Simple and rapid peptide nanoprobe biosensor for the detection of Legionellaceae.

This study demonstrates the development of a sensitive, specific, and quantitative peptide-based nanoprobe prototype assay for the detection of Legionellaceae in a simple way and in a short time. In this work, proteases present in the culture supernatants of Legionella spp. were used as a biomarker. Fluorogenic peptide substrates, specific to Legionella strains culture supernatant proteases, were identified. Peptidases produced a significant increase in the fluorescence intensity following the cleavage of the dipeptide fluorogenic substrates. The specific substrates were identified and coupled with carboxyl-terminated nano-magnetic particles (NMPs). On the other hand, the C-terminal was conjugated with the cysteine residue to covalently integrate with a gold sensing platform via the Au-S linkage. Four different sensors were fabricated from the four specific substrates, which were treated with the protesase of six different species of Legionella. In the presence of specific protease, the peptide sequence is digested and the magnetic nanobeads moved out of the gold surface, resulting in the apparence of gold color. One of the nanoprobes sensitivity detects as low as 60 CFU mL-1 of Legionella anisa, Legionella micdadei, and Fluoribacter dumoffii. The cross-reactivity of the sensors was tested using other closely associated bacterial species and no significant cross-reactivity of the sensors was found. It is envisaged that this assay could be useful for screening purposes or might be supportive for the fast and easy detection of Legionella protease activity for water monitoring purposes.

Translation and Validation of the TOR-BSST© into Brazilian Portuguese for Adults with Stroke.

Brazil has a higher rate of dysphagia in stroke patients compared to developed countries, but does not have a fully validated method for early identification of dysphagia in this population. The aim of this study is to translate the TOR-BSST© into Brazilian Portuguese and assess the newly translated version for reliability and validity with Brazilian adult patients with stroke. The translation of the TOR-BSST© followed a multi-step process, according to the International Quality of Life Assessment project. For validation, we included patients with age ≥ 18 years and stroke diagnosis confirmed by neuroimaging and tolerance for videofluoroscopic swallowing assessment. The BR-PTfinal TOR-BSST© was administered by two trained screeners within two hours of videofluoroscopy. All assessors were independent and blinded. Estimates for reliability used the intraclass correlation coefficient (ICC) and for accuracy both sensitivity (SN) and negative predictive (NP) values were used, along with 95% confidence intervals (CI). Sixty patients were enrolled and tested for a mean (SD) of 14.4 (6.9) days from last seen normal. Of all the patients, 41 (68.3%) failed the BR-PTfinal TOR-BSST© and 21 (35%) were scored to have dysphagia on videofluoroscopy, of which 11 (52.4%) had mild dysphagia. The overall reliability between screeners was satisfactory (ICC = 0.59; 95% CI 0.32 to 0.76). The SN and NP values for the BR-PTfinal TOR-BSST© were 85.7% (95% CI 0.62-0.96) and 84.2% (95% CI 0.72-0.95), respectively. The TOR-BSST© was successfully translated to Brazilian Portuguese with the BR-PTfinal TOR-BSST© proven to have high sensitivity and negative predictive values when compared to gold standard videofluoroscopy.

Noise Sources and Requirements for Confocal Raman Spectrometers in Biosensor Applications.

Raman spectroscopy probes the biochemical composition of samples in a non-destructive, non-invasive and label-free fashion yielding specific information on a molecular level. Nevertheless, the Raman effect is very weak. The detection of all inelastically scattered photons with highest efficiency is therefore crucial as well as the identification of all noise sources present in the system. Here we provide a study for performance comparison and assessment of different spectrometers for confocal Raman spectroscopy in biosensor applications. A low-cost, home-built Raman spectrometer with a complementary metal-oxide-semiconductor (CMOS) camera, a middle price-class mini charge-coupled device (CCD) Raman spectrometer and a laboratory grade confocal Raman system with a deeply cooled CCD detector are compared. It is often overlooked that the sample itself is the most important "optical" component in a Raman spectrometer and its properties contribute most significantly to the signal-to-noise ratio. For this purpose, different representative samples: a crystalline silicon wafer, a polypropylene sample and E. coli bacteria were measured under similar conditions using the three confocal Raman spectrometers. We show that biosensor applications do not in every case profit from the most expensive equipment. Finally, a small Raman database of three different bacteria species is set up with the middle price-class mini CCD Raman spectrometer in order to demonstrate the potential of a compact setup for pathogen discrimination.

