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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.

Amyloid-β oligomers (AβO) are important diagnostic markers for Alzheimer's disease (AD) and potential therapeutic targets for treating it. Herein, a simple and label-free electrochemical biosensor is presented for the specific recognition of AβO based on the binding of these biomarkers to ssDNA aptamer receptors. The ssDNA aptamer self-assembles to gold rod electrodes through Au-S interactions and binds specifically to AβO. The novel aptasensor shows a wide linear concentration range from 0.1 to 500 nM with a low detection limit of 0.03 nM as derived from electrochemical impedance spectroscopy invesastigations. Furthermore, owing to the high selectivity among Aβ species, this label-free sensor was used to monitor the process of Aβ protein aggregation, which was validated by atomic force microscope analysis. In addition, the aptasensor can be used to detect AβO in artificial CSF with satisfying accuracy. To our knowledge, it is first label-free aptasensor for an AβO assay based on EIS that works in artificial CSF and can be used for monitoring Aβ protein aggregation. These features, together with its easy fabrication, operation convenience, and effective regeneration, make our new aptasensor a promising tool for the early detection of AD and drug development by monitoring Aβ plaques degradation.

Mercury and its compounds are emitted during industrial processes and are extremely harmful for eco systems and human health. Therefore, the detection of mercury ions (Hg2+) in our living and working environment is of great importance for the society and especially for the health of human beings. Here we demonstrate a proof of concept nanoparticle stripe sensor for highly sensitive and selective detection of Hg2+. This sensor is based on the changes of the charge transport between the neighboring nanoparticles in the nanoparticle stripe. The addition of Hg2+ induces a chelation between Hg2+ and carboxylic groups on the surface modification molecules and thus facilitates the charge transport, causing an increase of conductivity in the nanoparticle stripe. These nanoparticle stripes with a few layers in height and several micrometers in width possess large surface area, which increases their exposure to ions and improves the ability to detect Hg2+ at low concentrations. Besides, we studied the effect of molecular length on the sensitivity of the sensor. It is shown that the length of surface modification molecules is positively correlated with the sensitivity of the sensor. The fabricated devices exhibit a detection limit as low as 0.1 nM and a specific response towards Hg2+ ions.

Malaria infection remains a significant risk for much of the population of tropical and subtropical areas, particularly in developing countries. Therefore, it is of high importance to develop sensitive, accurate and inexpensive malaria diagnosis tests. Here, we present a novel aptamer-based electrochemical biosensor (aptasensor) for malaria detection by impedance spectroscopy, through the specific recognition between a highly discriminatory DNA aptamer and its target Plasmodium falciparum lactate dehydrogenase (PfLDH). Interestingly, due to the isoelectric point (pI) of PfLDH, the aptasensor response showed an adjustable detection range based on the different protein net-charge at variable pH environments. The specific aptamer recognition allows sensitive protein detection with an expanded detection range and a low detection limit, as well as a high specificity for PfLDH compared to analogous proteins. The specific feasibility of the aptasensor is further demonstrated by detection of the target PfLDH in human serum. Furthermore, the aptasensor can be easily regenerated and thus applied for multiple usages. The robustness, sensitivity, and reusability of the presented aptasensor make it a promising candidate for point-of-care diagnostic systems.

Power-line harmonic interference and fixed-frequency noise peaks may cause stripe-artifacts in ultra-low field (ULF) magnetic resonance imaging (MRI) in an unshielded environment and in a conductively shielded room. In this paper we describe an adaptive suppression method to eliminate these artifacts in MRI images. This technique utilizes spatial correlation of the interference from different positions, and is realized by subtracting the outputs of the reference channel(s) from those of the signal channel(s) using wavelet analysis and the least squares method. The adaptive suppression method is first implemented to remove the image artifacts in simulation. We then experimentally demonstrate the feasibility of this technique by adding three orthogonal superconducting quantum interference device (SQUID) magnetometers as reference channels to compensate the output of one 2nd-order gradiometer. The experimental results show great improvement in the imaging quality in both 1D and 2D MRI images at two common imaging frequencies, 1.3 kHz and 4.8 kHz. At both frequencies, the effective compensation bandwidth is as high as 2 kHz. Furthermore, we examine the longitudinal relaxation times of the same sample before and after compensation, and show that the MRI properties of the sample did not change after applying adaptive suppression. This technique can effectively increase the imaging bandwidth and be applied to ULF MRI detected by either SQUIDs or Faraday coil in both an unshielded environment and a conductively shielded room.

