Enhancing the substrate selectivity of enzyme mimetics in biosensing and bioassay: Novel approaches.

A substantial development in nanoscale materials possessing catalytic activities comparable with natural enzymes has been accomplished. Their advantages were owing to the excellent sturdiness in an extreme environment, possibilities of their large-scale production resulting in higher profitability, and easy manipulation for modification. Despite these advantages, the main challenge for artificial enzyme mimetics is the lack of substrate selectivity where natural enzymes flourish. This review addresses this vital problem by introducing substrate selectivity strategies to three classes of artificial enzymes: molecularly imprinted polymers, nanozymes (NZs), and DNAzymes. These rationally designed strategies enhance the substrate selectivity and are discussed and exemplified throughout the review. Various functional mechanisms associated with applying enzyme mimetics in biosensing and bioassays are also given. Eventually, future directives toward enhancing the substrate selectivity of biomimetics and related challenges are discussed and evaluated based on their efficiency and convenience in biosensing and bioassays.

An inexpensive UV-LED photoacoustic based real-time sensor-system detecting exhaled trace-acetone.

In this research we present a low-cost system for breath acetone analysis based on UV-LED photoacoustic spectroscopy. We considered the end-tidal phase of exhalation, which represents the systemic concentrations of volatile organic compounds (VOCs) - providing clinically relevant information about the human health. This is achieved via the development of a CO2-triggered breath sampling system, which collected alveolar breath over several minutes in sterile and inert containers. A real-time mass spectrometer is coupled to serve as a reference device for calibration measurements and subsequent breath analysis. The new sensor system provided a 3σ detection limit of 8.3 ppbV and an NNEA of 1.4E-9 Wcm-1Hz-0.5. In terms of the performed breath analysis measurements, 12 out of 13 fell within the error margin of the photoacoustic measurement system, demonstrating the reliability of the measurements in the field.

A sub-ppbv-level Acetone and Ethanol Quantum Cascade Laser Based Photoacoustic Sensor - Characterization and Multi-Component Spectra Recording in Synthetic Breath.

Trace gas analysis in breath is challenging due to the vast number of different components. We present a highly sensitive quantum cascade laser based photoacoustic setup for breath analysis. Scanning the range between 8263 and 8270 nm with a spectral resolution of 48 pm, we are able to quantify acetone and ethanol within a typical breath matrix containing water and CO2. We photoacoustically acquired spectra within this region of mid-infra-red light and prove that those spectra do not suffer from non-spectral interferences. The purely additive behavior of a breath sample spectrum was verified by comparing it with the independently acquired single component spectra using Pearson and Spearman correlation coefficients. A previously presented simulation approach is improved and an error attribution study is presented. With a 3σ detection limit of 6.5 ppbv in terms of ethanol and 250 pptv regarding acetone, our system is among the best performing presented so far.

Combining amperometry and mass spectrometry as a dual detection approach for capillary electrophoresis.

Dual detection concepts (DDCs) are becoming more and more popular in analytical chemistry. In this work, we describe a novel DDC for capillary electrophoresis (CE) consisting of an amperometric detector (AD) and a mass spectrometer (MS). This detector combination has a good complementarity as the AD exhibits high sensitivity, whereas the MS provides excellent selectivity. Both detectors are based on a destructive detection principle, making a serial detector arrangement impossible. Thus, for the realization of the DDC, the CE flow was divided into two parts with a flow splitter. The DDC was characterized in a proof-of-concept study with ferrocene derivates and a nonaqueous background electrolyte. We could show that splitting the CE flow was a suitable method for the instrumental realization of the DDC consisting of two destructive detectors. By lowering the height of the AD compared to the MS, it was possible to synchronize the detector responses. Additionally, for the chosen model system, we confirmed that the AD was much more reproducible and had lower limits of detection (LODs) than the MS. The LODs were identical for the DDC and the single-detection arrangements, indicating no sensitivity decrease due to the CE flow splitting. The DDC was successfully applied to determine the drug and doping agent trimetazidine.

Dead volume-free flow splitting in capillary electrophoresis.

