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.

Fast-Track, One-Step E. coli Detection: A Miniaturized Hydrogel Array Permits Specific Direct PCR and DNA Hybridization while Amplification.

A timesaving and convenient method for bacterial detection based on one-step, one-tube deoxyribonucleic acid (DNA) hybridization on hydrogel array while target gene amplification is described. The hydrogel array is generated by a fast one-pot synthesis, where N,N'-dimethylacrylamide/polyethyleneglycol(PEG1900 )-bisacrylamide mixture polymerizes via radical photoinitiation by visible light within 20 min concomitant with in situ capture probe immobilization. These DNA-functionalized hydrogel droplets arrayed on a planar glass surface are placed in the polymerase chain reaction (PCR) mixture during the thermal amplification cycles. The bacterial cells can be implemented in a direct PCR reaction, omitting the need for prior template DNA extraction. The resulting fluorescence signal is immediately detectable after the end of the PCR (1 h) following one short washing step by microscopy. Therefore a valid signal can be reached within 1.5 h including 10 min for pipetting and placement of the tubes and chips. The performance of this novel hydrogel DNA array was successfully proven with varying cell numbers down to a limit of 10(1) Escherichia coli cells.

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.

Non-instrumented DNA isolation, amplification and microarray-based hybridization for a rapid on-site detection of devastating Phytophthora kernoviae.

A rapid and simple instrument-free detection system was developed for the identification of the plant pathogen Phytophthora kernoviae (P. kernoviae). The on-site operable analysis steps include magnetic particle based DNA isolation, helicase-dependent amplification (HDA) and chip-based DNA hybridization. The isothermal approach enabled the convenient amplification of the yeast GTP-binding protein (Ypt1) target gene in a miniaturized HDA-zeolite-heater (HZH) by an exothermic reaction. The amplicon detection on the chip was performed under room temperature conditions ­ either by successive hybridization and enzyme binding or by a combined step. A positive signal is displayed by enzymatically generated silver nanoparticle deposits, which serve as robust endpoint signals allowing an immediate visual readout. The hybridization assay enabled the reliable detection of 10 pg µL(-1) target DNA. This is the first report of an entirely electricity-free, field applicable detection approach for devastating Phytophthora species, exemplarily shown for P. kernoviae.

Label-free detection of Phytophthora ramorum using surface-enhanced Raman spectroscopy.

In this study, we report on a novel approach for the label-free and species-specific detection of the plant pathogen Phytophthora ramorum from real samples using surface enhanced Raman scattering (SERS). In this context, we consider the entire analysis chain including sample preparation, DNA isolation, amplification and hybridization on SERS substrate-immobilized adenine-free capture probes. Thus, the SERS-based detection of target DNA is verified by the strong spectral feature of adenine which indicates the presence of hybridized target DNA. This property was realized by replacing adenine moieties in the species-specific capture probes with 2-aminopurine. In the case of the matching capture and target sequence, the characteristic adenine peak serves as an indicator for specific DNA hybridization. Altogether, this is the first assay demonstrating the detection of a plant pathogen from an infected plant material by label-free SERS employing DNA hybridization on planar SERS substrates consisting of silver nanoparticles.

KRAS mutation screening by chip-based DNA hybridization--a further step towards personalized oncology.

The use of predictive biomarkers can help to improve therapeutic options for the individual cancer patient. For the treatment of colon cancer patients with anti-EGFR-based drugs, the KRAS mutation status has to be determined to pre-select responders that will benefit from this medication. Amongst others, array-based tests have been established for profiling of the KRAS mutation status. Within this article we describe an on-chip hybridization technique to screen therapeutic relevant KRAS codon 12 mutations. The DNA chip-based platform enables the reliable discrimination of selected mutations by allele-specific hybridization. Here, silver deposits represent robust endpoint signals that allow for a simple naked eye rating. With the here presented assay concept a precise identification of heterozygous and homozygous KRAS mutations, even against a background of up to 95% wild-type DNA, was realizable. The applicability of the test was successfully proven for various cancer cell lines as well as clinical tumour samples. Thus, the chip-based DNA hybridization technique seems to be a promising tool for KRAS mutation analysis to further improve personalized cancer treatment.

Easy daylight fabricated hydrogel array for colorimetric DNA analysis.

