demonstrate that opening of capsules at moderate light intensities does not impair the cellular metabolism, whereas capsule opening at high light intensities ultimately leads to cell death.

Improvement of conversion efficiency for multi-junction solar cells by incorporation of Au nanoclusters.

We studied the photoluminescence (PL) and photovoltaic current-voltage characteristics of the three-junction InGaP/InGaAs/Ge solar cells by depositing Au nanoclusters on the cell surface. The increases of the PL intensity and short-circuit current after incorporation of Au nanoclusters are evident. An increase of 15.3% in energy conversion efficiency (from 19.6 to 22.6%) is obtained for the three-junction solar cells in which Au nanoclusters have been incorporated. We suggest that the increased light trapping due to radiative scattering from Au nanoclusters is responsible for improving the performance of the three-junction solar cells.

Photoelectrochemical signal chain based on quantum dots on gold--sensitive to superoxide radicals in solution.

A photoelectrochemical signal chain sensitive to the presence of superoxide radicals was developed on the basis of CdSe/ZnS quantum dots which were immobilized on gold electrodes using a dithiol compound. The conditions of photo current generation under illumination have been characterized with respect to the dependence on the applied electrode potential, the wavelength of the light beam and the stability of the measurement. Because of photoexcitation electron-hole pair generation is enforced in the nanoparticles enhancing the conductivity of the quantum dot layer. This was independently verified by impedance measurements. In order to observe direct electron transfer with the redox protein cytochrome c different surface modifications of the quantum dots were investigated-mercaptopropionic acid, mercaptosuccinic acid and mercaptopyridine. Varying superoxide concentrations in solution can be detected by an enhanced conversion of superoxide-reduced cytochrome c and thus by an enhanced photo current at the quantum dot modified electrode. The electrode was found to be sensitive to higher nanomolar concentrations of the radical.

Gel electrophoresis of gold-DNA nanoconjugates.

Gold-DNA conjugates were investigated in detail by a comprehensive gel electrophoresis study based on 1200 gels. A controlled number of single-stranded DNA of different length was attached specifically via thiol-Au bonds to phosphine-stabilized colloidal gold nanoparticles. Alternatively, the surface of the gold particles was saturated with single stranded DNA of different length either specifically via thiol-Au bonds or by nonspecific adsorption. From the experimentally determined electrophoretic mobilities, estimates for the effective diameters of the gold-DNA conjugates were derived by applying two different data treatment approaches. The first method is based on making a calibration curve for the relation between effective diameters and mobilities with gold nanoparticles of known diameter. The second method is based on Ferguson analysis which uses gold nanoparticles of known diameter as reference database. Our study shows that effective diameters derived from gel electrophoresis measurements are affected with a high error bar as the determined values strongly depend on the method of evaluation, though relative changes in size upon binding of molecules can be detected with high precision. Furthermore, in this study, the specific attachment of DNA via gold-thiol bonds to Au nanoparticles is compared to nonspecific adsorption of DNA. Also, the maximum number of DNA molecules that can be bound per particle was determined.

Gold nanoparticles quench fluorescence by phase induced radiative rate suppression.

The fluorescence quantum yield of Cy5 molecules attached to gold nanoparticles via ssDNA spacers is measured for Cy5-nanoparticle distances between 2 and 16 nm. Different numbers of ssDNA per nanoparticle allow to fine-tune the distance. The change of the radiative and nonradiative molecular decay rates with distance is determined using time-resolved photoluminescence spectroscopy. Remarkably, the distance dependent quantum efficiency is almost exclusively governed by the radiative rate.

Cytotoxicity of nanoparticle-loaded polymer capsules.

Cytotoxic effects of micrometer-sized polymer capsules composed out of alternating layers of polystyrenesulfonate (PSS) and polyallylamine hydrochloride (PAH) on a fibroblast cell line have been investigated with an adhesion assay. For the purpose of visualization with fluorescence nanometer-sized CdTe nanoparticles have been embedded in the walls of the capsules. Similar to free CdTe nanoparticles, toxic Cd-ions are also released from CdTe nanoparticles that have been embedded in capsules. At high capsule concentrations, the capsules start to sediment on top of the cells and thus impair cell viability.