In recent years, several dual detection concepts (DDCs) for CE were developed, which consisted of at least one nondestructive detector. For these DDCs, a linear detector arrangement could be used, which is not possible when both detectors are destructive. To overcome this problem, we developed a concept for the splitting of the CE stream utilizing commercially available flow splitters (FSs) that allow the parallel positioning of two destructive detectors. In this proof-of-concept study, T- and Y-shaped FSs were characterized regarding their suitability for DDCs. To keep it simple, a UV detector (UV) and a C4 D were used for the characterization. The model system consisted of an acetonitrile-based background electrolyte and the two model substances, (ferrocenylmethyl)trimethylammonium iodide and caffeine. CE hyphenated to a UV detector (CE-UV) measurements revealed that the split ratio was about 50% for both FSs. CE-C4 D was used to evaluate the peak shape in front of and behind the FSs. These measurements showed that there was no significant peak broadening introduced by the FSs. Additionally, there were no changes in the LODs in front of and behind the FSs. Furthermore, the flexibility of the new FS approach allowed the usage of capillaries with different ids (25-75 µm) for injection and detection.

An Algorithmic Approach to Compute the Effect of Non-Radiative Relaxation Processes in Photoacoustic Spectroscopy.

Successful transfer of photoacoustic gas sensors from laboratory to real-life applications requires knowledge about potential cross-sensitivities towards environmental and gas matrix changes. Multi-dimensional calibration in case of cross-sensitivities can become very complex or even unfeasible. To address this challenge, we present a novel algorithm to compute the collision based non-radiative efficiency and phase lag of energy relaxation on a molecular level (CoNRad) for photoacoustic signal calculation. This algorithmic approach allows to calculate the entire relaxation cascade of arbitrarily complex systems, yielding a theoretical photoacoustic signal. In this work the influence of varying bulk compositions, i.e. nitrogen (N2), oxygen (O2) and water (H2O) on the photoacoustic signal during methane (CH4) detection is demonstrated. The applicability of the algorithm to other photoacoustic setups is shown exemplary by applying it to the relaxational system investigated in [1]. Hayden et al. examined the effect of water on photoacoustic carbon monoxide (CO) detection.

Catalyst Formation and In Operando Monitoring of the Electrocatalytic Activity in Flow Reactors.

Flow reactors are of increasing importance and have become crucial devices due to their wide application in chemical synthesis, electrochemical hydrogen evolution reaction (HER), or electrochemical waste water treatment. In many of these applications, catalyst materials such as transition-metal chalcogenides (TMCs) for the HER, provide the desired electrochemical reactivity for the HER. Generally, the flow electrolyzers' performance is evaluated as the overall output, but the decrease in activity of the electrolyzer is due to localized failure of the catalyst. Herein, we present a method for the spatially resolved (tens of micrometers) In Operando analysis of the catalytic activity under real operation conditions as well as the localized deposition of the catalyst in an operating model flow reactor. For these purposes, scanning electrochemical microscopy was applied for MoSx catalyst deposition and for localized tracking of the TMC activity with a resolution of 25 µm. This approach offers detailed information about the catalytic performance and should find broad application for the characterization and optimization of flow reactor catalysis under real operational conditions.

Enhanced resolution of generator-collector studies of enzymatic structures by means of hydrodynamic scanning electrochemical microscopy.

In this report, the effects of forced convection on scanning electrochemical microscopy (SECM) studies of enzymes in the context of the generator-collector mode (G/C mode) were investigated. Forced convection was generated via an electrical high precision stirrer integrated into the electrochemical cell. Circular spots of glucose oxidase were immobilized on a gold support serving as model substrate. The diffusion layer of enzymatically generated H2O2 was characterized recording probe scan curves (PSCs) in z-direction. Furthermore, the enzyme-modified surfaces were investigated via constant-height SECM imaging in feedback mode and in G/C mode. For methodical comparison all sets of experiments were performed in quiescent solution (conventional approach) and with forced convection, respectively. In contrast to a growing diffusion layer without forced convection by applying forced convection, a constant diffusion layer of produced H2O2 was observed. Hence, via hydrodynamic SECM time-independent images within a reasonable time scale of SECM measurements in G/C mode were enabled and their resolution was enhanced.

