Fatty acid distribution and polymorphism in solid lipid particles of milkfat and long chain omega-3 fatty acids.

Saturated fatty acid-containing lipids, such as milkfat, may protect long chain polyunsaturated fatty acids in fish oil when blended together into solid lipid particles (SLPs). One of the main challenges of SLPs is structural polymorphism, which can lead to expulsion of the protected component during prolonged storage. To investigate this phenomenon, the change in thermal and crystalline behaviours, and fatty acid distribution, were analysed in SLPs of fish oil and milkfat during storage at different temperatures for up to 28 days. X-ray diffraction analysis showed changes in molten and crystalline states occurred even at -22 °C. Room temperature (21 °C) storage led to more than 45% molten state but SLPs retained their initial shape. Confocal Raman Spectroscopy of the SLPs showed the distribution of fatty acids was not uniform, with 10 µm outermost layer of predominantly saturated fatty acids likely responsible for the intact SLP shape and stability of the core.

Disposable Coverslip for Rapid Throughput Screening of Malaria Using Attenuated Total Reflection Spectroscopy.

Malaria is considered to be one of the most catastrophic health issues in the whole world. Vibrational spectroscopy is a rapid, robust, label-free, inexpensive, highly sensitive, nonperturbative, and nondestructive technique with high diagnostic potential for the early detection of disease agents. In particular, the fingerprinting capability of attenuated total reflection spectroscopy is promising as a point-of-care diagnostic tool in resource-limited areas. However, improvements are required to expedite the measurements of biofluids, including the drying procedure and subsequent cleaning of the internal reflection element to enable high throughput successive measurements. As an alternative, we propose using an inexpensive coverslip to reduce the sample preparation time by enabling multiple samples to be collectively dried together under the same temperature and conditions. In conjunction with partial least squares regression, attenuated total reflection spectroscopy was able to detect and quantify the parasitemia with root mean square error of cross-validation and R2 values of 0.177 and 0.985, respectively. Here, we characterize an inexpensive, disposable coverslip for the high throughput screening of malaria parasitic infections and thus demonstrate an alternative approach to direct deposition of the sample onto the internal reflection element.

Ultraviolet/Visible and Near-Infrared Dual Spectroscopic Method for Detection and Quantification of Low-Level Malaria Parasitemia in Whole Blood.

The scourge of malaria infection continues to strike hardest against pregnant women and children in Africa and South East Asia. For global elimination, testing methods that are ultrasensitive to low-level ring-staged parasitemia are urgently required. In this study, we used a novel approach for diagnosis of malaria infection by combining both electronic ultraviolet-visible (UV/vis) spectroscopy and near infrared (NIR) spectroscopy to detect and quantify low-level (1-0.000001%) ring-staged malaria-infected whole blood under physiological conditions uisng Multiclass classification using logistic regression, which showed that the best results were achieved using the extended wavelength range, providing an accuracy of 100% for most parasitemia classes. Likewise, partial least-squares regression (PLS-R) analysis showed a higher quantification sensitivity (R2 = 0.898) for the extended spectral region compared to UV/vis and NIR (R2 = 0.806 and 0.556, respectively). For quantifying different-stage blood parasites, the extended wavelength range was able to detect and quantify all thePlasmodium falciparum accurately compared to testing each spectral component separately. These results demonstrate the potential of a combined UV/vis-NIR spectroscopy to accurately diagnose malaria-infected patients without the need for elaborate sample preparation associated with the existing mid-IR approaches.

Efficient long-range conduction in cable bacteria through nickel protein wires.

Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures.

Infrared Based Saliva Screening Test for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID-19. We propose a portable infrared spectrometer with purpose-built transflection accessory for rapid point-of-care detection of COVID-19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM-IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS-CoV-2 by RT-qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT-qPCR. Herein, we demonstrate a proof-of-concept high throughput infrared COVID-19 test that is rapid, inexpensive, portable and utilizes sample self-collection thus minimizing the risk to healthcare workers and ideally suited to mass screening.

A Near-Infrared "Matchbox Size" Spectrometer to Detect and Quantify Malaria Parasitemia.

New point-of-care diagnostic approaches for malaria that are sensitive to low parasitemia, easy to use in a field setting, and affordable are urgently required to meet the World Health Organization's objective of reducing malaria cases and related life losses by 90% globally on or before 2030. In this study, an inexpensive "matchbox size" near-infrared (NIR) spectrophotometer was used for the first time to detect and quantify malaria infection in vitro from isolated dried red blood cells using a fingerpick volume of blood. This the first study to apply a miniaturized NIR device to diagnose a parasitic infection and identify marker bands indicative of malaria infection in the NIR region. An NIR device has many advantages including wavelength accuracy and repeatability, speed, resolution, and a greatly improved signal-to-noise ratio compared to existing spectroscopic options. Using multivariate data analysis, we discriminated control red blood cells from infected cells and established the limit of detection of the technique. Principal component analysis displayed a good separation between the infected and uninfected RBCs, while partial least-squares regression analysis yielded a robust parasitemia prediction with root-mean-square error of prediction values of 0.446 and 0.001% for the higher and lower parasitemia models, respectively. The R2 values of the higher and lower parasitemia models were 0.947 and 0.931, respectively. Finally, an estimated parasitemia detection limit of 0.00001% and a qunatification limit of 0.001% was achieved; to ascertain the true efficacy of the technique for point-of-care screening, clinical studies using large patient numbers are required, which is the subject of future studies.

