In order to detect biomolecules, different approaches using for instance biological, spectroscopic or imaging techniques are established. Due to the broad variety of these methods, this review is focused on surface enhanced Raman spectroscopy (SERS) as an analytical tool in biomolecule detection. Here, the molecular specificity of Raman spectroscopy is combined with metallic nanoparticles as sensor platform, which enhances the signal intensity by several orders of magnitude. Within this article, the characterization of diverse biomolecules by means of SERS is explained and moreover current application fields are presented. The SERS intensity and as a consequence thereof the reliable detection of the biomolecule of interest is effected by distance, orientation and affinity of the molecule towards the metal surface. Furthermore, the great capability of the SERS technique for cutting-edge applications like pathogen detection and cancer diagnosis is highlighted. We wish to motivate by this comprehensive and critical summary researchers from various scientific background to create their own ideas and schemes for a SERS-based detection and analysis of biomolecules.

Cell division cycle protein 45 (Cdc45) plays a critical role in DNA replication to ensure that chromosomal DNA is replicated only once per cell cycle. We analysed the expression of human Cdc45 in proliferating and nonproliferating cells. Our findings show that Cdc45 protein is absent from long‐term quiescent, terminally differentiated and senescent human cells, although it is present throughout the cell cycle of proliferating cells. Moreover, Cdc45 is much less abundant than the minichromosome maintenance (Mcm) proteins in human cells, supporting the concept that origin binding of Cdc45 is rate limiting for replication initiation. We also show that the Cdc45 protein level is consistently higher in human cancer‐derived cells compared with primary human cells. Consequently, tumour tissue is preferentially stained using Cdc45‐specific antibodies. Thus, Cdc45 expression is tightly associated with proliferating cell populations and Cdc45 seems to be a promising candidate for a novel proliferation marker in cancer cell biology.

Cdc45 is an essential cellular protein that functions in both the initiation and elongation of DNA replication. Here, we analyzed the localization of human Cdc45 and its interactions with other proteins during the cell cycle. Human Cdc45 showed a diffuse distribution in G1 phase, a spot-like pattern in S and G2, and again a diffuse distribution in M phase of the cell cycle. The co-localization of Cdc45 with active replication sites during S phase suggested that the human Cdc45 protein was part of the elongation complex. This view was corroborated by findings that Cdc45 interacted with the elongating DNA polymerases δ δ δ δ and ε ε ε ε , with Psf2, which is a component of the GINS complex as well as with Mcm5 and 7, subunits of the putative replicative DNA helicase complex. Hence, Cdc45 may play an important role in elongation of DNA replication by bridging the processive DNA polymerases δ δ δ δ and ε ε ε ε with the replicative helicase in the elongating machinery.

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.

No abstract