The fabrication of 3D hydrogel microarrays for DNA analytics that allow simple visual signal readout for on-site applications is described. A convenient one-step polymerization of the hydrogel including in situ capture oligonucleotide immobilization is accomplished by using N,N'-dimethylacrylamide/polyethylene glycol (PEG1900 )-bisacrylamide monomers. The implementation of an acylphosphine-oxide photoinitiator even allows polymerization at daylight, whereas other approaches require exposure with light in the UV-range. This minimizes the risk of UV-caused DNA damages within the capture DNA-strand that could adversely affect the subsequent hybridization step. The porous network of these gel segments allows DNA as well as protein penetration. Thus, the successful in-gel DNA hybridization is monitored by the deposition of silver nanoparticles. These metal particles allow naked eye signal readout.

Chip-on-foil devices for DNA analysis based on inkjet-printed silver electrodes.

For a rapid on-site diagnosis of pathogens, low-cost chip-based devices are of great interest. Here, we report the successful fabrication of inkjet printed silver electrodes on polymer foils as disposable chips for molecular DNA analytics. In order to manufacture these electrode structures, silver nanoparticle inks were inkjet printed onto planar polypropylene substrates. Due to the low thermal stability of the foils, substrate preserving sintering techniques, including low temperature thermal sintering and low pressure argon plasma sintering, were implemented. Thus, sufficient electrical conductance of the printed structures at processing temperatures ≤100 °C was achieved. To test the applicability of the manufactured chips, specific capture DNA was immobilized within the gaps of the conductive electrode paths and hybridized in the next step with biotin-labeled target DNA. Subsequently, an enzymatically generated silver nanoparticle deposition was induced that bridges the electrode gap. This enabled both conductance measurement and gray value analysis as a fast, simple and robust electrical and optical read-out system. The proof-of-principle experiments successfully demonstrated the applicability of these convenient chip-on-foil devices for nucleic acid based pathogen detection.

Dry-reagent-based PCR as a novel tool for the rapid detection of Clostridium spp.

Improved conventional PCR techniques are required for the rapid on-site detection of human and animal diseases. In this context, a PCR method using dry-stored reagents intended for the detection of Clostridium spp. is presented. Basic PCR reagents (BSA, PCR buffer, MgCl2 and primers), which were dried on polyolefin matrices, showed stability at ambient temperatures for up to 10 months without any loss of functionality. An outstanding advantage of our amelioration is the elimination of PCR process errors caused by the improper storage and handling of liquid reagents. Moreover, our PCR-based amplification can be performed in less than 30 min, saving time compared with conventional detection methods. Thus, dry-reagent-based PCR is implementable in a suitcase-like modular device for the rapid on-site detection of microbial pathogens such as blackleg of ruminants caused by Clostridium chauvoei.

Interferon alpha-armed nanoparticles trigger rapid and sustained STAT1-dependent anti-viral cellular responses.

Interferon-α (IFNα) has enormous potential for anti-proliferative and anti-viral treatments. However, clinical success is still hampered due to its limited bioavailability and thus, lack of sustained modulation of disease-relevant protective programs. Consequently, we here examined whether IFNα immobilized on nanoscale ferromagnetic R-Chitosan carriers is capable of inducing rapid and sustained activation of STAT1 signaling. We report the spontaneous formation of a stable nanoparticle-IFNα protein corona, which was exploited to generate IFNα-loaded spheres, obviating the need to specifically couple the cytokine to the nanoparticles (NPs). Notably, comprehensive experimental approaches ensure that formation of the IFNα NP-corona does not affect the biological activity of the cytokine, as STAT1 signaling was efficiently activated. Employing human prostate cancer and melanoma cell models, we found that the intensity and duration of STAT1 phosphorylation as well as the downstream activation of pathobiologically relevant genes were dose and particle dependent. In comparison with free IFNα, IFNα-loaded spheres resulted in a more sustained biologically relevant STAT1 activation, demonstrated also by conferring innate cellular immunity against vesicular stomatitis virus (VSV) infection. For one, our study demonstrates the advantages of biodegradable IFNα-coated R-Chitosan NPs for controlled cytokine release, and thereby improved therapy. Second, we reveal that the permanent presence of IFNα and not just the initial STAT1 phosphorylation ensures sustained IFNα-dependent signaling.

Interactions of human Cdc45 with the Mcm2-7 complex, the GINS complex, and DNA polymerases delta and epsilon during S phase.

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 delta and epsilon, 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 delta and epsilon with the replicative helicase in the elongating machinery.