Rapid detection of the aspergillosis biomarker triacetylfusarinine C using interference-enhanced Raman spectroscopy.

Triacetylfusarinine C (TAFC) is a siderophore produced by certain fungal species and might serve as a highly useful biomarker for the fast diagnosis of invasive aspergillosis. Due to its renal elimination, the biomarker is found in urine samples of patients suffering from Aspergillus infections. Accordingly, non-invasive diagnosis from this easily obtainable body fluid is possible. Within our contribution, we demonstrate how Raman microspectroscopy enables a sensitive and specific detection of TAFC. We characterized the TAFC iron complex and its iron-free form using conventional and interference-enhanced Raman spectroscopy (IERS) and compared the spectra with the related compound ferrioxamine B, which is produced by bacterial species. Even though IERS only offers a moderate enhancement of the Raman signal, the employment of respective substrates allowed lowering the detection limit to reach the clinically relevant range. The achieved limit of detection using IERS was 0.5 ng of TAFC, which is already well within the clinically relevant range. By using an extraction protocol, we were able to detect 1.4 µg/mL TAFC via IERS from urine within less than 3 h including sample preparation and data analysis. We could further show that TAFC and ferrioxamine B can be clearly distinguished by means of their Raman spectra even in very low concentrations.

Raman Signal Enhancement Tunable by Gold-Covered Porous Silicon Films with Different Morphology.

The ease of fabrication, large surface area, tunable pore size and morphology as well surface modification capabilities of a porous silicon (PSi) layer make it widely used for sensoric applications. The pore size of a PSi layer can be an important parameter when used as a matrix for creating surface-enhanced Raman scattering (SERS) surfaces. Here, we evaluated the SERS activity of PSi with pores ranging in size from meso to macro, the surface of which was coated with gold nanoparticles (Au NPs). We found that different pore diameters in the PSi layers provide different morphology of the gold coating, from an almost monolayer to 50 nm distance between nanoparticles. Methylene blue (MB) and 4-mercaptopyridine (4-MPy) were used to describe the SERS activity of obtained Au/PSi surfaces. The best Raman signal enhancement was shown when the internal diameter of torus-shaped Au NPs is around 35 nm. To understand the role of plasmonic resonances in the observed SERS spectrum, we performed electromagnetic simulations of Raman scattering intensity as a function of the internal diameter. The results of these simulations are consistent with the obtained experimental data.

Nanobore fiber focus trap with enhanced tuning capabilities.

Confinement in fiber traps with two optical fibers facing one another relies on balancing the optical forces originating from the interaction of a scattering micro-object with the light beams delivered through the fibers. Here we demonstrate a novel type of dual fiber trap that involves the use of nanobore fibers, having a nano-channel located in the center of their fiber cores. This nano-element leads to a profound redistribution of the optical intensity and to considerably higher field gradients, yielding a trapping potential with greatly improved tuning properties compared to standard step-index fiber types. We evaluate the trap performance as a function of the fiber separation and find substantially higher stiffness for the nanobore fiber trap, especially in the range of short inter-fiber separations, while intermediate distances exhibit axial stiffness below that of the standard fiber. The results are in agreement with theoretical predictions and reveal that the exploitation of nanobore fibers allows for combinations of transverse and axial stiffness that cannot be accessed with common step-index fibers.

Interference-Enhanced Raman Spectroscopy as a Promising Tool for the Detection of Biomolecules on Raman-Compatible Surfaces.