Despite all efforts, malaria is still highly prevalent in tropical and developing countries. The “test, treat and track” policy of the World Health Organization (WHO) demands the development of affordable and highly sensitive malaria tests that discriminate between the two common malaria parasites, Plasmodium falciparum and Plasmodium vivax, to guide appropriate treatments. In response, we developed a flexible and disposable multielectrode array utilized as electrochemical malaria aptasensor. This multi-target aptasensor was modified by four different aptamer receptors, discriminating between P. falciparum and P. vivax infections via Plasmodium lactate dehydrogenase, expressed by both species, and histidine-rich protein 2, exclusively expressed by P. falciparum. The multi-target aptasensor detection was tested in blood samples spiked with target proteins, whole human blood spiked with P. falciparum, and P. falciparum in vitro cultures. A rigorous analysis revealed sensitivities of >75.0% for 0.001% parasitemia (50 parasites/µL) and a logic gate-based discrimination of Plasmodium infections, overcoming WHO standards. A cost analysis further substantiated the applicability of this multi-target aptasensor as a disposable point-of-care test for remote areas dealing with prevalent malaria parasite infections.

Here, we present a method to investigate cell adhesion on soft, non-conducting polymers that are implant candidate materials. Neuronal cells were grown on two elastomers (polydimethylsiloxane (PDMS) and Ecoflex®) and prepared for electron microscopy. The samples were treated with osmium tetroxide (OsO4) and uranylacetate (UrAc). Best results can be achieved when the polymers were coated with a thin iridium layer before the cell culture. This was done to emphasize the usage of soft polymers as supports for implant electrodes. A good contrast and the adhesion of the cells on soft polymers could be visualized.

Magnetic materials have been widely used for various electromagnetic actuators. However, there still exists a corrosion problem for most magnetic materials when used in a biological cell environment. The metal ions released can cause cytotoxicity. In this paper, we characterize the corrosion of a soft-magnetic FeCo alloy, which has been adopted in our magnetic tweezers, in several cell culture media. Obvious accelerated corrosion was observed in cardiomyocyte and neuronal cell media, but not in Dulbecco’s Modified Eagle Medium. To eliminate the corrosion we examined electrochemical deposition of polypyrrole as well as pyrolytical deposition of parylene C for passivation. It was found that the quality of polypyrrole deposition was insufficient in the area near the edges of the tweezers tips where they had been laser-cut. However, the parylene C coating exhibited excellent isolation properties. In addition, parylene C-coated magnetic parts can also withstand repeated high magnetic field application. The cell viability of in vitro cultures exposed to the passivated material was inspected by live/dead cell staining. Both cardiomyocyte and primary neuronal cells could be cultured on parylene C coated magnetic parts for up to three weeks.

The development rate of graphene-related research is tremendous. New methods of graphene growth and transfer are reported on a regular basis, trending towards large-scale. Nevertheless, the fabrication of high-yield and low-cost graphene devices is still challenging. In this work, we approach this problem from a technological point of view and propose a new, so-called “high-throughput transfer technique”. The technique allows a semi-automatic transfer of graphene films right at the desired places on a wafer. We demonstrate the applicability of our method by aligning 52 graphene devices on a 4-inch wafer using only 4 cm2 of graphene. The overall yield of this process is over 90%.