Investigation of the Electrooxidation of Thymine on Screen-Printed Carbon Electrodes by Hyphenation of Electrochemistry and Mass Spectrometry.

The electrooxidation of thymine on screen-printed carbon electrodes was investigated utilizing different complementary instrumental approaches. The potential-dependent product profile was obtained by recording real-time mass voltammograms. Electrochemical flow cells with integrated disposable electrodes were directly coupled with mass spectrometry to facilitate a very fast detection of electrogenerated species. Thymine dimers were found at a potential of about 1.1 V in ammonium acetate (pH 7.0) and 1.25 V in ammonium hydrogen carbonate electrolyte (pH 8.0). Electrochemistry-capillary electrophoresis-mass spectrometry measurements revealed that two isobaric isomers of a dimeric oxidation product were formed. Separations at different time intervals between end of oxidation and start of separation showed that these were hydrated over time. An investigation of the pKa values by changing the separation conditions in electrochemistry-capillary electrophoresis-ultraviolet-visible spectroscopy measurements allowed for further characterization of the primary oxidation products. The results showed that both isomers exhibited two deprotonation steps. The oxidation products were further characterized by high-performance liquid chromatography-tandem mass spectrometry. Based on the obtained data, the main oxidation products of thymine in aqueous solution could most likely be identified as N(1)-C(5') and N(1)-C(6') linked dimer species evolving into the corresponding dimer hydrates over time. The presented methods for online characterization of electrochemically pretreated samples showed that not only mass spectrometric data can be obtained by electrochemistry-mass spectrometry but also further characterizations such as the investigation of product stability and the pH-dependent protonation or deprotonation behavior are possible. This is valid not only for stable oxidation products but also for intermediates, as analysis can be carried out within a short time scale. Thus, a vast amount of valuable experimental data can be acquired, which can help in understanding electrooxidation processes.

Scanning Electrochemical Microscopy of Electrically Heated Wire Substrates.

We report a new configuration for enhancing the performance of scanning electrochemical microscopy (SECM) via heating of the substrate electrode. A flattened Pt microwire was employed as the substrate electrode. The substrate was heated by an alternating current (AC), resulting in an increased mass transfer between the wire surface and the bulk solution. The electrochemical response of the Pt wire during heating was investigated by means of cyclic voltammetry (CV). The open circuit potential (OCP) of the wire was recorded over time, while varied heating currents were applied to investigate the time needed for establishing steady-state conditions. Diffusion layer studies were carried out by performing probe approach curves (PACs) for various measuring modes of SECM. Finally, imaging studies of a heated substrate electrode surface, applying feedback, substrate generation/tip collection (SG/TC), and the competition mode of SECM, were performed and compared with room temperature results.

Selectivity enhancement in capillary electrophoresis by means of two-dimensional separation or dual detection concepts.

For the identification and quantification of analytes in complex samples, highly selective analytical strategies are required. The selectivity of single separation techniques such as gas chromatography (GC), liquid chromatography (LC), or capillary electrophoresis (CE) with common detection principles can be enhanced by hyphenating orthogonal separation techniques but also by using complementary detection systems. In this review, two-dimensional systems containing CE in at least one dimension are reviewed, namely LC-CE or 2D CE systems. Particular attention is paid to the aspect of selectivity enhancement due to the orthogonality of the different separation mechanisms. As an alternative concept, dual detection approaches are reviewed using the common detectors of CE such as UV/VIS, laser-induced fluorescence, capacitively coupled contactless conductivity (C4D), electrochemical detection, and mass spectrometry. Special emphasis is given to dual detection systems implementing the highly flexible C4D as one detection component. Selectivity enhancement can be achieved in case of complementarity of the different detection techniques.

High-resolution polymer high performance liquid chromatography: Application of a saw tooth gradient for the separation of various polymers.

Currently, a lot of research effort in polymer analysis by liquid chromatographic techniques, including size exclusion chromatography (SEC), polymer HPLC or liquid chromatography at critical conditions, is done aiming to improve separation performance. In this study, novel gradient protocols were investigated primarily based on gradient polymer elution chromatography (GPEC). Starting with linear gradients and stepwise gradients a new periodic saw tooth gradient profile was developed and optimized. Optimum settings for the saw tooth gradient design were evaluated by design of experiments (DoE) based on Taguchi's methodology for various types of stationary phases. The gain of peak resolution was dependent on the effective gradient step height. The optimized protocol enabled high-resolution polymer HPLC (HRP-HPLC) separations with common HPLC instruments. The quality of separation was evaluated by heart-cut fraction collection of HRP-HPLC and subsequent determination of the individual fractions by SEC or MALDI-ToF mass spectrometry. Finally, different types of polymers, such as PVC, PDMS, PMMA, or PPG, were studied with the new method and a universal applicability was shown.

New Electrochemical Flow-Cell Configuration Integrated into a Three-Dimensional Microfluidic Platform: Improving Analytical Application in the Presence of Air Bubbles.

A newly configured electrochemical flow cell to be used for (end-channel) amperometric detection in a microfluidic device is presented. The design was assembled to place the reference electrode in a separated compartment, isolated from the flow in the microchannel, while the working and counter electrodes remain in direct contact with both compartments. Moreover, a three-dimensional coil-shaped microfluidic device was fabricated using a nonconventional protocol. Both devices working in association enabled us to solve the drawback caused by the discrete injection when the automatic micropipette was used. The high performance of the proposed electrochemical flow cell was demonstrated after in situ modifying the surface of the platinum working electrode with surfactant (e.g., using Tween 20 at 0.10%). As the reference electrode remained out of contact with the flowing solution, there was no trouble by air bubble formation (generated by accidental insertion or by presence of surfactants) throughout the measurements. This device was characterized regarding its analytical performance by evaluating the amperometric detection of acetaminophen, enabling determination from 6.60 to 66.0 µmol L-1. This issue is important since at high concentration (e.g., as assessed in clinical analysis) the acetaminophen is known to passivate the working electrode surfaces by electrogenerated products, impairing the accuracy of the electrochemical measurements.

Methodical studies of the simultaneous determination of anions and cations by IC×CE-MS using arsenic species as model analytes.

The separation of the constituents of complex sample mixtures is a challenging task in analytical chemistry. Multidimensional separation systems are widely used to enhance the peak capacity. The comprehensive hyphenation of ion chromatography (IC) and capillary electrophoresis (CE) is promising because the two most important instrumental techniques in ion analysis are combined. In this report a new configuration for capillary anion chromatography is presented enabling the simultaneous IC×CE analysis of anions and cations using a switching valve. Electrospray ionization mass spectrometry (MS) was used for detection. A mixture of organic and inorganic arsenic species served as a model system. The coupling of anion chromatography to CE-MS was done via a modulator enabling periodical injections of the IC effluent into the CE. The injection parameters of the modulator were studied taking into account the complex transport situation. Graphical abstract ᅟ.

Adaption of a parallel-path poly(tetrafluoroethylene) nebulizer to an evaporative light scattering detector: Optimization and application to studies of poly(dimethylsiloxane) oligomers as a model polymer.

The adaption of an parallel-path poly(tetrafluoroethylene)(PTFE) ICP-nebulizer to an evaporative light scattering detector (ELSD) was realized. This was done by substituting the originally installed concentric glass nebulizer of the ELSD. The performance of both nebulizers was compared regarding nebulizer temperature, evaporator temperature, flow rate of nebulizing gas and flow rate of mobile phase of different solvents using caffeine and poly(dimethylsiloxane) (PDMS) as analytes. Both nebulizers showed similar performances but for the parallel-path PTFE nebulizer the performance was considerably better at low LC flow rates and the nebulizer lifetime was substantially increased. In general, for both nebulizers the highest sensitivity was obtained by applying the lowest possible evaporator temperature in combination with the highest possible nebulizer temperature at preferably low gas flow rates. Besides the optimization of detector parameters, response factors for various PDMS oligomers were determined and the dependency of the detector signal on molar mass of the analytes was studied. The significant improvement regarding long-term stability made the modified ELSD much more robust and saved time and money by reducing the maintenance efforts. Thus, especially in polymer HPLC, associated with a complex matrix situation, the PTFE-based parallel-path nebulizer exhibits attractive characteristics for analytical studies of polymers.

