SERS-based sensor for the detection of sexually transmitted pathogens in the male swab specimens: A new approach for clinical diagnosis.

The surface-enhanced Raman scattering (SERS) has been widely tested for its usefulness in microbiological studies, providing many information-rich spectra which are a kind of 'whole-organism fingerprint' and enabling identification of bacterial species. Here we show, previously not considered, the comprehensive SERS-chemometric analysis of five bacterial pathogens, namely Neisseria gonorrhoeae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Haemophilus ducreyi, all being responsible for sexually transmitted diseases (STDs). In the designed biosensor, the direct, intrinsic format of the spectroscopic analysis was adopted for the SERS-based screening of gonorrhea and chlamydiosis due to vibrational analysis of men's urethra swabs. Our experiments demonstrated that the applied method enables identification the individual species of the Neisseria genus with high accuracy. In order to differentiate the sexually transmitted pathogens and to classify the clinical samples of male urethra swabs, three multivariate methods were used. In the external validation the created models correctly classified the men's urethra swabs with prediction accuracy reaching 89% for SIMCA and 100% for PLS-DA. As a result, the developed protocol enables: (i) simple and non-invasive analysis of clinical samples (the collection of urethra swabs specimens could be carried out at different points of care, such as doctor's office); (ii) fast analysis (<15 min); (iii) culture-free identification; (iv) sensitive and reliable SERS-based diagnosis of STD. The simplicity of the developed detection procedure, supported by high sensitivity, reproducibility, and specificity, open a new path in the improvement of the point-of-care applications.

In Search of Spectroscopic Signatures of Periodontitis: A SERS-Based Magnetomicrofluidic Sensor for Detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.

Recently, Porphyromonas gingivalis, the keystone pathogen implicated in the development of gum disease (periodontitis), was detected in the brains of Alzheimer's disease patients, opening up a fascinating possibility that it is also involved in the pathobiology of this neurodegenerative illness. To verify this hypothesis, an unbiased, specific, and sensitive method to detect this pathogen in biological specimens is needed. To this end, our interdisciplinary studies demonstrate that P. gingivalis can be easily identified by surface-enhanced Raman scattering (SERS). Moreover, based on SERS measurements, P. gingivalis can be distinguished from another common periodontal pathogen, Aggregatibacter actinomycetemcomitans, and also from ubiquitous oral Streptococcus spp. The results were confirmed by principal component analysis (PCA). Furthermore, we have shown that different P. gingivalis and A. actinomycetemcomitans strains can easily adsorb to silver-coated magnetic nanoparticles (Fe2O3@AgNPs). Thus, it is possible to magnetically separate investigated bacteria from other components of a specimen using the microfluidic chip. To obtain additional enhancement of the Raman signal, the NPs adsorbed to bacterial cells were magnetically attracted to the Si/Ag SERS platform. Afterward, the SERS spectra could be recorded. Such a time-saving procedure can be very helpful in rapid medical diagnostics and thus in starting the appropriate pharmacological therapy to prevent the development of periodontitis and associated comorbidities, e.g., Alzheimer's disease.

Nanoplasmonic sensor for foodborne pathogens detection. Towards development of ISO-SERS methodology for taxonomic affiliation of Campylobacter spp.

According to EU summary report on zoonoses, zoonotic agents and food-borne outbreaks in 2017, Campylobacter was the most commonly reported gastrointestinal bacterial pathogen in humans in the EU. Unfortunately, the standard methods for the detection of thermotolerant Campylobacter spp. in foods are time-consuming. Additionally, the qualified staff is obligatory. For this reason, new methods of pathogens detection are needed. The present work demonstrates that surface-enhanced Raman scattering (SERS) is a reliable and fast method for detection of Campylobacter spp. in food samples. The proposed method combines the SERS measurements performed on an Ag/Si substrate with two initial steps of the ISO standard procedure. Finally, the principal component analysis (PCA) allows for statistical classification of the studied bacteria. By applying the proposed ISO-SERS-PCA method in the case of Campylobacter bacteria the total detection time may be reduced from 7 to 8 days required by ISO method to 3 to 4 days in the case of SERS-based approach.

Correction to: Sources of variability in SERS spectra of bacteria: comprehensive analysis of interactions between selected bacteria and plasmonic nanostructures.

The authors would like to call the reader's attention to the fact that unfortunately following information was missing in the original article: "Evelin Witkowska is supported by the Foundation of Polish Science (FNP)."

Sources of variability in SERS spectra of bacteria: comprehensive analysis of interactions between selected bacteria and plasmonic nanostructures.

