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Keywords
Frequency mixing magnetic detection (FMMD) has been widely utilized as a measurement technique in magnetic immunoassays. It can also be used for the characterization and distinction (also known as “colourization”) of different types of magnetic nanoparticles (MNPs) based on their core sizes. In a previous work, it was shown that the large particles contribute most of the FMMD signal. This leads to ambiguities in core size determination from fitting since the contribution of the small-sized particles is almost undetectable among the strong responses from the large ones. In this work, we report on how this ambiguity can be overcome by modelling the signal intensity using the Langevin model in thermodynamic equilibrium including a lognormal core size distribution fL(dc,d0,σ) fitted to experimentally measured FMMD data of immobilized MNPs. For each given median diameter d0, an ambiguous amount of best-fitting pairs of parameters distribution width σ and number of particles Np with R2 > 0.99 are extracted. By determining the samples’ total iron mass, mFe, with inductively coupled plasma optical emission spectrometry (ICP-OES), we are then able to identify the one specific best-fitting pair (σ, Np) one uniquely. With this additional externally measured parameter, we resolved the ambiguity in core size distribution and determined the parameters (d0, σ, Np) directly from FMMD measurements, allowing precise MNPs sample characterization.
Biologically sensitive field-effect devices (BioFEDs) advantageously combine the electronic field-effect functionality with the (bio)chemical receptor’s recognition ability for (bio)chemical sensing. In this review, basic and widely applied device concepts of silicon-based BioFEDs (ion-sensitive field-effect transistor, silicon nanowire transistor, electrolyte-insulator-semiconductor capacitor, light-addressable potentiometric sensor) are presented and recent progress (from 2019 to early 2021) is discussed. One of the main advantages of BioFEDs is the label-free sensing principle enabling to detect a large variety of biomolecules and bioparticles by their intrinsic charge. The review encompasses applications of BioFEDs for the label-free electrical detection of clinically relevant protein biomarkers, deoxyribonucleic acid molecules and viruses, enzyme-substrate reactions as well as recording of the cell acidification rate (as an indicator of cellular metabolism) and the extracellular potential.
The light-addressable potentiometric sensor (LAPS) is an electrochemical sensor with a field-effect structure to detect the variation of the Nernst potential at its sensor surface, the measured area on which is defined by illumination. Thanks to this light-addressability, the LAPS can be applied to chemical imaging sensor systems, which can visualize the two-dimensional distribution of a particular target ion on the sensor surface. Chemical imaging sensor systems are expected to be useful for analysis of reaction and diffusion in various electrochemical and biological samples. Recent developments of LAPS-based chemical imaging sensor systems, in terms of the spatial resolution, measurement speed, image quality, miniaturization and integration with microfluidic devices, are summarized and discussed.
Realization of a calorimetric gas sensor on polyimide foil for applications in aseptic food industry
(2010)
Realisation of a calorimetric gas sensor on polyimide foil for applications in aseptic food industry
(2012)
A calorimetric gas sensor is presented for the monitoring of vapour-phase H2O2 at elevated temperature during sterilisation processes in aseptic food industry. The sensor was built up on a flexible polyimide foil (thickness: 25 μm) that has been chosen due to its thermal stability and low thermal conductivity. The sensor set-up consists of two temperature-sensitive platinum thin-film resistances passivated by a layer of SU-8 photo resist and catalytically activated by manganese(IV) oxide. Instead of an active heating structure, the calorimetric sensor utilises the elevated temperature of the evaporated H2O2 aerosol. In an experimental test rig, the sensor has shown a sensitivity of 4.78 °C/(%, v/v) in a H2O2 concentration range of 0%, v/v to 8%, v/v. Furthermore, the sensor possesses the same, unchanged sensor signal even at varied medium temperatures between 210 °C and 270 °C of the gas stream. At flow rates of the gas stream from 8 m3/h to 12 m3/h, the sensor has shown only a slightly reduced sensitivity at a low flow rate of 8 m3/h. The sensor characterisation demonstrates the suitability of the calorimetric gas sensor for monitoring the efficiency of industrial sterilisation processes.
Hydrogen peroxide (H₂O₂), a strong oxidizer, is a commonly used sterilization agent employed during aseptic food processing and medical applications. To assess the sterilization efficiency with H₂O₂, bacterial spores are common microbial systems due to their remarkable robustness against a wide variety of decontamination strategies. Despite their widespread use, there is, however, only little information about the detailed time-resolved mechanism underlying the oxidative spore death by H₂O₂. In this work, we investigate chemical and morphological changes of individual Bacillus atrophaeus spores undergoing oxidative damage using optical sensing with trapping Raman microscopy in real-time. The time-resolved experiments reveal that spore death involves two distinct phases: (i) an initial phase dominated by the fast release of dipicolinic acid (DPA), a major spore biomarker, which indicates the rupture of the spore’s core; and (ii) the oxidation of the remaining spore material resulting in the subsequent fragmentation of the spores’ coat. Simultaneous observation of the spore morphology by optical microscopy corroborates these mechanisms. The dependence of the onset of DPA release and the time constant of spore fragmentation on H₂O₂ shows that the formation of reactive oxygen species from H₂O₂ is the rate-limiting factor of oxidative spore death.
