Jennifer A. Dougan

Enhanced oligonucleotide–nanoparticle conjugate stability using thioctic acid modified oligonucleotides

Metallic nanoparticles of gold functionalized with oligonucleotides conventionally use a terminal thiol modification and have been used in a wide range of applications. Although readily available, the oligonucleotide–nanoparticle conjugates prepared in this way suffer from a lack of stability when exposed to a variety of small molecules or elevated temperatures. If silver is used in place of gold then this lack of stability is even more pronounced. In this study we report the synthesis of highly stabilized oligonucleotide–nanoparticle conjugates using a simple oligonucleotide modification. A modified solid support was used to generate 3′-thioctic acid modified oligonucleotides by treatment with an N-hydroxysuccimidyl ester of thioctic acid. Unusually, both gold and silver nanoparticles have been investigated in this study and show that these disulphide-modified oligonucleotide probes offer significant improvements in nanoparticle stability when treated with dithiothreitol (DTT) compared with monothiol analogues. This is a significant advance in oligonucleotide–nanoparticle conjugate stability and for the first time allows silver nanoparticles to be prepared that are more stable than standard gold-thiol functionalized nanoparticles. This opens up the possibility of using silver nanoparticles functionalized with oligonucleotides as an alternative to gold.

Simultaneous detection and quantification of three bacterial meningitis pathogens by SERS

Bacterial meningitis is well known for its rapid onset and high mortality rates, therefore rapid detection of bacteria found in cerebral spinal fluid (CSF) and subsequent effective treatment is crucial. A new quantitative assay for detection of three pathogens that result in bacterial meningitis using a combination of lambda exonuclease (λ-exonuclease) and surface enhanced Raman scattering (SERS) is reported. SERS challenges current fluorescent-based detection methods in terms of both sensitivity and more importantly the detection of multiple components in a mixture, which is becoming increasingly more desirable for clinical diagnostics. λ-Exonuclease is a processive enzyme that digests one strand of double stranded DNA bearing a terminal 5′-phosphate group. The new assay format involves the simultaneous hybridisation of two complementary DNA probes (one containing a SERS active dye) to a target sequence followed by λ-exonuclease digestion of double stranded DNA and SERS detection of the digestion product. Three meningitis pathogens were successfully quantified in a multiplexed test with calculated limits of detection in the pico-molar range, eliminating the need for time consuming culture based methods that are currently used for analysis. Quantification of each individual pathogen in a mixture using SERS is complex, however, this is the first report that this is possible using the unique spectral features of the SERS signals combined with partial least squares (PLS) regression. This is a powerful demonstration of the ability of this SERS assay to be used for analysis of clinically relevant targets with significant advantages over existing approaches and offers the opportunity for future deployment in healthcare applications.

Detection of SERS active labelled DNA based on surface affinity to silver nanoparticles

Developments in specific DNA detection assays have been shown to be increasingly beneficial for molecular diagnostics and biological research. Many approaches use optical spectroscopy as an assay detection method and, owing to the sensitivity and molecular specificity offered, surface enhanced Raman scattering (SERS) spectroscopy has become a competitively exploited technique. This study utilises SERS to demonstrate differences in affinity of dye labelled DNA through differences in electrostatic interactions with silver nanoparticles. Results show clear differences in the SERS intensity obtained from single stranded DNA, double stranded DNA and a free dye label and demonstrate surface attraction is driven through electrostatic charges on the nucleotides and not the SERS dye. It has been further demonstrated that, through optimisation of experimental conditions and careful consideration of sequence composition, a DNA detection method with increased sample discrimination at lower DNA concentrations can be achieved.