In recent years, optogenetic approaches have significantly advanced the experimental repertoire of cellular and functional neuroscience. Yet, precise and reliable methods for specific expression of optogenetic tools remain challenging. In this work, we studied the transduction efficiency of seven different adeno-associated virus (AAV) serotypes in primary cortical neurons and revealed recombinant (r) AAV6 to be the most efficient for constructs under control of the cytomegalovirus (CMV) promoter. To further specify expression of the transgene, we exchanged the CMV promoter for the human synapsin (hSyn) promoter. In primary cortical-glial mixed cultures transduced with hSyn promoter-containing rAAVs, expression of ChR2opt (a Channelrhodopsin-2 variant) was limited to neurons. In these neurons action potentials could be reliably elicited upon laser stimulation (473nm). The use of rAAV serotype alone to restrict expression to neurons results in a lower transduction efficiency than the use of a broader transducing serotype with specificity conferred via a restrictive promoter. Cells transduced with the hSyn driven gene expression were able to elicit action potentials with more spatially and temporally accurate illumination than neurons electrofected with the CMV driven construct. The hSyn promoter is particularly suited to use in AAVs due to its small size. These results demonstrate that rAAVs are versatile tools to mediate specific and efficient transduction as well as functional and stable expression of transgenes in primary cortical neurons.

Bioinspired molecular complexes that mimic the enzymatic catalysis of redox transformations offer a versatile platform for the development of non-enzymatic mediatorless sensors with high sensitivity, selectivity, and robustness without the use of precious metals. The aim of this study was to prepare and investigate biomimetic sensors based on the electrocatalytic reduction of hydrogen peroxide and oxygen by a series of immobilized complexes of iron and manganese with porphyrin macrocycles for the detection of hydrogen peroxide and glucose. The influence of substitution of the macroheterocyclic ligand, composition of the adsorption solution, Nafion membrane, and amino acids on the properties of the sensors was studied. Optimized sensor function is based on the electrogenerated reduced form of Mn(II) meso-tetra(N-methyl-4-pyridyl) porphyrin as a catalyst and allows high sensitivity of the hydrogen peroxide detection of 1.8 A M−1 cm−2 and 0.071 A M−1 cm−2 to be achieved in the lower and higher concentration ranges, respectively, with a low detection limit of 5⋅10−7 M at physiological pH 7.4 and in the presence of oxygen. The MnTMPyP electrode was investigated as an electrochemical transducer in the glucose-oxidase-based biosensor. The sensors were successfully applied for the detection of hydrogen peroxide and glucose in human serum samples. Along with a simple fabrication procedure and robustness of the sensor, the biomimetic electrocatalytic properties of the MnTMPyP complex facilitate excellent performance of the proposed sensors for hydrogen peroxide and glucose determination in biological media, emphasizing the importance of bioinspired electrocatalytic metalloporphyrin complexes for the development of sensors and point-of-care devices.

Magnetic field fluctuations in our unshielded urban laboratory can reach hundreds of nT in the noisy daytime and is only a few nT in the quiet midnight. The field fluctuation causes the Larmor frequency fL to drift randomly for several Hz during the unshielded ultra-low field (ULF) nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) measurements, thus seriously spoiling the averaging effect and causing imaging artifacts. By using an active compensation (AC) technique based on the spatial correlation of the low-frequency magnetic field fluctuation, the field fluctuation can be suppressed to tens of nT, which is a moderate situation between the noisy daytime and the quiet midnight. In this paper, the effect of the field fluctuation on ULF MRI measurements was investigated. The 1D and 2D MRI signals of a water phantom were measured using a second-order low-Tc superconducting quantum interference device (SQUID) in three fluctuation cases: severe fluctuation (noisy daytime), moderate fluctuation (daytime with AC) and minute fluctuation (quiet midnight) when different gradient fields were applied. When the active compensation is applied or when the frequency encoding gradient field Gx reaches a sufficiently strong value in our measurements, the image artifacts become invisible in all three fluctuation cases. Therefore it is feasible to perform ULF-MRI measurements in unshielded urban environment without imaging artifacts originating from magnetic fluctuations by using the active compensation technique and/or strong gradient fields.