Metabolic activation stimulates acid secretion and expression of matrix degrading proteases in human osteoblasts.

BACKGROUND: Both cellular and matrix components of healthy bone are permanently renewed in a balanced homoeostasis. Osteoclastic bone resorption involves the expression of vacuolar-type ATPase proton pumps (vATPase) on the outer cell membrane and the secretion of matrix degrading proteases. Osteoblasts modulate the deposition of bone mineral components and secrete extracellular matrix proteins. OBJECTIVES: To investigate the ability of osteoblasts and osteosarcoma to secrete acid and express matrix degrading proteases upon metabolic activation. To examine also the potential contribution of vATPases to proton secretion expressed on osteoblasts. METHODS: Osteoblasts were isolated from trabecular bone and characterised by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Proton secretion was analysed by a cytosensor microphysiometer. RESULTS: Osteoblasts not only express matrix degrading proteases upon stimulation with tumour necrosis factor or with phorbol ester but they also secrete protons upon activation. Proton secretion by osteoblasts is associated partially with proton pump ATPases. CONCLUSION: These data suggest that, in addition to monocyte derived osteoclasts, cytokine activated mesenchymal osteoblasts and osteosarcoma cells may contribute to the acidic milieu required for bone degradation.

Spatially resolved monitoring of cellular metabolic activity with a semiconductor-based biosensor.

Metabolic activity of cultured cells can be monitored by measuring changes in the pH of the surrounding medium caused by metabolic products such as protons, carbon dioxide or lactic acid. Although many systems designed for this purpose have been reported, almost all of them are based on bulk measurements, where the average metabolic activity of all cells in contact with the device is recorded. Here, we report on a novel biosensor, based on a modified light-addressable potentiometric sensor (LAPS) device, which enables the metabolic activity of cultured cells to be measured with spatial resolution. This is demonstrated here by detecting the differential sensitivity to a cholinergic receptor agonist of two different co-cultured cellular populations. By making simultaneous measurements of the metabolic activity of different cell types seeded on different segments of one sensor, this device not only provides a rapid means of assessing cellular specificity of pharmaceutical compounds but also has the potential of being used to non-invasively monitor humoral as well as synaptic communication between different cell populations in co-culture. The temporal and spatial resolution of the device were investigated and are discussed.

Effects of semiconductor substrate and glia-free culture on the development of voltage-dependent currents in rat striatal neurones.

An essential requirement for successful long-term coupling between neuronal assemblies and semiconductor devices is that the neurones must be able to fully develop their electrogenic repertoire when growing on semiconductor (silicon) substrates. While it has for some time been known that neurones may be cultured on silicon wafers insulated with SiO2 and Si3N4, an electrophysiological characterisation of their development under such conditions is lacking. The development of voltage-dependent membrane currents, especially of the rapid sodium inward current underlying the action potential, is of particular importance because the conductance change during the action potential determines the quality of cell-semiconductor coupling. We have cultured rat striatal neurones on either glass coverslips or silicon wafers insulated with SiO2 and Si3N4 using both serum-containing and serum-free media. We here report evidence that not only serum-free culture media but also growth on semiconductor surfaces may negatively affect the development of voltage-dependent currents in neurones. Furthermore, using surface-charge measurements with the atomic force microscope, we demonstrate a reduced negativity of the semiconductor surface compared to glass. The reduced surface charge may affect cellular development through an effect on the binding and/or orientation of extracellular matrix proteins, such as laminin. Our findings therefore suggest that semiconductor substrates are not entirely equivalent to glass in terms of their effects on neuronal cell growth and differentiation.

Metabolic activation stimulates acid production in synovial fibroblasts.

