New insights for using self-assembly materials to improve the detection stability in label-free DNA-chip and immuno-sensors

This paper examines reliable advancements in low-cost DNA- and immuno-chips. Capacitance detection was successfully chosen to develop label-free bio-chips. Probe immobilization was rigorously investigated in order to obtain reliable capacitance measurements. Protein probes immobilized by using usual alkanethiols or thiolated ssDNA probes directly immobilized on gold do not allow sufficient stable capacitance measurements. New alkanethiols improved with ethylene–glycol function are shown in this paper to be more suitable materials for capacitive bio-chip development. Atomic Force Microscopy, Quartz Crystal Microbalance, and Capacitance Measurements were used to demonstrate that ethylene–glycol alkanethiols allow high time stability, smaller errors in detection, and improved ideal behaviour of the sensing surfaces. Measured capacitance is in the range of 8–11 nF/mm² for antibody layers and close to 6 nF/mm² for DNA probes. It is in the range of 10–12 nF/mm² and of 4–6 nF/mm² for antigen and DNA detection, respectively. The percentage error in detection is highly improved and it is in the range of 11–37% and of 0,23–0,82% for antigen and DNA, respectively. The reproducibility is also improved and it is close to 0,44% for single spot measurements on ethylene–glycol alkanethiols. A molecular theory attributing these improvements to water molecules strongly coordinated by ethylene–glycol functional groups and to solution ions not entering into probe films is finally proposed.     

A disposable and cost efficient microfluidic device for the rapid chip-based electrical detection of DNA

Requirements for a point-of-care device are an easy and robust read-out and – above all – a simple handling. We integrated an established robust electrical read-out for DNA-chips into a microfluidic device, thereby creating an automated analysis system that combines the necessary steps for a chip-based analysis. It is based on the electrical detection of biotin-labeled DNA in a gap between two microstructured electrodes on the surface of a DNA-chip. The biotin serves as binding molecule for streptavidin-conjugated horseradish peroxidase. A following enzyme-induced silver deposition bridges the gap by a conductive layer. The miniaturized chip gives the possibility to realize a durable system suitable for point-of-care applications.To enable an initial automation, all corresponding process steps were executed in a miniaturized silicone flow cell. The required defined temperatures for the hybridization and the washing steps can be adjusted by a heating foil.This paper characterizes the performance of the flow cell based system in terms of reaction speed and analysis time, sensitivity as well as specificity, and the comparison to a conventional system, without flow cell. These first steps of automation and integration will help to realize a laboratory-independent bioanalytical tool, for the use outside of specialized laboratories for fast analysis of different chemical and biological applications.     

Electrical DNA-chip-based identification of different species of the genus Kitasatospora

The identification of different Kitasatospora strains has been shown with a DNA-chip based on an electrical readout scheme. The 16S-23S rDNA internal transcribed spacer region of these Actinomycetes was used for identification. Two different capture probes per strain were immobilized on the chip. The capture probes were spotted on a DNA-chip with electrode structures for an electrical DNA detection. A biotinylated PCR product of the 16S-23S rDNA region was incubated on the chips and bound to its complementary capture sequences. Followed by a gold nanoparticle or enzyme labeling and a deposition of silver, the binding of the PCR product was detected by an increase of the measured conductivity on the chip. To show the applicability of this detection system, four strains of Kitasatospora were chosen for an identification using the DNA-chip with electrical detection. Each strain was clearly identified using the system. Concentrations of the polymerase chain reaction (PCR) products within the range of 1 ng/ml to 1 μg/ml were detected and identified. These tests are the first application of this novel electrical detection scheme for the identification and classification of microorganisms. The presented results show that the DNA-chip with electrical detection can be used for a robust and cost-efficient DNA analysis.     

Comparison of an oligo-chip based assay with PCR method to measure the prevalence of tick-borne pathogenic bacteria in central Slovakia

Ticks are well-known vectors for a wide range of pathogenic microorganisms. We examined the presence of Rickettsia spp., Anaplasma spp., Borrelia spp., Coxiella burnetii and Francisella tularensis in Ixodes ricinus ticks collected in central Slovakia using oligo-chip based assay. Rickettsiae were detected in 5.6% of examined ticks. Borreliae and anaplasmae were identified in 2.1% and 2.8% ticks, respectively. All tested samples were negative for presence of Coxiella burnetii and Francisella tularensis. All these results were compared with those obtained by PCR analysis, and a close correlation between them was found. In addition, rickettsiae of spotted fever group (SFG), Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato were found in ticks using genera or species-specific PCR methods. They are circulating in 10 out of 18 studied localities.     

Universal Labeling Chemistry for Nucleic Acid Detection on DNA-Arrays

Abstract

We show here a new and efficient aqueous chemistry for labeling of any class of nucleic acids for their detection on DNA chip. The labels contain a diazo function as reactive moiety and biotin as detectable unit. The highly selective reaction of diazo group on the phosphate does not disrupt base pairing recognition and hybridization specificity.