Infrared Spectroscopy of Blood.

The magnitude of infectious diseases in the twenty-first century created an urgent need for point-of-care diagnostics. Critical shortages in reagents and testing kits have had a large impact on the ability to test patients with a suspected parasitic, bacteria, fungal, and viral infections. New point-of-care tests need to be highly sensitive, specific, and easy to use and provide results in rapid time. Infrared spectroscopy, coupled to multivariate and machine learning algorithms, has the potential to meet this unmet demand requiring minimal sample preparation to detect both pathogenic infectious agents and chronic disease markers in blood. This focal point article will highlight the application of Fourier transform infrared spectroscopy to detect disease markers in blood focusing principally on parasites, bacteria, viruses, cancer markers, and important analytes indicative of disease. Methodologies and state-of-the-art approaches will be reported and potential confounding variables in blood analysis identified. The article provides an up to date review of the literature on blood diagnosis using infrared spectroscopy highlighting the recent advances in this burgeoning field.

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20-100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.

Vibrational Spectroscopy as a Sensitive Probe for the Chemistry of Intra-Phase Bacterial Growth.

Bacterial growth in batch cultures occurs in four phases (lag, exponential/log, stationary and death phase) that differ distinctly in number of different bacteria, biochemistry and physiology. Knowledge regarding the growth phase and its kinetics is essential for bacterial research, especially in taxonomic identification and monitoring drug interactions. However, the conventional methods by which to assess microbial growth are based only on cell counting or optical density, without any insight into the biochemistry of cells or processes. Both Raman and Fourier transform infrared (FTIR) spectroscopy have shown potential to determine the chemical changes occurring between different bacterial growth phases. Here, we extend the application of spectroscopy and for the first time combine both Raman and FTIR microscopy in a multimodal approach to detect changes in the chemical compositions of bacteria within the same phase (intra-phase). We found a number of spectral markers associated with nucleic acids (IR: 964, 1082, 1215 cm-1; RS: 785, 1483 cm-1), carbohydrates (IR: 1035 cm-1; RS: 1047 cm-1) and proteins (1394 cm-1, amide II) reflecting not only inter-, but also intra-phase changes in bacterial chemistry. Principal component analysis performed simultaneously on FTIR and Raman spectra enabled a clear-cut, time-dependent discrimination between intra-lag phase bacteria probed every 30 min. This demonstrates the unique capability of multimodal vibrational spectroscopy to probe the chemistry of bacterial growth even at the intra-phase level, which is particularly important for the lag phase, where low bacterial numbers limit conventional analytical approaches.

Rapid Approach for Detection of Antibiotic Resistance in Bacteria Using Vibrational Spectroscopy.

Here, we applied vibrational spectroscopy to investigate the drug response following incubation of S. aureus with oxacillin. The main focus of this work was to identify the chemical changes caused by oxacillin over time and to determine the feasibility of the spectroscopic approach to detect antimicrobial resistance. The oxacillin-induced changes in the chemical composition of susceptible bacteria, preceding (and leading to) the inhibition of growth, included an increase in the relative content of nucleic acids, alteration in the α-helical/ß-sheet protein ratio, structural changes in carbohydrates (observed via changes in the band at 1035 cm-1), and significant thickening of the cell wall. These observations enabled a dose-dependent discrimination between susceptible bacteria incubated with and without oxacillin after 120 min. In methicillin resistant strains, no spectral differences were observed between cells, regardless of drug exposure. These results pave the way for a new, rapid spectroscopic approach to detect drug resistance in pathogens, based on their early positive/negative drug response.

Influence of the Sample Preparation Method in Discriminating Candida spp. Using ATR-FTIR Spectroscopy.

