Robert J. Stokes

Introducing dip pen nanolithography as a tool for controlling stem cell behaviour: unlocking the potential of the next generation of smart materials in regenerative medicine.

Reproducible control of stem cell populations, regardless of their original source, is required for the true potential of these cells to be realised as medical therapies, cell biology research tools and in vitro assays. To date there is a lack of consistency in successful output when these cells are used in clinical trials and even simple in vitro experiments, due to cell and material variability. The successful combination of single chemistries in nanoarray format to control stem cell, or any cellular behaviour has not been previously reported. Here we report how homogenously nanopatterned chemically modified surfaces can be used to initiate a directed cellular response, particularly mesenchymal stem cell (MSC) differentiation, in a highly reproducible manner without the need for exogenous biological factors and heavily supplemented cell media. Successful acquisition of these data should lead to the optimisation of cell selective properties of materials, further enhancing the role of nanopatterned substrates in cell biology and regenerative medicine. The successful design and comparison of homogenously molecularly nanopatterned surfaces and their direct effect on human MSC adhesion and differentiation are reported in this paper. Planar gold surfaces were patterned by dip pen nanolithography (DPN) to produce arrays of nanodots with optimised fixed diameter of 70 nanometres separated by defined spacings, ranging from 140 to 1000 nm with terminal functionalities of simple chemistries including carboxyl, amino, methyl and hydroxyl. These nanopatterned surfaces exhibited unprecedented control of initial cell interactions and subsequent control of cell phenotype and offer significant potential for the future.

Surface-enhanced Raman scattering spectroscopy as a sensitive and selective technique for the detection of folic acid in water and human serum.

Surface-enhanced Raman scattering (SERS) is shown to give linear and sensitive concentration-dependent detection of folic acid using silver nanoparticles created via ethylene-diaminetetraacetic acid (EDTA) reduction. Optical detection by SERS overcomes the primary limitation of photodissociation encountered during the application of other shorter wavelength ultraviolet (UV)/near-UV techniques such as fluorescence based microscopy. The SERS approach in water-based samples was demonstrated and optimized using several longer wavelengths of excitation (514.5, 632.8, and 785 nm). Excitation in the green (514.5 nm) was found to achieve the best balance between photodissociation and SERS efficiency. Linear concentration dependence was observed in the range of 0.018 to 1 μM. The importance of folic acid in a clinical setting and the potential applications of this technique in a biological environment are highlighted. We demonstrate the potential to transfer this technique to real biological samples by the detection of folic acid in human serum samples by SERS.

Synthesis of unique nanostructures with novel optical properties using oligonucleotide mixed-metal nanoparticle conjugates

Oligonucleotide-gold-nanoparticle (OGN) conjugates are now recognized as both powerful tools for ultrasensitive detection and building blocks for the controlled creation of nanostructures (see image). The first use of both OGN and oligonucleotide-silver nanoparticle (OSN) conjugates are used to create mixed-metal nanostructures with novel optical properties. (Abstract from: http://www3.interscience.wiley.com/journal/121357719/abstract)

Immunoassay for P38 MAPK using surface enhanced resonance Raman spectroscopy (SERRS)

A micro-bead sandwich assay for P38 mitogen-activated protein kinase using surface enhanced resonance Raman spectroscopy (SERRS) detection is reported. Monoclonal capture antibodies were immobilised on a solid phase of magnetic micro-beads with secondary detection using a rhodamine-labelled antibody. Quantitative SERRS detection of the secondary antibody was possible with a limit of detection of 9.5 x 10(-12) mol dm(-3). The sandwich assay was quantitative and sensitive to 6 ng ml(-1). The mechanism of the SERRS detection in the immunoassay was investigated. The addition of SERRS aggregating agents causes the dissociation of the immuno-complex from the magnetic beads. Scanning electron microscopy images indicate that the colloidal suspension rather than adsorbed silver nanoparticles on the beads provide the SERRS signals, that the aggregate size is partially controlled and that there is some inhomogeneity in the distribution of organic matter on the nanoscale.

In situ detection of pterins by SERS.