Electrochemical aptamer-based (E-AB) sensors represent an emerging class of recently developed sensors. However, numerous of these sensors are limited by a low surface density of electrode-bound redox-oligonucleotides which are used as probe. Here we propose to use the concept of electrochemical current rectification (ECR) for the enhancement of the redox signal of E-AB sensors. Commonly, the probe-DNA performs a change in conformation during target binding and enables a nonrecurring charge transfer between redox-tag and electrode. In our system, the redox-tag of the probe-DNA is continuously replenished by solution-phase redox molecules. A unidirectional electron transfer from electrode via surface-linked redox-tag to the solution-phase redox molecules arises that efficiently amplifies the current response. Using this robust and straight-forward strategy, the developed sensor showed a substantial signal amplification and consequently improved sensitivity with a calculated detection limit of 114nM for ATP, which was improved by one order of magnitude compared with the amplification-free detection and superior to other previous detection results using enzymes or nanomaterials-based signal amplification. To the best of our knowledge, this is the first demonstration of an aptamer-based electrochemical biosensor involving electrochemical rectification, which can be presumably transferred to other biomedical sensor systems.

Two types of gold nanostructures are discussed: charge(citrate)-stabilized and sterically(OA)-stabilized gold nanostructures and their assembly with heme-containing metalloproteins, cyt c and horseradish peroxidase (HRP) enzyme into a bioelectrochemically active nanoarchitecture as well as a sensor application. The assembly of the nanostructures on the thin-film gold electrodes and the immobilization and electrochemical properties of metalloproteins on these electrodes are presented. The nanostructured bioelectrochemical interfaces provide a good environment for the stable and reproducible immobilization of electroactive proteins. We show that both molecules preserve their functionalities (electrochemical and biocatalytic activities). The amount of electroactive proteins immobilized on the nanostructured electrode surfaces is significantly increased compared to the flat electrode surfaces. The kinetic parameters of the heterogeneous direct electron transfer reaction of cyt c on the nanostructured electrodes are compared. The thin-film gold electrodes modified with OANWs, OANPs, and citrate-stabilized NPs and covalently immobilized HRP exhibit an excellent catalytic activity toward the oxidation of hydrogen peroxide with a working concentration range from 20 μM to 500 μM, a sensitivity of 0.031 A M−1 cm−2 (RSD 0.005), 0.027 A M−1 cm−2 (RSD 0.004), and 0.022 A M−1 cm−2 (RSD 0.0035), and a detection limit of 5 μM, 8 μM, and 14 μM, respectively (RSDs near the detection limits were 9–12%). The HRP sensor characteristics are improved significantly compared to the flat thin-film sensors by using gold nanostructures. Our study shows that ultrathin gold nanowires and nanoparticles with two different types of stabilizing agents are promising materials for assembling biomolecules into functional nanoarchitectures for metalloprotein-based bioelectrochemical sensors.

Graphical abstractDisplay Omitted Study of interface between cells and 3D micro structures with Focused Ion Beam (FIB).Reduction of the curtaining artifacts of FIB sectioning.Investigation of cell-substrate interface and cell response to 3D structures. Coupling biological systems with electronic devices such as Multi Electrode Arrays (MEAs) has been largely investigated in the last years. The interface between cells and an engineered substrate varies due to the material and shape of the substrate. Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB) are suitable techniques for studying the interface at the nanoscale. Preserving cell structures and substrates morphology appears to be an important challenge while optimizing SEM and FIB for biological applications. Here, we describe a FIB sectioning method valid for cardiomyocytes-like cells (HL-1) on 3D metal microstructures, focusing on the reduction of mechanical artifacts such as curtaining phenomena.