The surface-enhanced Raman spectroscopy (SERS)-based analysis of bacteria suffers from the lack of a standard SERS detection protocol (type of substrates, excitation frequencies, and sampling methodologies) that could be employed throughout laboratories to produce repeatable and valuable spectral information. In this work, we have examined several factors influencing the spectrum and signal enhancement during SERS studies conducted on both Gram-negative and Gram-positive bacterial species: Escherichia coli and Bacillus subtilis, respectively. These factors can be grouped into those which are related to the structure and types of plasmonic systems used during SERS measurements and those that are associated with the culturing conditions, types of culture media, and method of biological sample preparation.

Detection of Circulating Tumor Cells Using Membrane-Based SERS Platform: A New Diagnostic Approach for 'Liquid Biopsy'.

The detection and monitoring of circulating tumor cells (CTCs) in blood is an important strategy for early cancer evidence, analysis, monitoring of therapeutic response, and optimization of cancer therapy treatments. In this work, tailor-made membranes (MBSP) for surface-enhanced Raman spectroscopy (SERS)-based analysis, which permitted the separation and enrichment of CTCs from blood samples, were developed. A thin layer of SERS-active metals deposited on polymer mat enhanced the Raman signals of CTCs and provided further insight into CTCs molecular and biochemical composition. The SERS spectra of all studied cells-prostate cancer (PC3), cervical carcinoma (HeLa), and leucocytes as an example of healthy (normal) cell-revealed significant differences in both the band positions and/or their relative intensities. The multivariate statistical technique based on principal component analysis (PCA) was applied to identify the most significant differences (marker bands) in SERS data among the analyzed cells and to perform quantitative analysis of SERS data. Based on a developed PCA algorithm, the studied cell types were classified with an accuracy of 95% in 2D PCA to 98% in 3D PCA. These results clearly indicate the diagnostic efficiency for the discrimination between cancer and normal cells. In our approach, we exploited the one-step technology that exceeds most of the multi-stage CTCs analysis methods used and enables simultaneous filtration, enrichment, and identification of the tumor cells from blood specimens.

Steel Wire Mesh as a Thermally Resistant SERS Substrate.

In this paper, we present novel type of Surface-enhanced Raman spectroscopy (SERS) platform, based on stainless steel wire mesh (SSWM) covered with thin silver layer. The stainless steel wire mesh, typically used in chemical engineering industry, is a cheap and versatile substrate for SERS platforms. SSWM consists of multiple steel wires with diameter of tens of micrometers, which gives periodical structure and high stiffness. Moreover, stainless steel provides great resistance towards organic and inorganic solvents and provides excellent heat dissipation. It is worth mentioning that continuous irradiation of the laser beam over the SERS substrate can be a source of significant increase in the local temperature of metallic nanostructures, which can lead to thermal degradation or fragmentation of the adsorbed analyte. Decomposition or fragmentation of the analysed sample usually causea a significant decrease in the intensity of recorded SERS bands, which either leads to false SERS responses or enables the analysis of spectral data. To our knowledge, we have developed for the first time the thermally resistant SERS platform. This type of SERS substrate, termed Ag/SSWM, exhibit high sensitivity (Enhancement Factor (EF) = 106) and reproducibility (Relative Standard Deviation (RSD) of 6.4%) towards detection of p-mercaptobenzoic acid (p-MBA). Besides, Ag/SSWM allows the specific detection and differentiation between Gram-positive and Gram-negative bacterial species: Escherichia coli and Bacillus subtilis in label-free and reproducible manner. The unique properties of designed substrate overcome the limitations associated with photo- and thermal degradation of sensitive bacterial samples. Thus, a distinctive SERS analysis of all kinds of chemical and biological samples at high sensitivity and selectivity can be performed on the developed SERS-active substrate.

Strain-level typing and identification of bacteria - a novel approach for SERS active plasmonic nanostructures.

One of the potential applications of surface-enhanced Raman spectroscopy (SERS) is the detection of biological compounds and microorganisms. Here we demonstrate that SERS coupled with principal component analysis (PCA) serves as a perfect method for determining the taxonomic affiliation of bacteria at the strain level. We demonstrate for the first time that it is possible to distinguish different genoserogroups within a single species, Listeria monocytogenes, which is one of the most virulent foodborne pathogens and in some cases contact with which may be fatal. We also postulate that it is possible to detect additional proteins in the L. monocytogenes cell envelope, which provide resistance to benzalkonium chloride and cadmium. A better understanding of this infectious agent could help in selecting the appropriate pharmaceutical product for enhanced treatment. Graphical abstract ᅟ.

Genus- and species-level identification of dermatophyte fungi by surface-enhanced Raman spectroscopy.