Surface enhanced Raman scattering (SERS) has been used to detect specific pterin molecules at sub-nanomolar concentrations. SERS is fast becoming a widely used technique for the sensitive and specific detection of multiple analytes. The information-rich and concentration-dependent spectra obtained from SERS make the technique ideally placed for high speed, low cost analysis of almost any analyte. Further, to show the feasibility of SERS in the detection of biologically relevant targets, a synthetic pterin analogue of the naturally occurring pterin cofactor, tetrahydrobiopterin, has been detected at a series of concentrations and the method used for the successful detection of the synthetic pterin in mouse serum. In this analysis, spectroscopic collection was optimized for water-based pteridine derivatives using two visible wavelengths of excitation (514.5 and 632.8 nm) and differing mesoscopic metal nanoparticles allowing the limits of detection to be calculated.

Functionalized nanoparticles for bioanalysis by SERRS

Metallic nanoparticles can be used as basic materials for a wide variety of purposes including building blocks for nanoassemblies, substrates for enhanced spectroscopies such as fluorescence and Raman and as labels for biomolecules. In the present paper, we report how silver and gold nanoparticles can be functionalized with specific biomolecular probes to interact in a specific manner with a target molecule to provide a change in the properties of the nanoparticles which can be measured to indicate the molecular recognition event. Examples of this approach include DNA hybridization to switch on SERRS (surface-enhanced resonance Raman scattering) when a specific target sequence is present, the use of nanoparticles for in vivo SERRS imaging and the use of nanoparticles functionalized with antibodies to provide a new type of immunoassay. These examples indicate how nanoparticles can be used to provide highly sensitive and informative data from a variety of biological systems when used in combination with SERRS.

Analysis of intracellular enzyme activity by surface enhanced Raman scattering

Dysfunctional intracellular enzymatic activity is believed to be an underlying cause of a myriad of diseases. We present the first use of surface enhanced Raman scattering (SERS) as a detection technique capable of reporting intracellular activity of a specific enzyme. Careful choice of reagents allowed the preparation of high resolution cellular activity maps highlighting the specific conversion of the commonly used ELISA reagent 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal), by wild type β-galactosidase enzymes. Further, through co-addition of X-Gal substrate and inhibitors we were able to demonstrate that intracellular substrate conversion occurred predominantly through an enzymatically specific pathway. The data presented therefore supports the application of SERS probes as sensitive, specific sensors of biochemical activity and demonstrates the use of SERS probes for the first time as beacons capable of high resolution subcellular localisation of native enzymes.

Functionalized nanoparticles for nucleic acid sequence analysis using optical spectroscopies.

SERRS (surface-enhanced resonance Raman scattering) is a vibrational spectroscopy which allows extremely sensitive and selective detection of labelled DNA sequences with detection limits which rival, and in most cases surpass, that of fluorescence. SERRS relies on a visible chromophore adsorbing on to an enhancing surface. DNA itself is not SERRS-active, as it lacks a suitable visible chromophore and has poor adsorption properties on to the surfaces used for enhancement. The surface normally used for enhancement in these sorts of studies are metallic nanoparticles and, through modification of DNA probes by the addition of suitable SERRS labels, signals can be obtained that are highly sensitive and very selective. The aggregation state of the nanoparticles is critical to the sensitivity, and, in the present paper, we show how straightforward detection of labelled DNA probes can be achieved using SERRS in a quantitative manner and with a variety of different commercially available labels. In a second approach, we show how the properties of aggregation to turn on the SERRS effect can be exploited through DNA hybridization to give identification of a particular DNA sequence. This approach lends itself to closed-tube formats and is a promising way forward for molecular diagnostics using SERRS.

Imaging inflammation in real time—future of nanoparticles

The detection of subclinical early inflammation in autoimmune diseases is an important but currently technically demanding approach to direct initial diagnosis and subsequent choice of therapy. Recent advances in imaging using NP provides the potential to detect cellular recruitment, vascular activation or leakage at a subclinically stage of disease and may provide predictive “biomarkers” of future pathogenesis. The NP used are either untargeted and taken up by phagocytic cells, or are linked to a ligand, targeting localisation to the site of inflammation. Techniques, varying from MRI and fluorescence to Raman spectroscopy are being employed. In this short review, we summarise many of the recent developments in the field of NP imaging related to inflammation.

Dip-pen nanolithography and serrs as synergic techniques

We demonstrate the powerful combination of dip-pen nanolithography (DPN) performed on non-flat plasmonic gold surfaces and subsequent detection by surface enhanced resonance Raman scattering (SERRS).