This paper describes ultrathin gold nanowires (NWs) and nanoparticles (NPs) prepared by oleylamine (OA) synthesis and their assembly with horseradish peroxidase enzyme (HRP) for bioelectrochemical sensing of hydrogen peroxide for the first time. The immobilization of oxidoreductase enzyme HRP on the electrodes modified with OA gold nanostructures (OANSs) is discussed. The HRP-sensor characteristics, namely sensitivity, working concentration range, sensor-to-sensor and measurement-to-measurement reproducibility as well as long-term stability, are improved significantly compared to the planar thin-film sensors by using OANSs. The thin-film gold electrodes modified with OANWs and OANPs exhibit a catalytic activity towards oxidation of hydrogen peroxide with a working concentration range from 20 µM to 500 µM, a sensitivity of 0.031 A M(-1) cm(-2) (RSD 0.046) and 0.027 A M(-1) cm(-2) (RSD 0.045), and a detection limit of 5 µM and 8 µM, respectively (RSD near the detection limits was 9-12%). Our study shows that ultrathin gold nanowires and nanoparticles prepared by oleylamine synthesis are prospective materials to assemble biomolecules into functional nanoarchitectures for enzyme-based bioelectrochemical sensors, metalloprotein bioelectronics, and energy research.

Transport properties of single-walled carbon nanotube (CNT) structures with Pd contacts were studied using noise spectroscopy. The high values of the mobility and low noise level are characteristic of high-quality CNT material. The detailed analysis of the transport and noise properties of the CNT structure with back gate topography allows us to study the transport determined by Schottky barriers and by pure CNT channel conductivity and to establish their separate contribution to the total conductivity of the structure. It was demonstrated that at small gate overdrive the main source of flicker noise is related to the Schottky barriers of the CNT–FETs. With increasing gate voltage, the magnitude of flicker noise decreases and at a certain gate voltage it is only determined by the transport properties of carbon nanotubes with a noise level lower by one order of magnitude. In contrast to previous studies where flicker noise determined the excess noise of CNT-based structures, we registered generation–recombination noise components in our structures and studied their behavior in a wide temperature range. This allowed us to investigate the origin of traps capturing the carriers, which considerably affects the noise and transport properties of CNT structures.

The performance of the direct readout schemes for dc SQUID, Additional Positive Feedback (APF), noise cancellation (NC) and SQUID bootstrap circuit (SBC), have been studied in conjunction with planar SQUID magnetometers. In this paper, we examine the NC technique applied to a niobium SQUID gradiometer module with an Nb wire-wound antenna connecting to a dual-loop SQUID chip with an integrated voltage feedback circuit for suppression of the preamplifier noise contribution. The sensitivity of the SQUID gradiometer module is measured to be about 1 fT/(cm √Hz) in the white noise range in a magnetically shielded room. Using such gradiometer, both MCG and MEG signals are recorded.

We have synthesized photocrosslinkable benzophenone copolymer, Poly(St- co -MBz), and fabricated cell-repellent patterns of Poly(St- co -MBz) on covalently bound poly- d -lysine (PDL) layer via the photocrosslinking. We have successfully obtained fine grid line pattern with line width of 3 μm and fine neurite, presumably axon, patterns with excellent pattern fidelity. We found that benzophenone unit can be crosslinked under the exposure of UV (with the intensity of ∼77 mW/cm 2 at 280 nm and ∼60 mW/cm 2 at 365 nm) without photo-oxidative damage to PDL, poly- l -lysine, and polyethyleneimine.