Biomedical applications of magnetic nanoparticles (MNP) fundamentally rely on the particles’ magnetic relaxation as a response to an alternating magnetic field. The magnetic relaxation complexly depends on the interplay of MNP magnetic and physical properties with the applied field parameters. It is commonly accepted that particle core size is a major contributor to signal generation in all the above applications, however, most MNP samples comprise broad distribution spanning nm and more. Therefore, precise knowledge of the exact contribution of individual core sizes to signal generation is desired for optimal MNP design generally for each application. Specifically, we present a magnetic relaxation simulation-driven analysis of experimental frequency mixing magnetic detection (FMMD) for biosensing to quantify the contributions of individual core size fractions towards signal generation. Applying our method to two different experimental MNP systems, we found the most dominant contributions from approx. 20 nm sized particles in the two independent MNP systems. Additional comparison between freely suspended and immobilized MNP also reveals insight in the MNP microstructure, allowing to use FMMD for MNP characterization, as well as to further fine-tune its applicability in biosensing.
Persistent infection with high-risk human papillomaviruses (hrHPV) can result in the formation of anogenital cancers. As hrHPV proteins E6 and E7 are required for cancer initiation and maintenance, they are ideal targets for immunotherapeutic interventions. Previously, we have described the development of DNA vaccines for the induction of HPV16 E6 and E7 specific T cell immunity. These vaccines consist of ‘gene-shuffled’ (SH) versions of HPV16 E6 and E7 that were fused to Tetanus Toxin Fragment C domain 1 (TTFC) and were named TTFC-E6SH and TTFC-E7SH. Gene-shuffling was performed to avoid the risk of inducing malignant transformation at the vaccination site. Here, we describe the preclinical safety evaluation of these candidate vaccines by analysis of their transforming capacity in vitro using established murine fibroblasts (NIH 3T3 cells) and primary human foreskin keratinocytes (HFKs). We demonstrate that neither ectopic expression of TTFC-E6SH and TTFC-E7SH alone or in combination enabled NIH 3T3 cells to form colonies in soft agar. In contrast, expression of HPV16 E6WT and E7WT alone or in combination resulted in effective transformation. Similarly, retroviral transduction of HFKs from three independent donors with both TTFC-E6SH and TTFC-E7SH alone or in combination did not show any signs of immortalization. In contrast, the combined expression of E6WT and E7WT induced immortalization in HFKs from all donors. Based on these results we consider it justified to proceed to clinical evaluation of DNA vaccines encoding TTFC-E6SH and TTFC-E7SH in patients with HPV16 associated (pre)malignancies.
Planar and three-dimensional (3D) interdigitated electrodes (IDE) with electrode digits separated by an insulating barrier of different heights were electrochemically characterized and compared in terms of their sensing properties. Due to the impact of the surface resistance, both types of IDE structures display a non-linear behavior in low-ionic strength solutions. The experimental data were fitted to an electrical equivalent circuit and interpreted taking into account the surface-charge-governed properties. The effect of a charged polyelectrolyte layer electrostatically assembled onto the sensor surface on the surface resistance in solutions with different KCl concentration is studied. In case of the same electrode footprint, 3D-IDEs show a larger cell constant and a higher sensitivity to molecular adsorption than that of planar IDEs. The obtained results demonstrate the potential of 3D-IDEs as a new transducer structure for a direct label-free sensing of charged molecules.
The subtilase family (S8), a member of the clan SB of serine proteases are ubiquitous in all kingdoms of life and fulfil different physiological functions. Subtilases are divided in several groups and especially subtilisins are of interest as they are used in various industrial sectors. Therefore, we searched for new subtilisin sequences of the family Bacillaceae using a data mining approach. The obtained 1,400 sequences were phylogenetically classified in the context of the subtilase family. This required an updated comprehensive overview of the different groups within this family. To fill this gap, we conducted a phylogenetic survey of the S8 family with characterised holotypes derived from the MEROPS database. The analysis revealed the presence of eight previously uncharacterised groups and 13 subgroups within the S8 family. The sequences that emerged from the data mining with the set filter parameters were mainly assigned to the subtilisin subgroups of true subtilisins, high-alkaline subtilisins, and phylogenetically intermediate subtilisins and represent an excellent source for new subtilisin candidates.