Raman spectroscopy in combination with appropriate sample preparation strategies, for example, enrichment of bacteria on metal surfaces, has been proven to be a promising approach for rapidly diagnosing infectious diseases. Unfortunately, the fabrication of the required chip substrates is usually very challenging due to the lack of feasible instruments that can be used for quality control in the surface modification process. The intrinsically weak Raman signal of the biomolecules, employed for the enrichment of the micro-organisms on the chip surface, does not allow for monitoring of the successful immobilization by means of a Raman spectroscopic approach. Within this contribution, we demonstrate how a simple modification of a plain aluminum surface enables enhancement (or a decrease, if desired) of the Raman signal of molecules deposited on that surface. The manipulation of the Raman signal strength is achieved via exploiting interference effects that occur, if the highly reflective aluminum surface is modified with thin layers of transparent dielectrics like aluminum oxide. The thicknesses of these layers were determined by theoretical considerations and calculations. For the first time, it is shown that the interference effects can be used for the detection of biomolecules as well by investigating the siderophore ferrioxamine B. The observed degree of enhancement was approximately 1 order of magnitude. Moreover, the employed aluminum/aluminum oxide layers have been thoroughly characterized using atomic force and scanning electron microscopy as well as X-ray reflectometry and UV-Vis measurements.

UV-Raman Spectroscopic Identification of Fungal Spores Important for Respiratory Diseases.

Fungal spores are one of several environmental factors responsible for causing respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), and aspergillosis. These spores also are able to trigger exacerbations during chronic forms of disease. Different fungal spores may contain different allergens and mycotoxins, therefore the health hazards are varying between the species. Thus, it is highly important quickly to identify the composition of fungal spores in the air. In this study, UV-Raman spectroscopy with an excitation wavelength of 244 nm was applied to investigate eight different fungal species implicated in respiratory diseases worldwide. Here, we demonstrate that darkly colored spores can be directly examined, and UV-Raman spectroscopy provides the information sufficient for classifying fungal spores. Classification models on the genus, species, and strain levels were built using a combination of principal component analysis and linear discriminant analysis followed by evaluation with leave-one-batch-out-cross-validation. At the genus level an accuracy of 97.5% was achieved, whereas on the species level four different Aspergillus species were classified with 100% accuracy. Finally, classifying three strains of Aspergillus fumigatus an accuracy of 89.4% was reached. These results demonstrate that UV-Raman spectroscopy in combination with innovative chemometrics allows for fast identification of fungal spores and can be a potential alternative to currently used time-consuming cultivation.

A droplet-based microfluidic chip as a platform for leukemia cell lysate identification using surface-enhanced Raman scattering.

A new approach is presented for cell lysate identification which uses SERS-active silver nanoparticles and a droplet-based microfluidic chip. Eighty-nanoliter droplets are generated by injecting silver nanoparticles, KCl as aggregation agent, and cell lysate containing cell constituents, such as nucleic acids, carbohydrates, metabolites, and proteins into a continuous flow of mineral oil. This platform enables accurate mixing of small volumes inside the meandering channels of the quartz chip and allows acquisition of thousands of SERS spectra with 785 nm excitation at an integration time of 1 s. Preparation of three batches of three leukemia cell lines demonstrated the experimental reproducibility. The main advantage of a high number of reproducible spectra is to apply statistics for large sample populations with robust classification results. A support vector machine with leave-one-batch-out cross-validation classified SERS spectra with sensitivities, specificities, and accuracies better than 99% to differentiate Jurkat, THP-1, and MONO-MAC-6 leukemia cell lysates. This approach is compared with previous published reports about Raman spectroscopy for leukemia detection, and an outlook is given for transfer to single cells. A quartz chip was designed for SERS at 785 nm excitation. Principal component analysis of SERS spectra clearly separates cell lysates using variations in band intensity ratios.

On site visual detection of Porphyromonas gingivalis related periodontitis by using a magnetic-nanobead based assay for gingipains protease biomarkers.

