Development of biologically relevant assays for the detection of disease DNA using SERS

DNA is the fundamental material responsible for storing the genetic coding required for the development of all living organisms. Since its discovery, there has been an intense amount of research into biorecognition events and the detection of DNA sequences coding for specific diseases. The development of the polymerase chain reaction (PCR) involved the amplification of small quantities of DNA allowing for subsequent analysis. However, fluorescence-based methods such as PCR have their limitations, for example the difficulties encountered when detecting multiple targets simultaneously. Therefore, there is a need for novel techniques that overcome these limitations associated with fluorescence-based methods. This research involves the use of SERS for the multiplex detection of target DNA, investigating the possible interactions between fluorescent dyes and DNA and SERS analysis of G-quadruplex formations. A SERS-based detection assay was designed for the simultaneous detection of three bacterial meningitis pathogens; Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae. By using SERS instead of fluorescence-based methods, multiplex detection was readily achieved and by using chemometric analysis it was the first report of pathogen quantification post-assay. To gain an understanding into interactions that can occur between fluorescent dyes (FAM and TAMRA), DNA and spermine, fluorescence and SERS studies were undertaken. Fluorescent studies gave an insight into the interactions that happen off the nanoparticle surface, while the SERS studies demonstrated the competitive interactions that occur between the nanoparticle surface and the two fluorescent dyes. These studies highlighted the consideration needed when selecting fluorescent dyes and target DNA sequences when designing a multiplex SERS assay. SERS was then applied to the detection of G-quadruplex formation. Previous reports used fluorescence-based methods, for example FID assays. Three ligands that selectively bind to and stabilise G-quadruplex DNA, previously used in fluorescence studies, were used and shown to have the ability to aggregate nanoparticles and act as Raman reporters. These ligands allowed for the design of the "on to off" SERS analysis of three G-quadruplex sequences.DNA is the fundamental material responsible for storing the genetic coding required for the development of all living organisms. Since its discovery, there has been an intense amount of research into biorecognition events and the detection of DNA sequences coding for specific diseases. The development of the polymerase chain reaction (PCR) involved the amplification of small quantities of DNA allowing for subsequent analysis. However, fluorescence-based methods such as PCR have their limitations, for example the difficulties encountered when detecting multiple targets simultaneously. Therefore, there is a need for novel techniques that overcome these limitations associated with fluorescence-based methods. This research involves the use of SERS for the multiplex detection of target DNA, investigating the possible interactions between fluorescent dyes and DNA and SERS analysis of G-quadruplex formations. A SERS-based detection assay was designed for the simultaneous detection of three bacterial meningitis pathogens; Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae. By using SERS instead of fluorescence-based methods, multiplex detection was readily achieved and by using chemometric analysis it was the first report of pathogen quantification post-assay. To gain an understanding into interactions that can occur between fluorescent dyes (FAM and TAMRA), DNA and spermine, fluorescence and SERS studies were undertaken. Fluorescent studies gave an insight into the interactions that happen off the nanoparticle surface, while the SERS studies demonstrated the competitive interactions that occur between the nanoparticle surface and the two fluorescent dyes. These studies highlighted the consideration needed when selecting fluorescent dyes and target DNA sequences when designing a multiplex SERS assay. SERS was then applied to the detection of G-quadruplex formation. Previous reports used fluorescence-based methods, for example FID assays. Three ligands that selectively bind to and stabilise G-quadruplex DNA, previously used in fluorescence studies, were used and shown to have the ability to aggregate nanoparticles and act as Raman reporters. These ligands allowed for the design of the "on to off" SERS analysis of three G-quadruplex sequences

Multiplex in vitro detection using SERS

The ability to detect multiple disease-related targets from a single biological sample in a quick and reliable manner is of high importance in diagnosing and monitoring disease. The technique known as surface enhanced Raman scattering (SERS) has been developed for the simultaneous detection of multiple targets present in biological samples. Advances in the SERS method have allowed for the sensitive and specific detection of biologically relevant targets, such as DNA and proteins, which could be useful for the detection and control of disease. This review focuses on the strengths of SERS for the detection of target molecules from complex mixtures and the clinical relevance of recent work combining SERS with multiplexed detection of biological targets