In this study we show an optical biosensor concept, based on elastic light scattering from sapphire microspheres. Transmitted and elastic scattering intensity of the microspheres (radius 500 μm, refractive index 1.77) on an optical fiber half coupler is analyzed at 1510 nm. The 0.43 nm angular mode spacing of the resonances is comparable to the angular mode spacing value estimated using the optical size of the microsphere. The spectral linewidths of the resonances are in the order of 0.01 nm, which corresponds to quality factors of approximately 105. A polydopamine layer is used as a functionalizing agent on sapphire microspherical resonators in view of biosensor implementation. The varying layer thickness on the microsphere is determined as a function of the resonance wavelength shift. It is shown that polymer functionalization has a minor effect on the quality factor. This is a promising step toward the development of an optical biosensor.
The presentation of enzymes on viral scaffolds has beneficial effects such as an increased enzyme loading and a prolonged reusability in comparison to conventional immobilization platforms. Here, we used modified tobacco mosaic virus (TMV) nanorods as enzyme carriers in penicillin G detection for the first time. Penicillinase enzymes were conjugated with streptavidin and coupled to TMV rods by use of a bifunctional biotin-linker. Penicillinase-decorated TMV particles were characterized extensively in halochromic dye-based biosensing. Acidometric analyte detection was performed with bromcresol purple as pH indicator and spectrophotometry. The TMV-assisted sensors exhibited increased enzyme loading and strongly improved reusability, and higher analysis rates compared to layouts without viral adapters. They extended the half-life of the sensors from 4 - 6 days to 5 weeks and thus allowed an at least 8-fold longer use of the sensors. Using a commercial budget-priced penicillinase preparation, a detection limit of 100 µM penicillin was obtained. Initial experiments also indicate that the system may be transferred to label-free detection layouts.
Penicillin detection by means of field-effect based sensors: EnFET, capacitive EIS sensor or LAPS?
(2001)
Penicillin detection by means of field-effect based sensors: EnFET, capacitive EIS sensor or LAPS?
(2000)
Optoelectronic Properties of Nanostructured Ensembles Controlled by Biomolecular Logic Systems
(2008)
Optimization of the immobilization of bacterial spores on glass substrates with organosilanes
(2016)
Spores can be immobilized on biosensors to function as sensitive recognition elements. However, the immobilization can affect the sensitivity and reproducibility of the sensor signal. In this work, three different immobilization strategies with organosilanes were optimized and characterized to immobilize Bacillus atrophaeus spores on glass substrates. Five different silanization parameters were investigated: nature of the solvent, concentration of the silane, silanization time, curing process, and silanization temperature. The resulting silane layers were resistant to a buffer solution (e.g., Ringer solution) with a polysorbate (e.g., Tween®80) and sonication.
Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN′ from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.
Optimization of passivation layers for corrosion protection of silicon-based microelectrode arrays
(2000)
A light-addressable potentiometric sensor (LAPS) is a field-effect-based potentiometric device, which detects concentration changes of an analyte solution on the sensor surface in a spatially resolved way. It uses a light source to generate electron–hole pairs inside the semiconductor, which are separated in the depletion region due to an applied bias voltage across the sensor structure and hence, a surface-potential-dependent photocurrent can be read out. However, depending on the beam angle of the light source, scattering effects can occur, which influence the recorded signal in LAPS-based differential measurements. To solve this problem, a novel illumination unit based on a field programmable gate array (FPGA) consisting of 16 small-sized tunable infrared laser-diode modules (LDMs) is developed. Due to the improved focus of the LDMs with a beam angle of only 2 mrad, undesirable scattering effects are minimized. Escherichia coli (E. coli) K12 bacteria are used as a test microorganism to study the extracellular acidification on the sensor surface. Furthermore, a salt bridge chamber is built up and integrated with the LAPS system enabling multi-chamber differential measurements with a single Ag/AgCl reference electrode.
The immobilization of NAD+-dependent dehydrogenases, in combination with a diaphorase, enables the facile development of multiparametric sensing devices. In this work, an amperometric biosensor array for simultaneous determination of ethanol, formate, d- and l-lactate is presented. Enzyme immobilization on platinum thin-film electrodes was realized by chemical cross-linking with glutaraldehyde. The optimization of the sensor performance was investigated with regard to enzyme loading, glutaraldehyde concentration, pH, cofactor concentration and temperature. Under optimal working conditions (potassium phosphate buffer with pH 7.5, 2.5 mmol L-1 NAD+, 2.0 mmol L-1 ferricyanide, 25 °C and 0.4% glutaraldehyde) the linear working range and sensitivity of the four sensor elements was improved. Simultaneous and cross-talk free measurements of four different metabolic parameters were performed successfully. The reliable analytical performance of the biosensor array was demonstrated by application in a clarified sample of inoculum sludge. Thereby, a promising approach for on-site monitoring of fermentation processes is provided.