Porphyromonas gingivalis (P. gingivalis) is a pathogen causing periodontitis. A rapid assay is described for the diagnosis of periodontal infections related to P. gingivalis. The method is making use of gingipains, a group of P. gingivalis specific proteases as a detection biomarker. Magnetic-nanobeads were labeled with gingipain-specific peptide substrates and immobilized on a gold biosensing platform via gold-thiol linkage. As a result of this, the color of the gold layer turns black. Upon cleavage of the immobilized substrates by gingipains, the magnetic-nanobeads-peptide fragments were attracted by a magnet so that the golden surface color becomes visible again. This assay is highly sensitive and specific. It is capable of detecting as little as 49 CFU·mL-1 of P. gingivalis within 30 s. Examination of periodontitis patients and healthy control saliva samples showed the potential of the assay. The simplicity and rapidity of the assay makes it an effective point-of-care device. Graphical abstract Schematic of the assay for the detection of P. gingivalis proteases as one of the promising biomarkers associated with periodontal diseases.

UV Absorption Spectroscopy in Water-Filled Antiresonant Hollow Core Fibers for Pharmaceutical Detection.

Due to a worldwide increased use of pharmaceuticals and, in particular, antibiotics, a growing number of these substance residues now contaminate natural water resources and drinking supplies. This triggers a considerable demand for low-cost, high-sensitivity methods for monitoring water quality. Since many biological substances exhibit strong and characteristic absorption features at wavelengths shorter than 300 nm, UV spectroscopy presents a suitable approach for the quantitative identification of such water-contaminating species. However, current UV spectroscopic devices often show limited light-matter interaction lengths, demand sophisticated and bulky experimental infrastructure which is not compatible with microfluidics, and leave large fractions of the sample analyte unused. Here, we introduce the concept of UV spectroscopy in liquid-filled anti-resonant hollow core fibers, with large core diameters and lengths of approximately 1 m, as a means to overcome such limitations. This extended light-matter interaction length principally improves the concentration detection limit by two orders of magnitude while using almost the entire sample volume-that is three orders of magnitude smaller compared to cuvette based approaches. By integrating the fibers into an optofluidic chip environment and operating within the lowest experimentally feasible transmission band, concentrations of the application-relevant pharmaceutical substances, sulfamethoxazole (SMX) and sodium salicylate (SS), were detectable down to 0.1 µM (26 ppb) and 0.4 µM (64 ppb), respectively, with the potential to reach significantly lower detection limits for further device integration.

Pioneering particle-based strategy for isolating viable bacteria from multipart soil samples compatible with Raman spectroscopy.

The study of edaphic bacteria is of great interest, particularly for evaluating soil remediation and recultivation methods. Therefore, a fast and simple strategy to isolate various bacteria from complex soil samples using poly(ethyleneimine) (PEI)-modified polyethylene particles is introduced. The research focuses on the binding behavior under different conditions, such as the composition, pH value, and ionic strength, of the binding buffer, and is supported by the characterization of the surface properties of particles and bacteria. The results demonstrate that electrostatic forces and hydrophobicity are responsible for the adhesion of target bacteria to the particles. Distinct advantages of the particle-based isolation strategy include simple handling, enrichment efficiency, and the preservation of viable bacteria. The presented isolation method allows a subsequent identification of the bacteria using Raman microspectroscopy in combination with chemometrical methods. This is demonstrated with a dataset of five different bacteria (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, Streptomyces tendae, and Streptomyces acidiscabies) which were isolated from spiked soil samples. In total 92% of the Raman spectra could be identified correctly.

Detection of Pseudomonas aeruginosa Metabolite Pyocyanin in Water and Saliva by Employing the SERS Technique.

Pyocyanin (PYO) is a metabolite specific for Pseudomonas aeruginosa. In the case of immunocompromised patients, it is currently considered a biomarker for life-threating Pseudomonas infections. In the frame of this study it is shown, that PYO can be detected in aqueous solution by employing surface-enhanced Raman spectroscopy (SERS) combined with a microfluidic platform. The achieved limit of detection is 0.5 µM. This is ~2 orders of magnitude below the concentration of PYO found in clinical samples. Furthermore, as proof of principle, the SERS detection of PYO in the saliva of three volunteers was also investigated. This body fluid can be collected in a non-invasive manner and is highly chemically complex, making the detection of the target molecule challenging. Nevertheless, PYO was successfully detected in two saliva samples down to 10 µM and in one sample at a concentration of 25 µM. This indicates that the molecules present in saliva do not inhibit the efficient adsorption of PYO on the surface of the employed SERS active substrates.

