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.     

Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in <Emphasis Type="Italic">Xenopus laevis</Emphasis> oocytes

Background  

Although numerous non-radioactive methods are in use to measure the catalytic activity of protein kinases, most require specialized equipment and reagents and are not sufficiently sensitive for the detection of endogenous kinase activity in biological samples. Kinases of the DYRK family have important functions in developmental and pathophysiological processes in eukaryotic organisms including mammals. We aimed to develop a highly sensitive, low-tech assay suitable to determine the activity of DYRK family kinases in tissues or cells from diverse sources.     

Detection of quorum sensing molecules in Burkholderia cepacia culture supernatants with enzyme-linked immunosorbent assays

The Burkholderia cepacia complex (Bcc) employs a quorum sensing (QS) mechanism which is a cell density-dependent bacterial communication system to regulate certain gene expressions. As with many other Gram-negative bacteria, Burkholderia cepacia species use (N-acyl-)homoserine lactones (AHLs or HSLs) as signalling molecules. Because of the essential role of QS in bacterial behavior, the aim of this study was to demonstrate the applicability of our in-house-developed enzyme-linked immunosorbent assays (ELISAs) for the detection of bacterial activities via HSLs in B. cepacia strain LA3 culture supernatants. For this purpose the previously developed monoclonal antibodies (mAbs) HSL1/2-2C10 and HSL1/2-4H5 were exploited. N-3-Oxo-decanoyl-L-homoserine lactone (3-oxo-C10-HSL) was used as main analyte throughout all experiments. With the bacterial culture medium (named ABC medium) a matrix effect in both ELISAs was visible (slight increase in optical density, shift in test midpoints (IC₅₀) and working ranges). For example, ELISA with mAb HSL1/2-2C10 and enzyme tracer HSL3-HRP (HSL derivative conjugated to horseradish peroxidase) had an IC₅₀ of 120 μg L⁻¹ for 3-oxo-C10-HSL in phosphate-buffered saline versus 372 μg L⁻¹ in ABC medium. A significant increase of HSLs in B. cepacia strain LA3 culture supernatants after 12 h to 48 h of growth was observed. Although the analytical result of these immunoassays cannot distinguish HSLs from homoserines (HSs), the appearance of these compounds can be easily followed. Hydrolysis and spiking experiments were carried out with these biological samples. According to our knowledge, these are the first immunoassays for the detection of quorum sensing molecules in biological culture supernatants. This study provides a cost-effective, fast, and sensitive analytical method for detection of HSLs/HSs in biological samples without complex sample preparation and will offer a quick idea about B. cepacia activities. The low sample amount requirement (less than 1 mL) constitutes a tremendous advantage for many analytical questions with biological samples.     

Microscale imaging of the preservation state of 5,000-year-old archaeological bones by synchrotron infrared microspectroscopy

Archaeological bone materials record characteristic markers of life in prehistoric times (dating, climate, environment, diet, human migration) in their isotopic and chemical composition in addition to palaeontological, archaeozoological, anthropological and palaeogenetic information. Thus, the discovery and conservation of archaeological bone materials is of great importance to get access to this information. However, archaeological materials are altered by different postmortem processes and it appears necessary to estimate if the archaeological information is still reliable or if it has been modified during burial. As archaeological bone materials present a high structural hierarchy at the micro- and nanoscale, changes induced by diagenetic phenomena have to be observed at these scales. One method for revealing post mortem changes of the bone structure and composition at the microscale is synchrotron radiation micro-FTIR imaging (SR micro-FTIR). Thus, thin sections of about 5,000-year-old archaeological bones have been analysed in transmission mode at the IRIS beamline (BESSY II, HZB Berlin) to determine markers of the state of bone preservation at the microscale. The archaeological bone material comes from station 19 of the Neolithic site of the Chalain Lake. By using SR micro-FTIR it was possible to image characteristic bone structures, e.g. osteons (the constitutive histological unit of cortical bone), using the absorption band ratios corresponding to different chemical bone constituents (collagen content and quality, phosphate crystallinity, carbonate content). These data allow us to precisely evaluate the state of preservation of a 5,000-year-old bone at the histological level.

Improved measurement of the hydrogen 1S-2S transition frequency

We have measured the 1S-2S transition frequency in atomic hydrogen via two-photon spectroscopy on a 5.8 K atomic beam. We obtain f(1S-2S) = 2,466,061,413,187,035 (10) Hz for the hyperfine centroid, in agreement with, but 3.3 times better than the previous result [M. Fischer et al., Phys. Rev. Lett. 92, 230802 (2004)]. The improvement to a fractional frequency uncertainty of 4.2 × 10(-15) arises mainly from an improved stability of the spectroscopy laser, and a better determination of the main systematic uncertainties, namely, the second order Doppler and ac and dc Stark shifts. The probe laser frequency was phase coherently linked to the mobile cesium fountain clock FOM via a frequency comb.     

Rudolf Kaiser — an Extraordinary Birthday

JPC – Journal of Planar Chromatography – Modern TLC -     

A New Family of Biuret Hydrolases Involved in S-Triazine Ring Metabolism

Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of biuret to allophanate and ammonia. The k(cat)/K(M) of 1.7 × 10(5) M(-1)s(-1) and the relatively low K(M) of 23 ± 4 μM together suggested that this enzyme acts uniquely on biuret physiologically. This is supported by the fact that of the 34 substrate analogs of biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for biuret. Biuret hydrolase does not react with carboxybiuret, the product of the enzyme immediately preceding biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway.