Thomas Schüler

The morphology of silver nanoparticles prepared by enzyme-induced reduction

Summary Silver nanoparticles were synthesized by an enzyme-induced growth process on solid substrates. In order to customize the enzymatically grown nanoparticles (EGNP) for analytical applications in biomolecular research, a detailed study was carried out concerning the time evolution of the formation of the silver nanoparticles, their morphology, and their chemical composition. Therefore, silver-nanoparticle films of different densities were investigated by using scanning as well as transmission electron microscopy to examine their structure. Cross sections of silver nanoparticles, prepared for analysis by transmission electron microscopy were additionally studied by energy-dispersive X-ray spectroscopy in order to probe their chemical composition. The surface coverage of substrates with silver nanoparticles and the maximum particle height were determined by Rutherford backscattering spectroscopy. Variations in the silver-nanoparticle films depending on the conditions during synthesis were observed. After an initial growth state the silver nanoparticles exhibit the so-called desert-rose or nanoflower-like structure. This complex nanoparticle structure is in clear contrast to the auto-catalytically grown spherical particles, which maintain their overall geometrical appearance while increasing their diameter. It is shown, that the desert-rose-like silver nanoparticles consist of single-crystalline plates of pure silver. The surface-enhanced Raman spectroscopic (SERS) activity of the EGNP structures is promising due to the exceptionally rough surface structure of the silver nanoparticles. SERS measurements of the vitamin riboflavin incubated on the silver nanoparticles are shown as an exemplary application for quantitative analysis.

Screen printing as cost-efficient fabrication method for DNA-chips with electrical readout for detection of viral DNA

The fast development in the field of DNA analytics is driven by the need for cost-effective and high-throughput methods for the detection of biomolecules. The detection of DNA using metal nanoparticles as labels is an interesting alternative to the standard fluorescence technique. Fluorescence is highly sensitive and broadly established, but shows limitations, for example instability of the signal and the requirement for sophisticated and high-cost equipment. A recently developed approach realizes a method for the electrical detection of DNA, based on the induction of silver nanoparticles growth in microelectrode gaps on the surface of a DNA-chip. This breakthrough towards robust and cost-effective detection was still hampered by the need for microstructured (and therefore expensive) substrates. We demonstrate that it is possible to utilize screen printed electrode structures for a chip-based electrical DNA detection. The electrode structures were produced on a glass substrate which made an additional optical readout possible. The screen printed structures show the required precision and are compatible with the applied biochemical protocols. A comparison with chip substrates produced by standard photolithography showed the same sensitivity and specificity for the screen printed chips. Screen printing of electrode structures for DNA-chip with electrical detection offers an interesting and cost-efficient possibility to produce DNA-chips with microstructured electrodes.

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.

Novel Bottom-Up SERS Substrates for Quantitative and Parallelized Analytics

Surface-enhanced Raman spectroscopy (SERS) is an emerging technology in the field of analytics. Due to the high sensitivity in connection with specific Raman molecular fingerprint information SERS can be used in a variety of analytical, bioanalytical, and biosensing applications. However, for the SERS effect substrates with metal nanostructures are needed. The broad application of this technology is greatly hampered by the lack of reliable and reproducible substrates. Usually the activity of a given substrate has to be determined by time-consuming experiments such as calibration or ultramicroscopic studies. To use SERS as a standard analytical tool, cheap and reproducible substrates are required, preferably with a characterization technique that does not interfere with the subsequent measurements. Herein we introduce an innovative approach to produce low-cost and large-scale reproducible substrates for SERS applications, which allows easy and economical production of micropatterned SERS active surfaces on a large scale. This approach is based on an enzyme-induced growth of silver nanostructures. The special structural feature of the enzymatically deposited silver nanoparticles prevents the breakdown of SERS activity even at high particle densities (particle density >60%) that lead to a conductive layer. In contrast to other approaches, this substrate exhibits a relationship between electrical conductivity and the resulting SERS activity of a given spot. This enables the prediction of the SERS activity of the nanostructure ensemble and therewith the controllable and reproducible production of SERS substrates of enzymatic silver nanoparticles on a large scale, utilizing a simple measurement of the electrical conductivity. Furthermore, through a correlation between the conductivity and the SERS activity of the substrates it is possible to quantify SERS measurements with these substrates.

Flexible Biochips for Detection of Biomolecules

Miniaturization of biosensors is envisaged by the development of biochips consisting of parallel microarray patterns of binding sites on rigid substrates, such as glass or silicon. Thin plastic substrates are promising flexible alternatives because of the possibility for large-area roll-to-roll manufacturing of disposable chips at lower costs. Mature optical lithography technology faces many challenges when used to pattern flexible foils as a result of the substrate instabilities, especially at higher temperatures. In this work, flexible biochips with gold electrode patterns were fabricated on thin polyethylene naphthalate (PEN) foils using photolithography. The gold electrode structures of the chips were manufactured by direct metal patterning and by lift-off processing. Both methodologies resulted in well-defined electrode patterns as concluded from optical microscopy and scanning electron microscopy (SEM) characterization and resistance measurements. The biochips were successfully employed for the electrical and optical detection of DNA molecules. The DNA detection was based on the immobilization of capture DNA between electrode gaps, hybridization with biotin-labeled target DNA, and enzymatic silver enhancement.