This paper demonstrates that surface-enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) can serve as a fast and reliable technique for detection and identification of dermatophyte fungi at both genus and species level. Dermatophyte infections are the most common mycotic diseases worldwide, affecting a quarter of the human population. Currently, there is no optimal method for detection and identification of fungal diseases, as each has certain limitations. Here, for the first time, we have achieved with a high accuracy, differentiation of dermatophytes representing three major genera, i.e. Trichophyton, Microsporum, and Epidermophyton. Two first principal components (PC), namely PC-1 and PC-2, gave together 97% of total variance. Additionally, species-level identification within the Trichophyton genus has been performed. PC-1 and PC-2, which are the most diagnostically significant, explain 98% of the variance in the data obtained from spectra of: Trichophyton rubrum, Trichophyton menatgrophytes, Trichophyton interdigitale and Trichophyton tonsurans. This study offers a new diagnostic approach for the identification of dermatophytes. Being fast, reliable and cost-effective, it has the potential to be incorporated in the clinical practice to improve diagnostics of medically important fungi.

SERS-based Immunoassay in a Microfluidic System for the Multiplexed Recognition of Interleukins from Blood Plasma: Towards Picogram Detection.

SERS-active nanostructures incorporated into a microfluidic device have been developed for rapid and multiplex monitoring of selected Type 1 cytokine (interleukins: IL-6, IL-8, IL-18) levels in blood plasma. Multiple analyses have been performed by using nanoparticles, each coated with different Raman reporter molecules: 5,5'-dithio-bis(2-nitro-benzoic acid) (DTNB), fuchsin (FC), and p-mercatpobenzoic acid (p-MBA) and with specific antibodies. The multivariate statistical method, principal component analysis (PCA), was applied for segregation of three different antigen-antibody complexes encoded by three Raman reporters (FC, p-MBA, and DTNB) during simultaneous multiplexed detection approach. To the best of our knowledge, we have also presented, for the first time, a possibility for multiplexed quantification of three interleukins: IL-6, IL-8, and IL-18 in blood plasma samples using SERS technique. Our method improves the detection limit in comparison to standard ELISA methods. The low detection limits were estimated to be 2.3 pg·ml-1, 6.5 pg·ml-1, and 4.2 pg·ml-1 in a parallel approach, and 3.8 pg·ml-1, 7.5 pg·ml-1, and 5.2 pg·ml-1 in a simultaneous multiplexed method for IL-6, IL-8, and IL-18, respectively. This demonstrated the sensitivity and reproducibility desirable for analytical examinations.

Surface-enhanced Raman spectroscopy introduced into the International Standard Organization (ISO) regulations as an alternative method for detection and identification of pathogens in the food industry.

We show that surface-enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) can serve as a fast, reliable, and easy method for detection and identification of food-borne bacteria, namely Salmonella spp., Listeria monocytogenes, and Cronobacter spp., in different types of food matrices (salmon, eggs, powdered infant formula milk, mixed herbs, respectively). The main aim of this work was to introduce the SERS technique into three ISO (6579:2002; 11290-1:1996/A1:2004; 22964:2006) standard procedures required for detection of these bacteria in food. Our study demonstrates that the SERS technique is effective in distinguishing very closely related bacteria within a genus grown on solid and liquid media. The advantages of the proposed ISO-SERS method for bacteria identification include simplicity and reduced time of analysis, from almost 144 h required by standard methods to 48 h for the SERS-based approach. Additionally, PCA allows one to perform statistical classification of studied bacteria and to identify the spectrum of an unknown sample. Calculated first and second principal components (PC-1, PC-2) account for 96, 98, and 90% of total variance in the spectra and enable one to identify the Salmonella spp., L. monocytogenes, and Cronobacter spp., respectively. Moreover, the presented study demonstrates the excellent possibility for simultaneous detection of analyzed food-borne bacteria in one sample test (98% of PC-1 and PC-2) with a goal of splitting the data set into three separated clusters corresponding to the three studied bacteria species. The studies described in this paper suggest that SERS represents an alternative to standard microorganism diagnostic procedures. Graphical Abstract New approach of the SERS strategy for detection and identification of food-borne bacteria, namely S. enterica, L. monocytogenes, and C. sakazakii in selected food matrices.

Polymer mat prepared via Forcespinning™ as a SERS platform for immobilization and detection of bacteria from blood plasma.

One of potential applications of nano- and microscale polymer fibers is SERS-active platforms for the detection of biological compounds and microorganisms. This paper demonstrates the polymer mat obtained with Forcespinning™ technique used to detect the bacteria from blood plasma. Forcespinning™ is a new method of manufacturing of polymer fibers which can be applied to variety of polymer materials, e.g. polyethylene, nylon, PA6 and others. The method is based on the centrifugal force to draw fiber from molten polymer, which allows tuning the diameter of the fiber from tens of nanometers up to micrometers. Wide range of diameters makes the forcespun polymer mat an excellent material to filter bacteria from fluids (e.g. blood plasma, water). Covering the mat with Au:Ag alloy turns it into a SERS platform able to immobilize, detect, and identify bacteria. We provide proof-of-concept, showing detection of S. aureus, P. aeruginosa, and S. Typhimurium from blood plasma.

