INB - Institut für Nano- und Biotechnologien
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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.
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.
Plant viruses are major contributors to crop losses and induce high economic costs worldwide. For reliable, on-site and early detection of plant viral diseases, portable biosensors are of great interest. In this study, a field-effect SiO2-gate electrolyte-insulator-semiconductor (EIS) sensor was utilized for the label-free electrostatic detection of tobacco mosaic virus (TMV) particles as a model plant pathogen. The capacitive EIS sensor has been characterized regarding its TMV sensitivity by means of constant-capacitance method. The EIS sensor was able to detect biotinylated TMV particles from a solution with a TMV concentration as low as 0.025 nM. A good correlation between the registered EIS sensor signal and the density of adsorbed TMV particles assessed from scanning electron microscopy images of the SiO2-gate chip surface was observed. Additionally, the isoelectric point of the biotinylated TMV particles was determined via zeta potential measurements and the influence of ionic strength of the measurement solution on the TMV-modified EIS sensor signal has been studied.
Photoelectrochemical (PEC) biosensors are a rather novel type of biosensors thatutilizelighttoprovideinformationaboutthecompositionofananalyte,enablinglight-controlled multi-analyte measurements. For enzymatic PEC biosensors,amperometric detection principles are already known in the literature. In con-trast, there is only a little information on H+-ion sensitive PEC biosensors. Inthis work, we demonstrate the detection of H+ions emerged by H+-generatingenzymes, exemplarily demonstrated with penicillinase as a model enzyme on atitanium dioxide photoanode. First, we describe the pH sensitivity of the sensorand study possible photoelectrocatalytic reactions with penicillin. Second, weshow the enzymatic PEC detection of penicillin.
A new functionalization method to modify capacitive electrolyte–insulator–semiconductor (EIS) structures with nanofilms is presented. Layers of polyallylamine hydrochloride (PAH) and graphene oxide (GO) with the compound polyaniline:poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PANI:PAAMPSA) are deposited onto a p-Si/SiO2 chip using the layer-by-layer technique (LbL). Two different enzymes (urease and penicillinase) are separately immobilized on top of a five-bilayer stack of the PAH:GO/PANI:PAAMPSA-modified EIS chip, forming a biosensor for detection of urea and penicillin, respectively. Electrochemical characterization is performed by constant capacitance (ConCap) measurements, and the film morphology is characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). An increase in the average sensitivity of the modified biosensors (EIS–nanofilm–enzyme) of around 15% is found in relation to sensors, only carrying the enzyme but without the nanofilm (EIS–enzyme). In this sense, the nanofilm acts as a stable bioreceptor onto the EIS chip improving the output signal in terms of sensitivity and stability.
Miniaturized electrolyte–insulator–semiconductor capacitors (EISCAPs) with ultrathin gate insulators have been studied in terms of their pH-sensitive sensor characteristics: three different EISCAP systems consisting of Al–p-Si–Ta2O5(5 nm), Al–p-Si–Si3N4(1 or 2 nm)–Ta2O5 (5 nm), and Al–p-Si–SiO2(3.6 nm)–Ta2O5(5 nm) layer structures are characterized in buffer solution with different pH values by means of capacitance–voltage and constant capacitance method. The SiO2 and Si3N4 gate insulators are deposited by rapid thermal oxidation and rapid thermal nitridation, respectively, whereas the Ta2O5 film is prepared by atomic layer deposition. All EISCAP systems have a clear pH response, favoring the stacked gate insulators SiO2–Ta2O5 when considering the overall sensor characteristics, while the Si3N4(1 nm)–Ta2O5 stack delivers the largest accumulation capacitance (due to the lower equivalent oxide thickness) and a higher steepness in the slope of the capacitance–voltage curve among the studied stacked gate insulator systems.
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.
Glucose oxidase (GOx) is an enzyme frequently used in glucose biosensors. As increased temperatures can enhance the performance of electrochemical sensors, we investigated the impact of temperature pulses on GOx that was drop-coated on flattened Pt microwires. The wires were heated by an alternating current. The sensitivity towards glucose and the temperature stability of GOx was investigated by amperometry. An up to 22-fold increase of sensitivity was observed. Spatially resolved enzyme activity changes were investigated via scanning electrochemical microscopy. The application of short (<100 ms) heat pulses was associated with less thermal inactivation of the immobilized GOx than long-term heating.
The treatment method to deactivate viable microorganisms from objects or products is termed sterilization. There are multiple forms of sterilization, each intended to be applied for a specific target, which depends on—but not limited to—the thermal, physical, and chemical stability of that target. Herein, an overview on the currently used sterilization processes in the global market is provided. Different sterilization techniques are grouped under a category that describes the method of treatment: radiation (gamma, electron beam, X-ray, and ultraviolet), thermal (dry and moist heat), and chemical (ethylene oxide, ozone, chlorine dioxide, and hydrogen peroxide). For each sterilization process, the typical process parameters as defined by regulations and the mode of antimicrobial activity are summarized. Finally, the recommended microorganisms that are used as biological indicators to validate sterilization processes in accordance with the rules that are established by various regulatory agencies are summarized.