OBJECTIVE: In rheumatoid arthritis (RA), synovial fibroblasts express proteases such as collagenases or cathepsins and inflammatory cytokines at elevated levels and so contribute to the inflammatory degradation process. Extracellular matrix degradation and cathepsin activity is dependent upon the presence of an acidic milieu. We examined whether activated synovial fibroblasts secrete acidic components. METHODS: Synovial fibroblasts were isolated and immortalized to study the mechanisms of metabolic activation. Naïve and immortalized fibroblasts were activated with different cytokines. The responses were investigated by immunoblot to detect Egr-1 and by a cytosensor microphysiometer analysis to evaluate acid secretion. Basic gene expression patterns were investigated in naïve and immortalized cells by RT-PCR analysis. RESULTS: We found RA synovial fibroblasts respond to different cytokines associated with the pathomechanisms of RA including interleukin 1, basic fibroblast growth factor, platelet derived growth factor, and tumor necrosis factor-alpha, with metabolic activation and enhanced secretion of acidic components. In addition, naive and SV40 TAg immortalized fibroblasts rapidly release acidic components after stimulation with phorbol ester or ionomycin as well. CONCLUSION: Activated synovial fibroblasts not only express inflammatory cytokines and matrix degrading proteases that are associated with the pathomechanisms of RA, but upon stimulation may release acidic components that lower pH and consequently enhance cathepsin activity and collagen solubilization.

Can the light-addressable potentiometric sensor (LAPS) detect extracellular potentials of cardiac myocytes?

The light-addressable potentiometric sensor (LAPS) measures localized photo-induced currents from a silicon wafer, which are dependent on the local surface potential and on the intensity of the light pointer. In this study the ability of the LAPS to record extracellular potentials of adherent cells was investigated. Time dependent LAPS photocurrent signals that correlated in time with contractions were recorded from beating cardiac myocytes cultured on LAPS surfaces. Signals could be recorded both when the LAPS was biased to working points where the photocurrent was maximally sensitive to potential changes and when it was biased to working points where the photocurrent was insensitive to changes in surface potential. Therefore, signals could not be predominantly created by changes in extracellular potential and might be related to mechanical contractions. One possible explanation might be, that the cell-induced modulation of photocurrents arose as a result of cell shape changes. Such alterations in cell shape might have focused and defocused the light pointer and, thus, modulated its intensity. To further test this hypothesis, height changes of beating cardiac myocytes were measured with an atomic force microscope (AFM). They were found to match well with signals derived from LAPS measurements. Therefore, it can be concluded, that LAPS signals were mainly determined by the periodic changes in shape of beating heart cells, and this interference precludes the measurements of extracellular electrophysiological potentials from these cells.

Mapping the mechanical pulse of single cardiomyocytes with the atomic force microscope.

The atomic force microscope (AFM) was used to analyse the contractile behaviour of embryonic chicken cardiomyocytes. The mechanical pulsing of cardiomyocytes was analysed by observing active single cells as well as cells in a confluent layer. When embedded in a confluent layer, owing to synchronisation, pulsing of the cells was often found to be very stable in terms of frequency and amplitude of the beat, including negative as well as positive amplitudes. Nevertheless, owing to movements of contraction centres within the layer, a flipping of the sign of the amplitude did sometimes also occur on a time scale of minutes. In contrast, single cells often changed between active periods of pulsing and periods of complete quietness. Also characteristic parameters like beat period and pulse amplitude were observed to be unstable. Finally, we combined the abilities of the AFM to image adherent single cells and to record locally beat amplitudes, to characterise the pulsing behaviour of single cells laterally resolved.

Electrically excitable normal rat kidney fibroblasts: A new model system for cell-semiconductor hybrids.

In testing various designs of cell-semiconductor hybrids, the choice of a suitable type of electrically excitable cell is crucial. Here normal rat kidney (NRK) fibroblasts are presented as a cell line, easily maintained in culture, that may substitute for heart or nerve cells in many experiments. Like heart muscle cells, NRK fibroblasts form electrically coupled confluent cell layers, in which propagating action potentials are spontaneously generated. These, however, are not associated with mechanical disturbances. Here we compare heart muscle cells and NRK fibroblasts with respect to action potential waveform, morphology, and substrate adhesion profile, using the whole-cell variant of the patch-clamp technique, atomic force microscopy (AFM), and reflection interference contrast microscopy (RICM), respectively. Our results clearly demonstrate that NRK fibroblasts should provide a highly suitable test system for investigating the signal transfer between electrically excitable cells and extracellular detectors, available at a minimum cost and effort for the experimenters.