Several studies have investigated the capacity of ATR-FTIR spectroscopy for fungal species discrimination. However, preparation methods vary among studies. This study aims to ascertain the effect of sample preparation on the discriminatory capacity of ATR-FTIR spectroscopy. Candida species were streaked to obtain colonies and spectra were collected from each preparation type, which included: (a) untreated colonies being directly transferred to the ATR crystal, (b) following washing and (c) following 24-h fixation in formalin. Spectra were pre-processed and principal component analysis (PCA) and K-means cluster analysis (KMC) were performed. Results showed that there was a clear discrimination between preparation types. Groups of spectra from untreated and washed isolates clustered separately due to intense protein, DNA and polysaccharide bands, whilst fixed spectra clustered separately due to intense polysaccharide bands. This signified that sample preparation had influenced the chemical composition of samples. Nevertheless, across preparation types, significant species discrimination was observed, and the polysaccharide (1200-900 cm-1) region was a common critical marker for species discrimination. However, different discriminatory marker bands were observed across preparation methods. Thus, sample preparation appears to influence the chemical composition of Candida samples; however, does not seem to significantly impact the species discrimination potential for ATR-FTIR spectroscopy.

Detection of Antimicrobial Resistance-Related Changes in Biochemical Composition of Staphylococcus aureus by Means of Atomic Force Microscopy-Infrared Spectroscopy.

The development of antimicrobial resistance (AMR) resulting from widespread antibiotic usage is occurring at an alarming pace, much faster than our understanding of the mechanisms behind resistance. Knowledge about resistance-related phenotypic and genotypic changes is critical for the development of new drugs. Here, we identify changes in the chemical composition of Staphylococcus aureus associated with the development of resistance to last resort drugs, vancomycin and daptomycin, using a novel, single cell, nanoscale technique, atomic force microscopy-infrared spectroscopy (AFM-IR), combined with chemometric analysis. We utilized paired clinical isolates, with the parent (susceptible) strain isolated prior to treatment and the daughter (resistant) strain obtained from the same patient after drug admission and clinical failure. We observed an increase in the amount of nonintracellular carbohydrates, indicating thickening or changes in the packing of the cell wall, as well as changes in the phospholipid content in relation to vancomycin resistance and daptomycin nonsusceptibility, respectively.

Whole-Organism Analysis by Vibrational Spectroscopy.

Vibrational spectroscopy has contributed to the understanding of biological materials for many years. As the technology has advanced, the technique has been brought to bear on the analysis of whole organisms. Here, we discuss advanced and recently developed infrared and Raman spectroscopic instrumentation to whole-organism analysis. We highlight many of the recent contributions made in this relatively new area of spectroscopy, particularly addressing organisms associated with disease with emphasis on diagnosis and treatment. The application of vibrational spectroscopic techniques to entire organisms is still in its infancy, but new developments in imaging and chemometric processing will likely expand in the field in the near future.

Determining the Age of Spoiled Milk from Dried Films Using Attenuated Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy.

Milk spoilage is an inevitable occurrence, which generates waste and can result in food poisoning. When milk spoils, the off-flavor and curdling are due to excessive proliferation of various bacteria which causes pH changes. Time, temperature, environment, and previous handling practice all affect the spoilage rate. There is a need for a fast reliable and accurate method that can identify in situ early spoilage of milk. Here we show the ability of attenuated total reflection Fourier transformed infrared spectroscopy (ATR FT-IR) in conjunction with multivariate data analysis to predict the age of milk. We found that dried films vastly increased the absorbance of important biomolecules within milk such as lipids, proteins, and sugars, compared to an unchanged milk sample. This allowed us to note the minor discrepancies that happened in spoilage. Spoilt milk was characterized by bands associated with increased lipids, proteins, and lactic acid and a decrease in carbohydrates. A semi-quantitative prediction model for milk spoilage at room temperature demonstrated ATR FT-IR spectroscopy can predict milk age with a root mean square error of prediction of approximately 14 h. The model showed poor performance in the first 40 h but the predictions improved significantly after this time. The experimental procedure proposed for detecting biomolecules within milk has the potential to improve common practice. Furthermore, the model would be a starting point for newer and improved methods to predict the spoilage date of milk, with potential commercial uses to reduce food waste and costs to the milk industry.

Raman spectroscopy as a tool for tracking cyclopropane fatty acids in genetically engineered Saccharomyces cerevisiae.

Cyclopropane fatty acids (CFAs) are a group of lipids with unique physical and chemical properties between those of saturated and monounsaturated fatty acids. The distinctive physicochemical characteristics of CFAs (e.g. oxidative stability, self-polymerization at high temperatures, etc.) results from the presence of a cyclopropane ring within their structure making them highly useful in industrial applications. CFAs are present in several species of plants and bacteria and are typically detected with standard lipid profiling techniques, such as gas or liquid chromatography. In this work we investigated several strains of S. cerevisiae, genetically modified to introduce the production of CFAs, in comparison to control strain using confocal Raman spectroscopy (CRS). The aim of our work was to demonstrate the potential of CRS not only to detect changes introduced due to the CFAs presence, but also to track CFAs within the cells. We present for the first time Raman and IR spectra of CFA standard (cis-9,10-methyleneoctadecanoic acid), completed with quantum chemical calculations and band assignment. We identified marker bands of CFA (e.g. 2992, 1222, 942 cm-1) attributed to the vibrations of the cyclopropyl ring. Furthermore, we analysed lipid bodies (LBs) from modified and control yeast using CRS imaging and identified multiple changes in size, number and composition of LBs from engineered strains. We observed a significant reduction in the degree of unsaturation of LBs using the ratio of bands located at 1660 cm-1 (ν(C[double bond, length as m-dash]C)) and 1448 cm-1 (δ(CH2)) in the modified cell lines. In addition, we were able to detect the presence of CFAs in LBs, using the established marker bands. CRS shows tremendous potential as technique to identify CFAs in lipid bodies providing a new way to track lipid production in genetically modified single yeast cells.