Highly efficient SERS-based detection of cerebrospinal fluid neopterin as a diagnostic marker of bacterial infection.

A highly efficient recognition unit based on surface-enhanced Raman spectroscopy (SERS) was developed as a promising, fast, and sensitive tool for detection of meningococcal meningitis, which is an extremely serious and often fatal disease of the nervous system (an inflammation of the lining around the brain and spinal cord). The results of this study confirmed that there were specific differences in SERS spectra between cerebrospinal fluid (CSF) samples infected by Neisseria meningitidis and the normal CSF, suggesting a potential role for neopterin in meningococcal meningitis detection and screening applications. To estimate the best performance of neopterin as a marker of bacterial infection, principal component analysis (PCA) was performed in a selected region (640-720 cm(-1)) where the most prominent SERS peak at 695 cm(-1) arising from neopterin was observed. The calculated specificity of 95 % and sensitivity of 98 % clearly indicate the effective diagnostic efficiency for differentiation between infected and control samples. Additionally, the limit of detection (LOD) of neopterin in CSF clinical samples was estimated. The level of neopterin was significantly higher in CSF samples infected by N. meningitidis (48 nmol/L), compared to the normal (control) group (4.3 nmol/L). Additionally, this work presents a new type of SERS-active nanostructure, based on polymer mats, that allows simultaneous filtration, immobilization, and enhancement of the Raman signal, enabling detection of spectra from single bacterial cells of N. meningitidis present in CSF samples. This provides a new possibility for fast and easy detection of bacteria in CSF and other clinical body fluids on a time scale of seconds. This method of detection produces consistent results faster and cheaper than traditional laboratory techniques, demonstrates the powerful potential of SERS for detection of disease, and shows the viability of future development in healthcare applications.

Detection of Hepatitis B virus antigen from human blood: SERS immunoassay in a microfluidic system.

A highly sensitive immunoassay utilizing surface-enhanced Raman scattering (SERS) has been developed with a new Raman reporter and a unique SERS-active substrate incorporated into a microfluidic device. An appropriately designed Raman reporter, basic fuchsin (FC), gives strong SERS enhancement and has the ability to bind both the antibody and gold nanostructures. The fuchsin-labeled immuno-Au nanoflowers can form a sandwich structure with the antigen and the antibody immobilized on the SERS-active substrate based on Au-Ag coated GaN. Our experimental results indicate that this SERS-active substrate with its strong surface-enhancement factor, high stability and reproducibility plays a crucial role in improving the efficiency of SERS immunoassay. This SERS assay was applied to the detection of Hepatitis B virus antigen (HBsAg) in human blood plasma. A calibration curve was obtained by plotting the intensity of SERS signal of FC band at 1178cm(-1) versus the concentration of antigen. The low detection limit for Hepatitis B virus antigen was estimated to be 0.01IU/mL. The average relative standard deviation (RSD) of this method is less than 10%. This SERS immunoassay gives exact results over a broad linear range, reflecting clinically relevant HBsAg concentrations. It also exhibits high biological specificity for the detection of Hepatitis B virus antigen.

Electrospun polymer mat as a SERS platform for the immobilization and detection of bacteria from fluids.

This work demonstrates the development of a new class of SERS substrates that allows for the simultaneous: (i) filtration of bacteria from any solution (blood, urine, water, or milk), (ii) immobilization of bacteria on the SERS platform, and (iii) enhancing the Raman signal of bacteria. The proposed platform is based on an electrospun polymer mat covered with a 90 nm layer of gold.

Nanostructured silver-gold bimetallic SERS substrates for selective identification of bacteria in human blood.

Surface-enhanced Raman spectroscopy (SERS) is a potentially important tool in the rapid and accurate detection of pathogenic bacteria in biological fluids. However, for diagnostic application of this technique, it is necessary to develop a highly sensitive, stable, biocompatible and reproducible SERS-active substrate. In this work, we have developed a silver-gold bimetallic SERS surface by a simple potentiostatic electrodeposition of a thin gold layer on an electrochemically roughened nanoscopic silver substrate. The resultant substrate was very stable under atmospheric conditions and exhibited the strong Raman enhancement with the high reproducibility of the recorded SERS spectra of bacteria (E. coli, S. enterica, S. epidermidis, and B. megaterium). The coating of the antibiotic over the SERS substrate selectively captured bacteria from blood samples and also increased the Raman signal in contrast to the bare surface. Finally, we have utilized the antibiotic-coated hybrid surface to selectively identify different pathogenic bacteria, namely E. coli, S. enterica and S. epidermidis from blood samples.