Dual detection for non-aqueous capillary electrophoresis combining contactless conductivity detection and mass spectrometry.

Coupling of two detectors is a powerful tool to enhance the overall analytical performance generating complementary information and overcoming the limitations of the single detectors. In this work, capacitively coupled contactless conductivity detection (C4D) and electrospray ionization mass spectrometry (ESI-MS) were coupled in conjunction with non-aqueous capillary electrophoresis (NACE). Non-aqueous electrolytes are highly compatible with ESI due to their volatility. Moreover, they exhibit low background conductivity, which is essential for the detection with C4D. A NACE-C4D-MS method was developed using an acetonitrile buffer containing 2 M HAc and 4 mM NH4Ac as background electrolyte. The influence of the inner diameter of the separation capillary on the C4D was studied and taken into account. A capillary with 50 µm inner diameter was found to be best suited. The complementarity of the two detectors was shown by determining a sample mixture containing choline, thiamine, nitrate, and chloride as well as bromide and acetylcholine as internal standards. The C4D was the detector of choice for the inorganic ions, which were not detectable with the MS whereas the MS had much lower limits of detections for the organic biomolecules. The method was applied on an extract of a food supplement containing the model analytes.

Scanning Electrochemical Microscopy with Forced Convection Introduced by High-Precision Stirring.

In the present report the well-known advantages of hydrodynamic mass transport in electrochemical systems are used in combination with scanning electrochemical microscopy (SECM). The hydrodynamic SECM system integrates a high-precision stirring device into the experimental setup. The well-defined stirring of the SECM electrolyte results in steady state diffusion layer characteristics in the vicinity of large substrate electrodes operated in chronoamperometric measuring mode. For a range of rotation frequencies of a rotating cylinder the thickness and the stability of the diffusion layer was studied by hydrodynamic SECM in the substrate generation/tip collection (SG/TC) as well as in the competition mode using ferrocene methanol as redox mediator. Different UME probe dimensions ranging from Pt diameters of 20 µm down to 0.6 µm were used. The smallest probe with dPt = 0.6 µm was found most suitable for these studies due to the almost convection-independent amperometric response associated with subµm electrodes. Additionally, preliminary studies of hydrodynamic SECM imaging of a 2 mm Pt disk electrode surface in the SG/TC mode based on in situ produced hydrogen as a mediator are presented. Comparative images measured in the conventional positive feedback mode in quiescent solution show that hydrodynamic SECM offers attractive complementary information.

Surfactant-free microemulsion electrokinetic chromatography (SF-MEEKC) with UV and MS detection - a novel approach for the separation and ESI-MS detection of neutral compounds.

Microemulsion electrokinetic chromatography (MEEKC) is a powerful tool to separate neutral species based on differences in their hydrophobic and hydrophilic properties. However, as a major drawback the conventionally used SDS based microemulsions are not compatible with electrospray ionization mass spectrometry (ESI-MS). In this work, a surfactant-free microemulsion (SFME) consisting of water, ethanol, and 1-octanol is used for surfactant-free microemulsion electrokinetic chromatography (SF-MEEKC). Ammonium acetate was added to the SFME enabling electrophoretic separations. The stability of SFMEs containing ammonium acetate was investigated using small-angle X-ray scattering and dynamic light scattering. A method for the separation of a model system of hydrophobic and hydrophilic neutral vitamins, namely the vitamins B2 and D3, and the cationic vitamin B1 was developed using UV/VIS detection. The influence of the ammonium acetate concentration on the separation performance was studied in detail. The method was characterized concerning reproducibility of migration times and peak areas and concerning the linearity of the calibration data. Furthermore, SF-MEEKC was coupled to ESI-MS investigating the compatibility between SFMEs and the ESI process. The signal intensities of ESI-MS measurements of the model analytes were comparable for SFMEs and aqueous systems. Finally, the vitamin D3 content of a drug treating vitamin D3 deficiency was determined by SF-MEEKC coupled to ESI-MS using 25-hydroxycholecalciferol as an internal standard. Graphical abstract The concept of surfactant-free microemulsion electrokinetic chromatography coupled to electrospray ionization mass spectrometry.