An acetoin biosensor based on a capacitive electrolyte–insulator–semiconductor (EIS) structure modified with the enzyme acetoin reductase, also known as butane-2,3-diol dehydrogenase (Bacillus clausii DSM 8716ᵀ), is applied for acetoin detection in beer, red wine, and fermentation broth samples for the first time. The EIS sensor consists of an Al/p-Si/SiO₂/Ta₂O₅ layer structure with immobilized acetoin reductase on top of the Ta₂O₅ transducer layer by means of crosslinking via glutaraldehyde. The unmodified and enzyme-modified sensors are electrochemically characterized by means of leakage current, capacitance–voltage, and constant capacitance methods, respectively.
Plant virus-like particles, and in particular, tobacco mosaic virus (TMV) particles, are increasingly being used in nano- and biotechnology as well as for biochemical sensing purposes as nanoscaffolds for the high-density immobilization of receptor molecules. The sensitive parameters of TMV-assisted biosensors depend, among others, on the density of adsorbed TMV particles on the sensor surface, which is affected by both the adsorption conditions and surface properties of the sensor. In this work, Ta₂O₅-gate field-effect capacitive sensors have been applied for the label-free electrical detection of TMV adsorption. The impact of the TMV concentration on both the sensor signal and the density of TMV particles adsorbed onto the Ta₂O₅-gate surface has been studied systematically by means of field-effect and scanning electron microscopy methods. In addition, the surface density of TMV particles loaded under different incubation times has been investigated. Finally, the field-effect sensor also demonstrates the label-free detection of penicillinase immobilization as model bioreceptor on TMV particles.
Coronavirus disease 2019 (COVID-19) is a novel human infectious disease provoked by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, no specific vaccines or drugs against COVID-19 are available. Therefore, early diagnosis and treatment are essential in order to slow the virus spread and to contain the disease outbreak. Hence, new diagnostic tests and devices for virus detection in clinical samples that are faster, more accurate and reliable, easier and cost-efficient than existing ones are needed. Due to the small sizes, fast response time, label-free operation without the need for expensive and time-consuming labeling steps, the possibility of real-time and multiplexed measurements, robustness and portability (point-of-care and on-site testing), biosensors based on semiconductor field-effect devices (FEDs) are one of the most attractive platforms for an electrical detection of charged biomolecules and bioparticles by their intrinsic charge. In this review, recent advances and key developments in the field of label-free detection of viruses (including plant viruses) with various types of FEDs are presented. In recent years, however, certain plant viruses have also attracted additional interest for biosensor layouts: Their repetitive protein subunits arranged at nanometric spacing can be employed for coupling functional molecules. If used as adapters on sensor chip surfaces, they allow an efficient immobilization of analyte-specific recognition and detector elements such as antibodies and enzymes at highest surface densities. The display on plant viral bionanoparticles may also lead to long-time stabilization of sensor molecules upon repeated uses and has the potential to increase sensor performance substantially, compared to conventional layouts. This has been demonstrated in different proof-of-concept biosensor devices. Therefore, richly available plant viral particles, non-pathogenic for animals or humans, might gain novel importance if applied in receptor layers of FEDs. These perspectives are explained and discussed with regard to future detection strategies for COVID-19 and related viral diseases.
Electrolyte-insulator-semiconductor (EIS) field-effect sensors belong to a new generation of electronic chips for biochemical sensing, enabling a direct electronic readout. The review gives an overview on recent advances and current trends in the research and development of chemical sensors and biosensors based on the capacitive field-effect EIS structure—the simplest field-effect device, which represents a biochemically sensitive capacitor. Fundamental concepts, physicochemical phenomena underlying the transduction mechanism and application of capacitive EIS sensors for the detection of pH, ion concentrations, and enzymatic reactions, as well as the label-free detection of charged molecules (nucleic acids, proteins, and polyelectrolytes) and nanoparticles, are presented and discussed.
The enantioselective synthesis of α-hydroxy ketones and vicinal diols is an intriguing field because of the broad applicability of these molecules. Although, butandiol dehydrogenases are known to play a key role in the production of 2,3-butandiol, their potential as biocatalysts is still not well studied. Here, we investigate the biocatalytic properties of the meso-butanediol dehydrogenase from Bacillus licheniformis DSM 13T (BlBDH). The encoding gene was cloned with an N-terminal StrepII-tag and recombinantly overexpressed in E. coli. BlBDH is highly active towards several non-physiological diketones and α-hydroxyketones with varying aliphatic chain lengths or even containing phenyl moieties. By adjusting the reaction parameters in biotransformations the formation of either the α-hydroxyketone intermediate or the diol can be controlled.
Extracellular acidification is a basic indicator for alterations in two vital metabolic pathways: glycolysis and cellular respiration. Measuring these alterations by monitoring extracellular acidification using cell-based biosensors such as LAPS plays an important role in studying these pathways whose disorders are associated with numerous diseases including cancer. However, the surface of the biosensors must be specially tailored to ensure high cell compatibility so that cells can represent more in vivo-like behavior, which is critical to gain more realistic in vitro results from the analyses, e.g., drug discovery experiments. In this work, O2 plasma patterning on the LAPS surface is studied to enhance surface features of the sensor chip, e.g., wettability and biofunctionality. The surface treated with O2 plasma for 30 s exhibits enhanced cytocompatibility for adherent CHO–K1 cells, which promotes cell spreading and proliferation. The plasma-modified LAPS chip is then integrated into a microfluidic system, which provides two identical channels to facilitate differential measurements of the extracellular acidification of CHO–K1 cells. To the best of our knowledge, it is the first time that extracellular acidification within microfluidic channels is quantitatively visualized as differential (bio-)chemical images.