Rapid Isolation and Identification of Pneumonia-Associated Pathogens from Sputum Samples Combining an Innovative Sample Preparation Strategy and Array-Based Detection.

With this study, an innovative and convenient enrichment and detection strategy for eight clinically relevant pneumonia pathogens, namely, Acinetobacter baumannii, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae is introduced. Bacteria were isolated from sputum samples with amine-modified particles exploiting pH-dependent electrostatic interactions between bacteria and the functionalized particle surface. Following this, an asymmetric polymerase chain reaction as well as subsequent stringent array-based hybridization with specific complementary capture probes were performed. Finally, results were visualized by an enzyme-induced silver nanoparticle deposition, providing stable endpoint signals and consequently an easy detection possibility. The assay was optimized using spiked samples of artificial sputum with different strains of the abovementioned bacterial species. Furthermore, actual patient sputum samples with S. pneumoniae were successfully analyzed. The presented approach offers great potential for the urgent need of a fast, specific, and reliable isolation and identification platform for important pneumonia pathogens, covering the complete process chain from sample preparation up to array-based detection within only 4 h.

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.

DNA hybridization assay at individual, biofunctionalized zinc oxide nanowires.

Reliable and efficient identification of DNA is a major goal in on-site diagnostics. One dimensional nanostructures like nanowires (NW) represent potential sensor structures due to their extreme surface-to-bulk ratio, enabling enhanced biomolecule binding which results in optimal signals. While silicon NW are already well studied, NW made from other materials with promising properties like ZnO are not yet established as NW sensor material for bioanalytics. Here we demonstrate the DNA functionalization of ZnO NW even at the single NW level and their successful application in a DNA hybridization assay.

Biofunctionalization of zinc oxide nanowires for DNA sensory applications.

We report on the biofunctionalization of zinc oxide nanowires for the attachment of DNA target molecules on the nanowire surface. With the organosilane glycidyloxypropyltrimethoxysilane acting as a bifunctional linker, amino-modified capture molecule oligonucleotides have been immobilized on the nanowire surface. The dye-marked DNA molecules were detected via fluorescence microscopy, and our results reveal a successful attachment of DNA capture molecules onto the nanowire surface. The electrical field effect induced by the negatively charged attached DNA molecules should be able to control the electrical properties of the nanowires and gives way to a ZnO nanowire-based biosensing device.