CE-UV/VIS and CE-MS for monitoring organic impurities during the downstream processing of fermentative-produced lactic acid from second-generation renewable feedstocks.

BACKGROUND: During the downstream process of bio-based bulk chemicals, organic impurities, mostly residues from the fermentation process, must be separated to obtain a pure and ready-to-market chemical. In this study, capillary electrophoresis was investigated for the non-targeting downstream process monitoring of organic impurities and simultaneous quantitative detection of lactic acid during the purification process of fermentatively produced lactic acid. The downstream process incorporated 11 separation units, ranging from filtration, adsorption and ion exchange to electrodialysis and distillation, and 15 different second-generation renewable feedstocks were processed into lactic acid. The identification of organic impurities was established through spiking and the utilization of an advanced capillary electrophoresis mass spectrometry system. RESULTS: A total of 53 % of the organic impurities were efficiently removed via bipolar electrodialysis; however, one impurity, pyroglutamic acid, was recalcitrant to separation. It was demonstrated that the presence of pyroglutamic acid disrupts the polymerization of lactic acid into poly lactic acid. Pyroglutamic acid was present in all lactic acid solutions, independent of the type of renewable resource or the bacterium applied. Pyroglutamic acid, also known as 5-oxoproline, is a metabolite in the glutathione cycle, which is present in all living microorganisms. pyroglutamic acid is found in many proteins, and during intracellular protein metabolism, N-terminal glutamic acid and glutamine residues can spontaneously cyclize to become pyroglutamic acid. Hence, the concentration of pyroglutamic acid in the lactic acid solution can only be limited to a certain amount. CONCLUSIONS: The present study proved the capillary electrophoresis system to be an important tool for downstream process monitoring. The high product concentration encountered in biological production processes did not hinder the capillary electrophoresis from separating and detecting organic impurities, even at minor concentrations. The coupling of the capillary electrophoresis with a mass spectrometry system allowed for the straightforward identification of the remaining critical impurity, pyroglutamic acid. Although 11 separation units were applied during the downstream process, the pyroglutamic acid concentration remained at 12,900 ppm, which was comparatively high. All organic impurities found were tracked by the capillary electrophoresis, allowing for further separation optimization.

Combining C(4) D and MS as a dual detection approach for capillary electrophoresis.

The hyphenation of two detectors in combination with separation techniques is a powerful tool to enhance the analytical information. In this work, we present for the first time the coupling of two important detectors for capillary electrophoresis (CE), namely capacitively coupled contactless conductivity detection (C(4) D) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The elaborated experimental protocol took into account the requirements of separation aspects and the compatibility with both detectors. ESI-TOF-MS requires background electrolytes (BGE) containing only volatile components such as ammonium acetate or formate. These, however, exhibit a rather high conductivity, which is disadvantageous for C(4) D. Thus, the selection of the BGE in an appropriate concentration was undertaken for the determination of various phenolic compounds serving as a model system. The chosen BGE was a 10 mM ammonium acetate/ammonia buffer with a pH of 9. This BGE was a compromise concerning the detection performance of both detectors. The LODs for m-cresol, m- and p-nitrophenol, and 2,4-dinitrophenol were 3.1 µM (C(4) D), 0.8 µM (MS), 0.8 µM (MS), and 1.5 µM (MS), respectively. Moreover, the overall separation efficiency was excellent illustrating that detector-induced band broadening can be neglected in the CE-C(4) D/MS system. The analytical characteristics for the determination of phenolic compounds show the suitability of this dual detection approach and demonstrate the complementary use of C(4) D and MS detection.