Stable dye-labelled oligonucleotide-nanoparticle conjugates for nucleic acid detection

Metallic nanoparticles functionalized with oligonucleotides are used for a number of nucleic acid detection strategies. However, oligonucleotide-nanoparticle conjugates suffer from a lack of stability when exposed to certain conditions associated with DNA detection assays. In this study, we report the synthesis of thiol and thioctic acid-modified oligonucleotide gold nanoparticle (OGNs) conjugates functionalized with a dye label and varying spacer groups. The thioctic acid-modified conjugates exhibit increased stability when treated with dithiothreitol (DTT) compared to the more commonly used thiol modification. When the dye labelled oligonucleotide nanoparticle conjugates are exposed to the same conditions there is a pronounced increase in the stability for both thioctic acid and thiol modified sequences. These results open up the possibility of simply using a dye label to enhance the stability of oligonucleotide-nanoparticle conjugates in DNA detection assays where the enhanced stability of the conjugate system can be advantageous in more complex biological environments.

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).

Improving the understanding of oligonucleotide–nanoparticle conjugates using DNA-binding fluorophores

A novel fluorescent-based method for characterisation of oligonucleotide-nanoparticle conjugates (ONPCs) is reported. We exploit the ability of the double-stranded DNA-binding dye, SYBR Green I, to develop a powerful analytical tool to investigate the melting properties and hybridisation behavior of ONPCs as well as their corresponding DNA-mediated assemblies.

DNA Sequence Detection Using Surface-Enhanced Resonance Raman Spectroscopy in a Homogeneous Multiplexed Assay

Detection of specific DNA sequences is central to modern molecular biology and also to molecular diagnostics where identification of a particular disease is based on nucleic acid identification. Many methods exist, and fluorescence spectroscopy dominates the detection technologies employed with different assay formats. This study demonstrates the use of surface-enhanced resonance Raman scattering (SERRS) to detect specific DNA sequences when coupled with modified SERRS-active probes that have been designed to modify the affinity of double- and single-stranded DNA for the surface of silver nanoparticles resulting in discernible differences in the SERRS which can be correlated to the specific DNA hybridization event. The principle of the assay lies on the lack of affinity of double-stranded DNA for silver nanoparticle surfaces; therefore, hybridization of the probe to the target results in a reduction in the SERRS signal. Use of locked nucleic acid (LNA) residues in the DNA probes resulted in greater discri...

Nanoparticle assembly for sensitive DNA detection using SERRS

SERRS (surface-enhanced resonance Raman scattering) is a vibrational technique, whereby a relatively weak Raman scattering effect is enhanced through the use of a visible chromophore and a roughened metal surface. The direct analysis of DNA by SERRS requires the modification of a nucleic acid sequence to incorporate a chromophore, and adsorption of the modified sequence on to a roughened metal surface. Aggregated metallic nanoparticles are commonly used in the analysis of dye-labelled DNA by SERRS, allowing for detection levels that rival those gained from standard fluorescence-based techniques. In the present paper, we report on how SERRS can be exploited for the analysis of clinically relevant DNA samples. We also report on the ability of nanoparticles to aggregate as the result of a biologically significant event, as opposed to the use of an external charge-modifying agent. The self-assembly of metallic nanoparticles is shown to be a promising new technique in the move towards extremely sensitive methods of DNA analysis by SERRS.

Sensitive molecular diagnostics using surface-enhanced resonance Raman scattering (SERRS)

Surface enhanced resonance Raman scattering (SERRS) is an analytical technique with several advantages over competitive techniques in terms of improved sensitivity and multiplexing. We have made great progress in the development of SERRS as a quantitative analytical method, in particular for the detection of DNA. SERRS is an extremely sensitive and selective technique which when applied to the detection of labelled DNA sequences allows detection limits to be obtained which rival, and in most cases, are better than fluorescence. Here the conditions are explored which will enable the successful detection of DNA using SERRS. The enhancing surface which is used is crucial and in this case suspensions of nanoparticles were used as they allow quantitative behaviour to be achieved and allow analogous systems to current fluorescence based systems to be made. The aggregation conditions required to obtain SERRS of DNA are crucial and herein we describe the use of spermine as an aggregating agent. The nature of the label which is used, be it fluorescent, positively or negatively charged also effects the SERRS response and these conditions are again explored here. We have clearly demonstrated the ability to identify the components of a mixture of 5 analytes in solution by using two different excitation wavelengths and also of a 6-plex using data analysis techniques. These conditions will allow the use of SERRS for the detection of target DNA in a meaningful diagnostic assay.