Bayesian modelling and quantification of Raman spectroscopy

Raman spectroscopy can be used to identify molecules such as DNA by the characteristic scattering of light from a laser. It is sensitive at very low concentrations and can accurately quantify the amount of a given molecule in a sample. The presence of a large, nonuniform background presents a major challenge to analysis of these spectra. To overcome this challenge, we introduce a sequential Monte Carlo (SMC) algorithm to separate each observed spectrum into a series of peaks plus a smoothly-varying baseline, corrupted by additive white noise. The peaks are modelled as Lorentzian, Gaussian, or pseudo-Voigt functions, while the baseline is estimated using a penalised cubic spline. This latent continuous representation accounts for differences in resolution between measurements. The posterior distribution can be incrementally updated as more data becomes available, resulting in a scalable algorithm that is robust to local maxima. By incorporating this representation in a Bayesian hierarchical regression model, we can quantify the relationship between molecular concentration and peak intensity, thereby providing an improved estimate of the limit of detection, which is of major importance to analytical chemistry

Bacterial meningitis pathogens identified in clinical samples using a SERS DNA detection assay

This communication reports the first demonstration of the detection of Streptococcus pneumoniae and Neisseria meningitidis bacterial DNA extracted from anonymous patient CSF samples and assesses the applicability of a previously developed SERS based DNA detection assay as a platform for the detection of multiple meningitis pathogens from clinical samples

Bioanalytical measurements enabled by surface-enhanced Raman scattering (SERS) probes

Since its discovery in 1974, surface-enhanced Raman scattering (SERS) has gained momentum as an important tool in analytical chemistry. SERS is used widely for analysis of biological samples, ranging from in vitro cell culture models, ex vivo tissue and blood samples, and direct in vivo application. New insights have been gained into biochemistry, with an emphasis on biomolecule detection, from small molecules such as glucose and amino acids to larger biomolecules such as DNA, proteins, and lipids. These measurements have increased our understanding of biological systems, and significantly, they have improved diagnostic capabilities. SERS probes display unique advantages in their detection sensitivity and multiplexing capability. We highlight key considerations that are required when performing bioanalytical SERS measurements, including sample preparation, probe selection, instrumental configuration, and data analysis. Some of the key bioanalytical measurements enabled by SERS probes with application to in vitro, ex vivo, and in vivo biological environments are discussed

Detection of cortisol in serum using quantitative resonance Raman spectroscopy

Measurement of cortisol in serum is used commonly as an indicator of stress and disease. Conventional analytical techniques have limited utility given that they remain largely laboratory based, they do not directly measure the deemed biologically active free cortisol, and there is no robust correlation between the free cortisol measurements within serum and saliva. It would therefore be desirable to measure both the free and total cortisol readily within the same matrix in a portable device in the field or at the bedside. This paper demonstrates the utility of a portable Raman approach to measure both the biological active free cortisol as well as total cortisol in human serum, compared to a laboratory-based chemiluminescence analysis technique. This alternative portable Raman method produced results that were consistent with results obtained from previous methods, which has the potential for further miniaturisation for point of test applications

You Missed a Spot! Disinfecting Shared Mobile Phones

The use of portable mobile devices has facilitated timely communication among healthcare team members. It\u27s now a common practice for hospital-owned mobile phones to be shared among healthcare employees from shift to shift. Despite the benefit of increased, timely communication between caregivers, sharing mobile devices can lead to the spread of healthcare-associated infections (HAIs). This article looks at the efficacy of two types of cleaning products on shared mobile phones carried by RNs at a 489-bed, Magnet-designated, Midwestern regional medical center. The cleaning methods evaluated were 70% isopropyl alcohol wipes and ethyl alcohol wipes

Bacteria on Shared Mobile Phones Can Lead to Infections

It\u27s now a common practice for hospital-owned mobile phones to be shared among healthcare employees from shift to shift. Despite the benefit of increased, timely communication between caregivers, sharing mobile devices can lead to the spread of healthcare-associated infections (HAIs) if they aren\u27t properly disinfected. The Guidelines for Disinfection and Sterilization in Healthcare Facilities describe non-critical environmental surfaces as items that are frequently touched by the hand and may pose a risk of secondary infection transmission