DNA detection using a triple readout optical/AFM/MALDI planar microwell plastic chip

A ready-to-spot disposable DNA chip for specific and sensitive detection of DNA was developed. Plastic copolymeric substrate chemistry was optimized to selectively couple the target DNA with the active chip surface. At the same time, the developed substrate limits the unspecific adsorption of probe DNA molecules or additional polar contaminants in the test samples to the chip surface. The combination of glycidyl and n-butyl methacrylates was found to best fit the requirements of the assay. The fabricated DNA microarrays have mechanical properties similar to those of the glass or silicon substrates and, at the same time, provide chemically reactive surfaces that do not require lengthy chemical modification. An additional advantage of the plastic microchip is its compatibility with different analytical readout techniques, such as mass spectrometry (MALDI-TOF/MS), optical detection (fluorescence and enzyme-induced metal deposition), and imaging techniques (atomic force microscopy). These multiple readout techniques have given us the ability to compare the sensitivity, selectivity, and robustness of current state-of-the-art bioanalytical methods on the same platform exemplified by successful DNA-based detection of human cytomegalovirus. The obtained sensitivity for enzymatically enhanced silver deposition (10(-15) M) surpasses that of conventional fluorescence readouts. In addition, the assays dynamic range (10(-6)-10(-15) M), reproducibility, and reliability of the DNA probe detection speaks for the silver deposition method. At compromised sensitivity (10(-9) M), the length of the DNA probes could be checked and, alternatively, DNA single point polymorphisms could be analyzed.

Printed conductive features for DNA chip applications prepared on PET without sintering

We present here an innovative and cheap alternative for the preparation of conductive tracks printed on flexible polymer substrates at room temperature. For this purpose, we applied a combination of a Tollens reagent-based silver deposition and printed mask, using an office laser printer. The as-prepared conductive structures were used for DNA chip fabrication. The great advantage of the presented method is that the conductive features can be fabricated in a facile and inexpensive way, while maintaining the high flexibility to tailor the design to its application. The DNA chips showed the same response as well as sensitivity compared to chips made conventionally by photolithography or screen printing.

Chip-based molecular diagnostics using metal nanoparticles

BACKGROUND Chip-based bioanalytical methods represent a promising approach for a highly parallel and robust analysis with minimal sample volumes. Key process parameters that can be decisive for certain applications are determined by the detection scheme utilized. OBJECTIVE This review addresses typical requirements of chip-based detection systems, especially for the emerging field of point-of-care diagnostics that make possible field detection with less-trained personnel, robust assays as well as low instrumentation costs. METHODS The use of metal nanoparticles as labels represents a promising approach. They exhibit a high stability in signal and new detection schemes that would allow for robustness and low-cost readout. RESULTS/CONCLUSION First examples of this kind have been established and are in the market, and more are in the development pipeline.

Easy characterization of SERS substrates of enzymatically produced silver nanoparticles and their applications in the area of bioanalytics

The broad application of surface-enhanced Raman spectroscopy (SERS) is greatly hampered by the lack of reliable and reproducible substrates; usually the activity of a given substrate has to be determined by time-consuming experiments such as calibration studies or ultramicroscopy. To use SERS as a standard analytical tool, cheap and reproducible substrates are required, preferably characterizable with a technique that does not interfere with the subsequent measurements. Here, we introduce an innovative approach to produce low cost and large scale reproducible substrates for SERS applications, which allows an easy and economical production of micropatterned SERS active surfaces based on an enzyme induced growth of silver nanostructures. The special structural feature of the enzymatically deposited silver nanoparticles prevents the breakdown of SERS activity even at high particle densities and exhibits a relationship between electrical conductivity and resulting SERS activity of a given spot. This enables the prediction of the SERS activity of the nanostructure ensemble and therewith the controllable and reproducible production of SERS substrates of enzymatic silver nanoparticles on a large scale. Furthermore, the presented substrate shows a high reproducibility and is appropriate for various applications.

Plasmonic nanostructures for biophotonic applications

Within this contribution we convincingly demonstrate that the enhancement of the intrinsically weak Raman signals through an interaction between an analyte molecule and enhanced electromagnetic fields in the vicinity of metallic nanostructured surfaces is an extremely potent tool in bioanalytical science because such a SERS approach comprises high sensitivity with molecular specificity. In particular innovative approaches to realize reproducible plasmonic nanostructures i.e. SERS substrates like e.g. lithographically produced nanostructured gold surfaces or the defined deposition of silver nanoparticles through an enzymatic reaction are introduced.