Susanne Pahlow

Isolation and identification of bacteria by means of Raman spectroscopy

Bacterial detection is a highly topical research area, because various fields of application will benefit from the progress being made. Consequently, new and innovative strategies which enable the investigation of complex samples, like body fluids or food stuff, and improvements regarding the limit of detection are of general interest. Within this review the prospects of Raman spectroscopy as a reliable tool for identifying bacteria in complex samples are discussed. The main emphasis of this work is on important aspects of applying Raman spectroscopy for the detection of bacteria like sample preparation and the identification process. Several approaches for a Raman compatible isolation of bacterial cells have been developed and applied to different matrices. Here, an overview of the limitations and possibilities of these methods is provided. Furthermore, the utilization of Raman spectroscopy for diagnostic purposes, food safety and environmental issues is discussed under a critical view.

Bioanalytical application of surface- and tip-enhanced Raman spectroscopy

Due to its fingerprint specificity and trace‐level sensitivity, surface‐enhanced Raman spectroscopy (SERS) is an attractive tool in bioanalytics. This review reflects the research in this highly interesting topic of the last 3–4 years. The detection of the SERS signature of biomolecules up to microorganisms and cells is introduced. Labeling using modified nanoparticles (SERS tags) is also introduced. In order to establish biomedical applications, SERS analysis is performed in complex matrices such as body fluids. Furthermore, the SERS technique is combined with other methods such as microfluidic devices for online monitoring and scanning probe microscopy (i.e. tip‐enhanced Raman spectroscopy, TERS) to investigate nanoscaled features. The present review illustrates the broad application fields of SERS and TERS in bioanalytics and shows the great potential of these methods for biomedical diagnostics.

Isolation and Enrichment of Pathogens with a Surface‐Modified Aluminium Chip for Raman Spectroscopic Applications

We developed a Raman-compatible chip for isolating microorganisms from complex media. The isolation of bacteria is achieved by using antibodies as capture molecules. Due to the very specific interaction with the targets, this approach is promising for isolation of bacteria even from complex matrices such as body fluids. Our choice of capture molecules also enabled the investigation of samples containing yet unidentified bacteria, as the antibodies can capture a large variety of bacteria based on their analogue cell wall surface structures. The capability of our system is demonstrated for a broad range of different Gram-positive and Gram-negative germs. Subsequent identification is done by recording Raman spectra of the bacteria. Further, it is shown that classification with chemometric methods is possible.

Clostridium spp. discrimination with a simple bead-based fluorescence assay

C. chauvoei is the causative agent of blackleg, an endogenous bacterial infection which usually affects cattle and other ruminants. Due to the fact that the symptoms of this severe disease are very similar to the phenotype caused by an infection with C. septicum, a reliable differentiation of C. chauvoei from other Clostridium spp. is mandatory. Traditional microbiological detection methods are time consuming and the proper specification is hampered by the overgrowing tendency of swarming C. septicum colonies when both species are in the clinical sample. Thus, there is an urgent need to improve and simplify the specific detection of C. chauvoei and C. septicum. We report an easy and fast Clostridium spp. discrimination method via a magnetic bead-based fluorescence assay. To that end, the target DNA was amplified using 16S-23S rDNA spacer region specific primers. These PCR products were employed to generate single-stranded capture probe DNA, which was immobilized on magnetic beads. Functionalized magnetic particles exhibit numerous advantages, like their simple manipulation in combination with a huge binding capacity of biomolecules and make therefore excellent biosensors. In this context, the discrimination between C. chauvoei and C. septicum was realized by means of hybridization with complementary detection probe DNA. Finally, fluorescence spectroscopy allowed the signal readout. With this approach a precise discrimination between C. chauvoei, C. septicum and C. carnis was accomplished.

Application of Vibrational Spectroscopy and Imaging to Point-of-Care Medicine: A Review:

Vibrational spectroscopy and imaging promise molecular information that can be rapidly acquired without the need for specialized stains or dyes, thereby potentially simplifying and speeding up necessary analyses for interventions in many facets of modern day healthcare. The salient characteristics of vibrational spectroscopy for molecular analyses, using non-perturbative optical measurements, and employing computational analysis of data, are especially useful near the point of care as assessments can be made with fewer reagents, under pressure of time and accuracy while not requiring extensive specialized human expertise. Significant technological development has occurred and many seminal proof of concept studies have been conducted to demonstrate the utility and vast potential of spectroscopic methods. Accordingly, a number of studies have focused on pushing the fundamental performance limits of spectroscopic methods while others have focused on specific problems where the use of vibrational spectroscopy promises to change the standard of care. Despite this impressive progress, however, the application area is still maturing and rapidly evolving. A vast array of potential applications continues to be assessed while others need further technological developments. In this review, we focus on recent developments that demonstrate potential for point of care impact and major trends that can lead, in turn, to improved spectroscopic technology. We provide focused examples of ‘‘case studies’’ and major trends in spectroscopic analyses ranging from in vivo measurements to that of ex vivo bodily fluids to extracted and processed tissues. In each case, the uniting theme is that information to the clinician is enabled closer to the patient, allowing for a shorter time between identification of the need for analyses and availability of information that guides care. Raman Spectroscopy for Intraoperative and In Vivo Diagnostics

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.

A Raman-Compatible Isolation Strategy for Human Pathogenic Bacteria in Tap Water Samples Relying on Siderophores

An innovative siderophore-based sample preparation strategy enabling the isolation of several Pseudomonas species from tap water samples is introduced. The bacterial cells are captured on a chip surface and subsequently identified via their Raman fingerprint.

Discrimination of clostridium species using a magnetic bead based hybridization assay

Clostridium chauvoei is the causative agent of blackleg, which is an endogenous bacterial infection. Mainly cattle and other ruminants are affected. The symptoms of blackleg are very similar to those of malignant edema, an infection caused by Clostridium septicum. [1, 2] Therefore a reliable differentiation of Clostridium chauvoei from other Clostridium species is required. Traditional microbiological detection methods are time consuming and laborious. Additionally, the unique identification is hindered by the overgrowing tendency of swarming Clostridium septicum colonies when both species are present. [1, 3, 4] Thus, there is a crucial need to improve and simplify the specific detection of Clostridium chauvoei and Clostridium septicum. Here we present an easy and fast Clostridium species discrimination method combining magnetic beads and fluorescence spectroscopy. Functionalized magnetic particles exhibit plentiful advantages, like their simple manipulation in combination with a large binding capacity of biomolecules. A specific region of the pathogenic DNA is amplified and labelled with biotin by polymerase chain reaction (PCR). These PCR products were then immobilized on magnetic beads exploiting the strong biotin-streptavidin interaction. The specific detection of different Clostridium species is achieved by using fluorescence dye labeled probe DNA for the hybridization with the immobilized PCR products. Finally, the samples were investigated by fluorescence spectroscopy. [5]

Chip-based Isolation of Pathogens for Subsequent Raman Spectroscopic Identification

A Raman chip, which enables isolation of a large variety of bacteria based on their analogue cell wall surface structures, is presented. Identification of the microorganisms is achieved using Raman microspectroscopy.

A Novel Bioassay for the Rapid Detection of E. coli

Susanne Pahlow, Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller-University, Jena, Germany, susanne.pahlow@ipht-jena.de Martha Schwarz, Institute of Photonic Technology, Jena, Germany, martha.schwarz@ipht-jena.de Karina Weber, Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller-University, Jena, Germany, karina.weber@ipht-jena.de Jurgen Popp, Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller-University, Jena, Germany, juergen.popp@ipht-jena.de