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.     

Investigation of the influence of voltage parameters on decay times in an ion mobility spectrometer with a pulsed non-radioactive electron source

Ion mobility spectrometry (IMS) is a well-known method for detecting hazardous compounds in air. Most ion mobility spectrometers use a radioactive source to provide electrons with high energy (5–50 keV) to ionize analytes in a series of chemical reactions. Instead of a radioactive source, we use a non-radioactive electron gun which can be operated in pulsed mode. Thus a delay time between ionization and ion extraction can be introduced which offers the possibility to use the signal decay characteristic of substances as a further discrimination parameter. The influence of voltages supplied to the reaction region and to the electron gun on signal intensities and decay times will be investigated in order to obtain further insight into the dependence of this signal decay on different experimental parameters and correspondingly into the underlying mechanisms.     

Synthesis and application of pyridine-based ambipolar hosts: control of charge balance in organic light-emitting devices by chemical structure modification

We studied the influence of a pyridine moiety versus a phenyl moiety when introduced in the molecular design of an ambipolar host. These pyridine-based host materials for organic light-emitting diodes (OLEDs) were synthesized in three to five steps from commercially available starting materials. The isomeric hosts have similar HOMO/LUMO energies; however, data from OLEDs fabricated using the above host materials demonstrate that small structural modification of the host results in significant changes in its carrier-transporting characteristics.     

Two-dimensional assembly of [Mn(III)₂Mn(II)₂] single-molecule magnets and [Cu(pic)₂] linking units (Hpic = picolinic acid)

In an attempt to develop novel coordination networks of SMMs, a Cu(II) picolinate complex has been used to coordinate S(T) = 9 tetranuclear Mn-based SMMs resulting in an intriguing 2D framework exhibiting a magnet-like behavior at low temperature.     

Redox electrodeposition polymers: adaptation of the redox potential of polymer-bound Os complexes for bioanalytical applications

The design of polymers carrying suitable ligands for coordinating Os complexes in ligand exchange reactions against labile chloro ligands is a strategy for the synthesis of redox polymers with bound Os centers which exhibit a wide variation in their redox potential. This strategy is applied to polymers with an additional variation of the properties of the polymer backbone with respect to pH-dependent solubility, monomer composition, hydrophilicity etc. A library of Os-complex-modified electrodeposition polymers was synthesized and initially tested with respect to their electron-transfer ability in combination with enzymes such as glucose oxidase, cellobiose dehydrogenase, and PQQ-dependent glucose dehydrogenase entrapped during the pH-induced deposition process. The different polymer-bound Os complexes in a library containing 50 different redox polymers allowed the statistical evaluation of the impact of an individual ligand to the overall redox potential of an Os complex. Using a simple linear regression algorithm prediction of the redox potential of Os complexes becomes feasible. Thus, a redox polymer can now be designed to optimally interact in electron-transfer reactions with a selected enzyme.     

LC-MS/MS screening method for designer amphetamines, tryptamines, and piperazines in serum

Since the late 1990s and early 2000s, derivatives of well-known designer drugs as well as new psychoactive compounds have been sold on the illicit drug market and have led to intoxications and fatalities. The LC-MS/MS screening method presented covers 31 new designer drugs as well as cathinone, methcathinone, phencyclidine, and ketamine which were included to complete the screening spectrum. All but the last two are modified molecular structures of amphetamine, tryptamine, or piperazine. Among the amphetamine derivatives are cathinone, methcathinone, 3,4-DMA, 2,5-DMA, DOB, DOET, DOM, ethylamphetamine, MDDMA, 4-MTA, PMA, PMMA, 3,4,5-TMA, TMA-6 and members of the 2C group: 2C-B, 2C-D, 2C-H, 2C-I, 2C-P, 2C-T-2, 2C-T-4, and 2C-T-7. AMT, DPT, DiPT, MiPT, DMT, and 5MeO-DMT are contained in the tryptamine group, BZP, MDBP, TFMPP, mCPP, and MeOPP in the piperazine group. Using an Applied Biosystems LC-MS/MS API 365 TurboIonSpray it is possible to identify all 35 substances. After addition of internal standards and mixed-mode solid-phase extraction the analytes are separated using a Synergi Polar RP column and gradient elution with 1 mM ammonium formate and methanol/0.1% formic acid as mobile phases A and B. Data acquisition is performed in MRM mode with positive electro spray ionization. The assay is selective for all tested substances. Limits of detection were determined by analyzing S/N-ratios and are between 1.0 and 5.0 ng/mL. Matrix effects lie between 65% and 118%, extraction efficiencies range from 72% to 90%.     

