Optimised Pre-Processing of Raman Spectra for Colorectal Cancer Detection Using High-Performance Computing.

Spectral pre-processing is an essential step in data analysis for biomedical diagnostic applications of Raman spectroscopy, allowing the removal of undesirable spectral contributions that could mask biological information used for diagnosis. However, due to the specificity of pre-processing for a given sample type and the vast number of potential pre-processing combinations, optimisation of pre-processing via a manual "trial and error" format is often time intensive with no guarantee that the chosen method is optimal for the sample type. Here we present the use of high-performance computing (HPC) to trial over 2.4 million pre-processing permutations to demonstrate the optimisation on the pre-processing of human serum Raman spectra for colorectal cancer detection. The effect of varying pre-processing order, using extended multiplicative scatter correction, spectral smoothing, baseline correction, binning and normalization was considered. Permutations were assessed on their ability to detect patients with disease using a random forest (RF) algorithm trained with 102 patients (510 spectra) and independently tested with a set of 439 patients (1317 spectra) in a primary care patient cohort. Optimising via HPC enables improved performance in diagnostic abilities, with sensitivity increasing by 14.6%, specificity increasing by 6.9%, positive predictive value increasing by 3.4%, and negative predictive value increasing by 2.4% when compared to a standard pre-processing optimisation. Ultimate values of these metrics are very important for diagnostic adoption, and once diagnostics demonstrate good accuracy these types of optimisations can make a significant difference to roll-out of a test and demonstrating advantages over existing tests. We also provide tips/recommendations for pre-processing optimisation without the use of HPC. From the HPC permutations, recommendations for appropriate parameter constraints for conducting a more basic pre-processing optimisation are also detailed, thus helping model development for researchers not having access to HPC.

Vibrational Spectroscopy: A Valuable Screening and Diagnostic Tool for Obstetric Disorders?

Preeclampsia (PE) is a common obstetric disorder typically affecting 2-8% of all pregnancies and can lead to several adverse obstetric outcomes for both mother and fetus with the greatest burden of severe outcomes in low middle-income countries (LMICs), therefore, screening for PE is vital. Globally, screening is based on maternal characteristics and medical history which are nonspecific for the disorder. In 2004, the World Health Organization acknowledged that no clinically useful test was able to predict the onset of PE, which prompted a universal search for alternative means of screening. Over the past decade or so, emphasis has been placed on the use of maternal characteristics in conjunction with biomarkers of disease combined into predictive algorithms, however these are yet to transition into the clinic and are cost prohibitive in LMICs. As a result, the screening paradigm for PE remains unchanged. It is evident that novel approaches are needed. Vibrational spectroscopy, specifically Raman spectroscopy and Fourier-transform infrared spectroscopy (FTIR), could provide better alternatives suited for implementation in low resource settings as no specialized reagents are required for conventional approaches and there is a drive to portable platforms usable in both urban and rual community settings. These techniques are based on light scattering and absorption, respectively, allowing detailed molecular analysis of samples to produce a unique molecular fingerprint of diseased states. The specificity of vibrational spectroscopy might well make it suited for application in other obstetric disorders such as gestational diabetes mellitus and obstetric cholestasis. In this review, we summarize current approaches sought as alternatives to current screening methodologies and introduce how vibrational spectroscopy could offer superior screening and diagnostic paradigms in obstetric care. Additionally, we propose a real benefit of such tools in LMICs where limited resources battle the higher prevalence of obstetric disorders.

A high-throughput serum Raman spectroscopy platform and methodology for colorectal cancer diagnostics.

Vibrational spectroscopic techniques such as Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR) have huge potential for the analysis of biological specimens. The techniques allow the user to gain label-free, non-destructive biochemical information about a given sample. Previous studies using vibrational spectroscopy with the specific application of diagnosing colorectal diseases such as cancer have mainly focused on in vivo or in vitro studies of tissue specimens using microscopy or probe based techniques. There have been few studies of vibrational spectroscopic techniques based on the analysis of blood serum for the advancement of colorectal cancer diagnostics. With growing interest in the field of liquid biopsies, this study presents the development of a high-throughput (HT) serum Raman spectroscopy platform and methodology and compares dry and liquid data acquisition of serum samples. This work considers factors contributing to translatability of the methodologies such as HT design, inter-user variability and sample handling effects on diagnostic capability. The HT Raman methods were tested on a pilot dataset of serum from 30 cancer patients and 30 matched control patients using statistical analysis via cross-validated PLS-DA with a maximum achieved a sensitivity of 83% and specificity of 83% for detecting colorectal cancer.

Investigation into the effects of surface stripping ZnO nanosheets.