Lab-on-a-Chip-Surface Enhanced Raman Scattering Combined with the Standard Addition Method: Toward the Quantification of Nitroxoline in Spiked Human Urine Samples.

The emergence of antibacterial resistance and the development of new drugs lead to a continuous change of guidelines for medical treatments. Hence, new analytical tools are required for the detection of drugs in biological fluids. In this study, the first surface enhanced Raman scattering (SERS) detection of nitroxoline (NTX) in purified water and in spiked human urine samples is reported. Insights concerning the nature of the molecule-metal interaction and its influence on the overall SERS signal are provided. Furthermore, three randomly collected urine samples originating from a healthy volunteer were spiked to assess the limit of detection (LOD), the limit of quantification (LOQ), and the linear dynamic range of the lab-on-a-chip SERS (LoC-SERS) method for NTX detection in human urine. The LOD is ∼3 µM (0.57 mg/L), LOQ ∼ 6.5 µM (1.23 mg/L) while the linear range is between 4.28 and 42.8 µM (0.81-8.13 mg/L). This covers the minimum inhibitory concentration (MIC) values of the most commonly encountered uropathogens. Finally, seven clinical samples having an "unknown" NTX concentration were simulated. The LoC-SERS technique combined with the standard addition method and statistical data analysis provided a good prediction of the unknown concentrations. Additionally, it is also demonstrated that the predictions carried out by multicurve resolution alternating least-squares (MCR-ALS) algorithm provides reliable results, and it is preferred to a univariate statistical approach.

Rapid Identification of Pseudomonas spp. via Raman Spectroscopy Using Pyoverdine as Capture Probe.

Pyoverdine is a substance which is excreted by fluorescent pseudomonads in order to scavenge iron from their environment. Due to specific receptors of the bacterial cell wall, the iron loaded pyoverdine molecules are recognized and transported into the cell. This process can be exploited for developing efficient isolation and enrichment strategies for members of the Pseudomonas genus, which are capable of colonizing various environments and also include human pathogens like P. aeruginosa and the less virulent P. fluorescens. A significant advantage over antibody based systems is the fact that siderophores like pyoverdine can be considered as "immutable ligands," since the probability for mutations within the siderophore uptake systems of bacteria is very low. While each species of Pseudomonas usually produces structurally unique pyoverdines, which can be utilized only by the producer strain, cross reactivity does occur. In order to achieve a reliable identification of the captured pathogens, further investigations of the isolated cells are necessary. In this proof of concept study, we combine the advantages of an isolation strategy relying on "immutable ligands" with the high specificity and speed of Raman microspectroscopy. In order to isolate the bacterial cells, pyoverdine was immobilized covalently on planar aluminum chip substrates. After capturing, single cell Raman spectra of the isolated species were acquired. Due to the specific spectroscopic fingerprint of each species, the bacteria can be identified. This approach allows a very rapid detection of potential pathogens, since time-consuming culturing steps are unnecessary. We could prove that pyoverdine based isolation of bacteria is fully Raman compatible and further investigated the capability of this approach by isolating and identifying P. aeruginosa and P. fluorescens from tap water samples, which are both opportunistic pathogens and can pose a threat for immunocompromised patients.

LOC-SERS: A Promising Closed System for the Identification of Mycobacteria.

A closed droplet based lab-on-a-chip (LOC) device has been developed for the differentiation of six species of mycobacteria, i.e., both Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM), using surface-enhanced Raman spectroscopy (SERS). The combination of a fast and simple bead-beating module for the disruption of the bacterial cell with the LOC-SERS device enables the application of an easy and reliable system for bacteria discrimination. Without extraction or further treatment of the sample, the obtained SERS spectra are dominated by the cell-wall component mycolic acid. For the differentiation, a robust data set was recorded using a droplet based LOC-SERS device. Thus, more than 2100 individual SERS spectra of the bacteria suspension were obtained in 1 h. The differentiation of bacteria using LOC-SERS provides helpful information for physicians to define the conditions for the treatment of individual patients.

Enhanced sensitivity in single-mode silicon nitride stadium resonators at visible wavelengths.

The marker-free and noninvasive detection of small traces of analytes in aqueous solution using integrated optical resonators is an emerging technique within bioanalytics. Here, we present a single-mode silicon-nitride stadium resonator operating at the red edge of the visible spectrum, showing sensitivities larger than 200 nm/RIU and transmission dips with extinction ratios of more than 15 dB. We introduce a mathematical model that allows analyzing of the resonator sensitivity using the properties of the guided mode only. Large geometric parameter scans using finite element simulations show that optimal sensing conditions are achieved for TM-polarized modes close to the modal cutoff. Due to its compactness and the short operation wavelength, we anticipate applications of our resonator for integrated bioanalytics.

Toward food analytics: fast estimation of lycopene and ß-carotene content in tomatoes based on surface enhanced Raman spectroscopy (SERS).

Carotenoids are molecules that play important roles in both plant development and in the well-being of mammalian organisms. Therefore, various studies have been performed to characterize carotenoids' properties, distribution in nature and their health benefits upon ingestion. Nevertheless, there is a gap regarding a fast detection of them at the plant phase. Within this contribution we report the results obtained regarding the application of surface enhanced Raman spectroscopy (SERS) toward the differentiation of two carotenoid molecules (namely, lycopene and ß-carotene) in tomato samples. To this end, an e-beam lithography (EBL) SERS-active substrate and a 488 nm excitation source were employed, and a relevant simulated matrix was prepared (by mixing the two carotenoids in defined percentages) and measured. Next, carotenoids were extracted from tomato plants and measured as well. Finally, a combination of principal component analysis and partial least squares regression (PCA-PLSR) was applied to process the data, and the obtained results were compared with HPLC measurements of the same extracts. A good agreement was obtained between the HPLC and the SERS results for most of the tomato samples.

Plasmonic nanostructures for surface enhanced spectroscopic methods.

A comprehensive review of theoretical approaches to simulate plasmonic-active metallic nano-arrangements is given. Further, various fabrication methods based on bottom-up, self-organization and top-down techniques are introduced. Here, analytical approaches are discussed to investigate the optical properties of isotropic and non-magnetic spherical or spheroidal particles. Furthermore, numerical methods are introduced to research complex shaped structures. A huge variety of fabrication methods are reviewed, e.g. bottom-up preparation strategies for plasmonic nanostructures to generate metal colloids and core-shell particles as well as complex-shaped structures, self-organization as well as template-based methods and finally, top-down processes, e.g. electron beam lithography and its variants as well as nanoimprinting. The review article is aimed at beginners in the field of surface enhanced spectroscopy (SES) techniques and readers who have a general interest in theoretical modelling of plasmonic substrates for SES applications as well as in the fabrication of the desired structures based on methods of the current state of the art.

Ciprofloxacin: pH-dependent SERS signal and its detection in spiked river water using LoC-SERS.

Monitoring the successful removal of antibiotics in waste and surface waters is of high interest to overcome the occurrence of antibacterial resistance in the ecosystem. Among the newly developed analytical methods, the lab-on-a-chip surface-enhanced Raman spectroscopic (LoC-SERS) technique has gained the interest of the scientific community in the last few years. Ciprofloxacin, a second-generation fluoroquinolone, is widely used and administered to patients in dosages up to 1000 mg. In addition, more than 50 % of the antibiotic is excreted in urine as the parental drug. Thus, ciprofloxacin in environmental samples may exceed the minimum inhibitory concentration (MIC) values. The present study aims to assess the potential of the LoC-SERS technique to detect the target analyte in spiked river water samples at MIC concentrations. As sample clean-up procedure, a simple filtration is proposed, while as SERS, active substrates silver nanoparticles prepared at room temperature are employed. Ciprofloxacin was successfully quantified in the 0.7-10 µM concentration range with data that were measured on two different days. Furthermore, because of the low solubility of the antibiotic at the neutral pH range, insights into the effect of pH on the SERS signal of the target molecule are also presented. Graphical Abstract Ciprofloxacin detected at MIC values by LOC-SERS.