Alignment of retention time obtained from multicapillary column gas chromatography used for VOC analysis with ion mobility spectrometry

Multicapillary column (MCC) ion mobility spectrometers (IMS) are increasingly in demand for medical diagnosis, biological applications and process control. In a MCC-IMS, volatile compounds are differentiated by specific retention time and ion mobility when rapid preseparation techniques are applied, e.g. for the analysis of complex and humid samples. Therefore, high accuracy in the determination of both parameters is required for reliable identification of the signals. The retention time in the MCC is the subject of the present investigation because, for such columns, small deviations in temperature and flow velocity may cause significant changes in retention time. Therefore, a universal correction procedure would be a helpful tool to increase the accuracy of the data obtained from a gas-chromatographic preseparation. Although the effect of the carrier gas flow velocity and temperature on retention time is not linear, it could be demonstrated that a linear alignment can compensate for the changes in retention time due to common minor deviations of both the carrier gas flow velocity and the column temperature around the MCC-IMS standard operation conditions. Therefore, an effective linear alignment procedure for the correction of those deviations has been developed from the analyses of defined gas mixtures under various experimental conditions. This procedure was then applied to data sets generated from real breath analyses obtained in clinical studies using different instruments at different measuring sites for validation. The variation in the retention time of known signals, especially for compounds with higher retention times, was significantly improved. The alignment of the retention time—an indispensable procedure to achieve a more precise identification of analytes—using the proposed method reduces the random error caused by small accidental deviations in column temperature and flow velocity significantly.     

GC/MS detection of central nervous tissue as specified BSE risk material in meat products and meat and bone meals: thermal stability of markers in comparison with immunochemistry and RT-PCR

Methods for the detection of central nervous tissue (CNT) are urgently needed in food control as a means for controlling strict adherence to both food labeling and banning of specified BSE risk material. Here, we report data on heat stability of the CNT markers neuron-specific enolase (NSE) in western blotting, glial fibrillary acidic protein (GFAP) in an enzyme linked immunoassay, mRNA_GFAP in a real-time PCR assay, and several fatty acids (C22:6, C24:0-OH, C24:1ω9/ω7, C24:1ω9-OH/ω7-OH, and C24:0) in gas chromatography mass spectrometry (GC/MS). The sample matrix, a standard material of emulsion-type sausage with varied contents of CNT (brain), was heat-treated in three studies: (1) routine meat technological heat treatment with low (85 °C, 30 min), medium (115 °C, 30 min), and high (133 °C, 30 min, 3 bar) heating of 72 anonymous samples from a blind trial; (2) heat treatment under experimental conditions (100, 110, …, 200 °C, 45 min); and (3) fractionized heating of central nervous system (up to three times) under moderate routine technological conditions (85, 100, and 115 °C, 30 min). The markers of the immunochemical methods showed a low GFAP or very low NSE temperature stability at medium and high temperature conditions. The real-time PCR assay gave inconsistent, non-quantitative results, which indicated an uncontrollable matrix effect. The relevant GC/MS markers (C24:0-OH, C24:1ω9/ω7, and C24:1ω9-OH/ω7-OH) proved to be extremely stable. Neither meat and bone meal conditions (133 °C) nor experimental heating (up to and above 140 °C) showed any reduction of GC/MS CNT quantification. On the contrary, a slight but significant increase was noted over a certain temperature range (120–140 °C) for most fatty acids, possibly due to an improved extractability of the fatty acids. We conclude that a quantitative approach is highly unreliable when using immunochemical methods; moreover, these methods might be basically prone to false-negative results depending on heat treatment and matrix composition. Therefore, antibodies with higher affinity to heat-treated CNT marker epitopes are needed. Relevant amounts of CNT (≥0.5%) in low- and medium-heated products would still be reliably detectable by the GFAP ELISA, which justifies its use as a screening method in official food control. The results obtained by the real-time PCR assay were contradictory to recently published data, indicating a need for further protocol optimization and collaborative trials. Up to date, the analytical approach using GC/MS is the only valid procedure as pertaining to heat stability and quantitative analysis; consequently, it should be recommended as the reference procedure in official food control for CNT detection in heat-treated meat products.     

Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro

Ketamine, a phencyclidine derivative, is used for induction of anesthesia, as an anesthetic drug for short term surgical interventions and in subanesthetic doses for postoperative pain relief. Ketamine undergoes extensive hepatic first-pass metabolism. Enantioselective capillary electrophoresis with multiple isomer sulfated β-cyclodextrin as chiral selector was used to identify cytochrome P450 enzymes involved in hepatic ketamine and norketamine biotransformation in vitro. The N-demethylation of ketamine to norketamine and subsequently the biotransformation of norketamine to other metabolites were studied via analysis of alkaline extracts of in vitro incubations of racemic ketamine and racemic norketamine with nine recombinantly expressed human cytochrome P450 enzymes and human liver microsomes. Norketamine was formed by CYP3A4, CYP2C19, CYP2B6, CYP2A6, CYP2D6 and CYP2C9, whereas CYP2B6 and CYP2A6 were identified to be the only enzymes which enable the hydroxylation of norketamine. The latter two enzymes produced metabolic patterns similar to those found in incubations with human liver microsomes. The kinetic data of ketamine N-demethylation with CYP3A4 and CYP2B6 were best described with the Michaelis–Menten model and the Hill equation, respectively. This is the first study elucidating the individual enzymes responsible for hydroxylation of norketamine. The obtained data suggest that in vitro biotransformation of ketamine and norketamine is stereoselective.