During the last decade protein electrochemistry at miniaturized electrodes has become important not only for functional studies of the charge transfer properties of redox proteins but also for fostering the development of sensitive biosensor and bioelectronic devices. One of the major challenges in this field is the directed coupling between electronic and biologically active components. A prerequisite for a fast and reversible electron transfer between electrode and protein is that the protein can be bound to the electrode in a favourable orientation. We examined electrostatic and bioaffinity-tag binding strategies for the directed immobilization of horse heart cytochrome c (cytc) on gold electrode surfaces to achieve this goal. Horse heart cytc was expressed in E. coli either as non-modified or genetically modified, i.e. histidine (his)-tag containing protein. The his-tags were introduced at defined positions at the N- or C-terminus of the polypeptide. It was our aim to generate tagged-versions of cytc that facilitate strong electronic coupling between protein and electrode and, at the same time, retain their catalytic and regulatory properties. The combination of different immobilization strategies, e.g. his-tag and electrostatic immobilization also opens new avenues for bivalent immobilization of proteins. This is of interest for molecular bioelectronic and biosensing applications where the proteins are immobilized between two crossing electrodes.

In order to improve signal-to-noise ratio of low-field Magnetic Resonance Imaging (MRI) measurements, a Permanent Magnet (PM) with a field of about 1 T was used as pre-polarizing field and a tuned high-Tc (HTS) rf Superconducting Quantum Interference Device (SQUID) acted as signal detector. Measurements were performed in a Magnetically Shielded Room (MSR) with the door open, while the PM was located outside. A manual mechanical transport system was used to repeatedly move sample from the PM to the measuring position beneath the SQUID. The two-dimensional LF-MRI of a sample tube of 0.5 ml tap water demonstrates the feasibility of this concept.

SQUID bootstrap circuit (SBC) is a SQUID direct readout operating in the voltage bias mode. It consists of two branches connected in parallel: SQUID-inductance La branch and inductance Lb-resistance Rb branch. Its bias voltage is connected to the input terminal of preamplifier. In the SBC the voltage noise from the preamplifier creates the current noise which can be modified by the parameters. Based on the equivalent circuit for SBC and its network equations we studied its characteristics and the effect of parameters on current noise due to biased voltage noise by numerical simulation. Results suggest that SBC current noise can be effectively reduced by modifying parameters, in optimal condition the current noise can be totally suppressed. The effect of resistance Rb on the excess noise is discussed.

Improvement of the interface contact between biological objects and electronic devices can significantly enhance the quality of electronic signal transfer. The surface of biosensor can be artificially modified in order to strengthen the adhesion of biological cells. We report on results of fabrication of micron and submicron golden spines by means of e-beam lithography and electroplating. The fabrication technique allows easy modification of the size and shape of golden spines by variation of processing parameters. The structures with different spine profiles and spacing have been fabricated for optimization of cell growth conditions. We present the results of growth of rat cortical neurons on the surface of spine modified samples. Well-defined cell guidance was established at the spine arrays. Furthermore, the results of transmission electron microscope and focused ion beam technique confirm the good adhesion between the spines and cell structures.

Substrate-bound gradients expressed in numerous spatio-temporal patterns play a crucial role during the development of complex neural circuits. A deeper understanding of the axon guidance mechanism is provided by studying the effect of a defined substrate-bound cue on a confined neural network. In this study, we constructed a discontinuous substrate-bound gradient to control neuronal cell position, the path of neurite growth, and axon directionality. A variety of gradient patterns, with slight changes in slope, width, and length were designed and fabricated by microcontact printing using laminin/poly-l-lysine (PLL) or PLL alone. The gradients were tested for neurite growth and their impact on axon guidance of embryonic rat cortical neurons. The neurite length was determined and the axon was evaluated by Tau-1 immunostaining. We found that the microgradients of laminin/PLL and PLL directed neurons' adhesion, differentially controlled the neurite growth, and guided up to 84% of the axons. The effect of the protein micropattern on axon guidance and neurite growth depended on the protein and geometric parameters used. Our approach proved to be very successful in guiding axons of single multipolar neurons with very high efficiency. It could thereby be useful to engineer defined neural networks for analyzing signal processing of functional circuits, as well as to unravel fundamental questions of the axon guidance mechanism.