ZnO nanosheets are polycrystalline nanostructures that are used in devices including solar cells and gas sensors. However, for efficient and reproducible device operation and contact behaviour the conductivity characteristics must be controlled and surface contaminants removed. Here we use low doses of argon bombardment to remove surface contamination and make reproducible lower resistance contacts. Higher doses strip the surface of the nanosheets altering the contact type from near-ohmic to rectifying by removing the donor-type defects, which photoluminescence shows to be concentrated in the near-surface. Controlled doses of argon treatments allow nanosheets to be customised for device formation.

Role of Raman spectroscopy and surface enhanced Raman spectroscopy in colorectal cancer.

Colorectal cancer (CRC) is the fourth most common cancer in the United Kingdom and is the second largest cause of cancer related death in the United Kingdom after lung cancer. Currently in the United Kingdom there is not a diagnostic test that has sufficient differentiation between patients with cancer and those without cancer so the current referral system relies on symptomatic presentation in a primary care setting. Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) are forms of vibrational spectroscopy that offer a non-destructive method to gain molecular information about biological samples. The techniques offer a wide range of applications from in vivo or in vitro diagnostics using endoscopic probes, to the use of micro-spectrometers for analysis of biofluids. The techniques have the potential to detect molecular changes prior to any morphological changes occurring in the tissue and therefore could offer many possibilities to aid the detection of CRC. The purpose of this review is to look at the current state of diagnostic technology in the United Kingdom. The development of Raman spectroscopy and SERS in clinical applications relation for CRC will then be discussed. Finally, future areas of research of Raman/SERS as a clinical tool for the diagnosis of CRC are also discussed.

Single-walled carbon nanotubes: differential genotoxic potential associated with physico-chemical properties.

Single-walled carbon nanotubes (SWCNTs) have recently attracted great attention because of their fibrous structure and high aspect ratio. Here the genotoxic potential of 400-800 nm, 1-3 µm and 5-30 µm SWCNT with respect to their geometry and surface characteristics was studied. Following thorough physico-chemical characterisation, human bronchial epithelial (BEAS-2B) and lymphoblastoid (MCL-5) cells were treated with SWCNT for 24 or 48 h. This showed significant increases in micronucleus frequency in a time- and dose-dependent manner in both cell types in the absence of cytotoxicity. Over the same dose range, only 1-3 µm SWCNT gave rise to significant increases in hprt point mutations at doses ≥25 µg/ml. Cellular 2,7-dichlorodihydrofluoresceindiacetate (DCFH-DA) fluorescence assay and RT-PCR for oxidative pathway gene profiling revealed a possible oxidative mechanism for the genotoxicity observed in the 1-3 µm SWCNT. Consequently, this study has demonstrated that SWCNT genotoxicity is dependent on its secondary structure under experimental conditions and oxidative stress alone cannot account for the observed damage.

Quantum dots for multiplexed detection and characterisation of prostate cancer cells using a scanning near-field optical microscope.

In this study scanning near-field optical microscopy (SNOM) has been utilised in conjunction with quantum dot labelling to interrogate the biomolecular composition of cell membranes. The technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. The technique has been applied to investigate cell-cell adhesion in human epithelial cells. This has been realised through immunofluorescence labelling of the cell-cell adhesion protein E-cadherin. Moreover, a dual labelling protocol has been optimised to facilitate a comparative study of the adhesion mechanisms and the effect of aberrant adhesion protein expression in both healthy and cancerous epithelial cells. This study reports clear differences in the morphology and phenotype of healthy and cancerous cells. In healthy prostate epithelial cells (PNT2), E-cadherin was predominantly located around the cell periphery and within filopodial extensions. The presence of E-cadherin appeared to be enhanced when cell-cell contact was established. In contrast, examination of metastatic prostate adenocarcinoma cells (PC-3) revealed no E-cadherin labelling around the periphery of the cells. This lack of functional E-cadherin in PC-3 cells coincided with a markedly different morphology and PC-3 cells were not found to form close cell-cell associations with their neighbours. We have demonstrated that with a fully optimised sample preparation methodology, multiplexed quantum dot labelling in conjunction with SNOM imaging can be successfully applied to interrogate biomolecular localisation within delicate cellular membranes.

Fluorescence imaging and investigations of directly labelled chromosomes using scanning near-field optical microscopy.

Scanning near-field optical microscopy (SNOM) has been successfully employed to generate high resolution (<100nm) fluorescence images of directly tagged human chromosomes. Direct tagging, fluorescence in-situ hybridisation processes (with and without amplification) are investigated and their fluorescence response to near-field excitation are compared. Using the simultaneous topography mode of SNOM, chromosome morphology was seen to differ as a result of the two processes; with chromatin collapse more extensive when the amplified direct tagging procedure was used. The results are discussed in the context of developing locus specific direct tags together with high resolution SNOM imaging for the observation of chromosome aberrations.