Single cell assessment of yeast metabolic engineering for enhanced lipid production using Raman and AFM-IR imaging.

BACKGROUND: Biodiesel is a valuable renewable fuel made from derivatized fatty acids produced in plants, animals, and oleaginous microbes. Of the latter, yeasts are of special interest due to their wide use in biotechnology, ability to synthesize fatty acids and store large amounts of triacylglycerols while utilizing non-food carbon sources. While yeast efficiently produce lipids, genetic modification and indeed, lipid pathway metabolic engineering, is usually required for cost-effective production. Traditionally, gas chromatography (GC) is used to measure fatty acid production and to track the success of a metabolic engineering strategy in a microbial culture; here we have employed vibrational spectroscopy approaches at population and single cell level of engineered yeast while simultaneously investigating metabolite levels in subcellular structures. RESULTS: Firstly, a strong correlation (r2 > 0.99) was established between Fourier transform infrared (FTIR) lipid in intact cells and GC analysis of fatty acid methyl esters in the differently engineered strains. Confocal Raman spectroscopy of individual cells carrying genetic modifications to enhance fatty acid synthesis and lipid accumulation revealed changes to the lipid body (LB), the storage organelle for lipids in yeast, with their number increasing markedly (up to tenfold higher); LB size was almost double in the strain that also expressed a LB stabilizing gene but considerable variation was also noted between cells. Raman spectroscopy revealed a clear trend toward reduced unsaturated fatty acid content in lipids of cells carrying more complex metabolic engineering. Atomic force microscopy-infrared spectroscopy (AFM-IR) analysis of individual cells indicated large differences in subcellular constituents between strains: cells of the most highly engineered strain had elevated lipid and much reduced carbohydrate in their cytoplasm compared with unmodified cells. CONCLUSIONS: Vibrational spectroscopy analysis allowed the simultaneous measurement of strain variability in metabolite production and impact on cellular structures as a result of different gene introductions or knockouts, within a lipid metabolic engineering strategy and these inform the next steps in comprehensive lipid engineering. Additionally, single cell spectroscopic analysis measures heterogeneity in metabolite production across microbial cultures under genetic modification, an emerging issue for efficient biotechnological production.

FT-IR- and Raman-based biochemical profiling of the early stage of pulmonary metastasis of breast cancer in mice.

The combination of FT-IR and Raman spectroscopies allowed the biochemical profiling of lungs in the early stage of pulmonary metastasis in the murine model of breast cancer. Histological staining was used as a reference. Raman spectroscopy was especially useful in the detection and semi-quantitative analysis of the vitamin A content in lung lipofibroblasts, whereas the IR technique provided semi-quantitative information on the contents of nucleic acids, carbohydrates including glycogen, and lipids as well as changes in the secondary structures of tissue proteins. Our spectroscopic results suggest that the early phase of metastasis in the lung is characterized by a decrease in the endogenous retinoid content in combination with a decrease in the content of glycogen and lipids.

In vivo atomic force microscopy-infrared spectroscopy of bacteria.

A new experimental platform for probing nanoscale molecular changes in living bacteria using atomic force microscopy-infrared (AFM-IR) spectroscopy is demonstrated. This near-field technique is eminently suited to the study of single bacterial cells. Here, we report its application to monitor dynamical changes occurring in the cell wall during cell division in Staphylococcus aureus using AFM to demonstrate the division of the cell and AFM-IR to record spectra showing the thickening of the septum. This work was followed by an investigation into single cells, with particular emphasis on cell-wall signatures, in several bacterial species. Specifically, mainly cell wall components from S. aureus and Escherichia coli containing complex carbohydrate and phosphodiester groups, including peptidoglycans and teichoic acid, could be identified and mapped at nanometre spatial resolution. Principal component analysis of AFM-IR spectra of six living bacterial species enabled the discrimination of Gram-positive from Gram-negative bacteria based on spectral bands originating mainly from the cell wall components. The ability to monitor in vivo molecular changes during cellular processes in bacteria at the nanoscale opens a new platform to study environmental influences and other factors that affect bacterial chemistry.