Here we describe a new analytical method for the detection of two influenza A viruses by nitrocellulose membrane and magnetic sensors that employ a special frequency mixing technique. The combination of the nitrocellulose membrane and magnetic bead detection permits a rapid assay procedure and excludes two steps (the development of color and the stop reaction) required for usual immunochemical detection methods such as ELISA. Quantitative virus detection was performed using magnetic beads conjugated with secondary antibody. The results were compared with conventional assay methods and with a dot-blot assay with fluorescence compound (FITC). Under optimum conditions, our new assay procedure is capable of detecting picograms of virus per well. This new method combining the nitrocellulose membrane and magnetic bead detection reduces analytical time and allows stable and repeatable analyses of samples in point-of-care applications.

There is a continuously growing scientific and technological interest to develop and improve the application of artificial sensors. Biological components which are capable to transduce neutral signals into specific, robust and reproducible indicators frame an attractive alternative to construct biohybrid sensors. Since naturally "occurring" biosensors are only sparsely compatible with artificial devices, genetic engineering of eukaryotic cells provides an attractive approach, where cells can be tailored such to detect target compounds with exquisite specificity and sensitivity. We have developed the prototype for a single-cell-based anion-selective biohybrid sensor. HEK293 cells were stably transfected with a gene encoding glycine receptor alpha(1) subunits. These cells were employed as transducers for glycine-evoked chloride currents in a concentration-dependent way. Cultured on substrate-integrated micro-devices, anionic membrane currents of cells were monitored extracellularly with field-effect transistors (FETs) and gold microelectrode arrays (MEAs). The results supported predictions of state-of-the-art models for cell-sensor coupling mechanisms and confirmed that extracellularly recorded anion currents cause similar signals, regardless whether obtained with field-effect transistors or microelectrodes. The whole-cell sensor successfully tracked glycine concentrations differing by three orders of magnitude. To our knowledge this contribution for the first time marks the functional characterization of an anion-selective biohybrid sensor.

We present a novel approach for large-scale silicon nanowire (SiNW) array fabrication for bioelectronic applications. Nanoimprint lithography was combined with standard CMOS processing on 4 in. SOI wafers in order to produce highly integrated arrays of silicon nanowire field-effect transistors (SiNW-FET). With a very smooth surface due to wet anisotropic etching, SiNW-FET arrays show a good electronic performance with a subthreshold slope of about 85 mV/decade. When applying a front-gate control of the wires via an electrochemical reference electrode, reliable electronic performance inside an electrolyte solution can be achieved. Our SiNW-FET sensors exhibit almost no electronic hysteresis on forward and backward bias sweeps. In this article the fabrication process, electronic and electrochemical characterizations and first biomolecular detection experiments are presented. For biodetection experiments we used a differential readout between molecule-free wires and wires carrying covalently attached biomolecules such as short, single-stranded DNA or biotin. With our SiNW-FET arrays a reliable detection of biomolecular layers can be achieved.

In the present paper, we demonstrate the electrochemical rectification of a redox current which is transferred between redox probes (ferricyanide) in solution and a gold electrode functionalized with the biomolecular redox mediator microperoxidase-11 (MP-11). MP-11 is the redox active, heme-containing domain of the biological electron shuttle cytochrome c (cyt c). In our system, a unidirectional current develops due to selective electron transport from the bio-functionalized electrode to ferricyanide such that MP-11 controls the read-out of our coupled redox system. The electrode was functionalized by adding a monolayer of undecanethiol (UDT) to promote the physisorption of MP-11 and inhibit the direct electron transfer between redox probe and electrode. The relative position of redox donator, mediator, and acceptor equilibrium potentials defines the charge transport and a potential-dependent electrochemical current rectification. The results of our investigations demonstrate that functional building blocks of proteins can be reassembled into new conceptual devices with operation modes deviating from their native function, which could prove highly useful in future design of biosensors and bioelectronic systems.

Transparent polymers are considered as alternative low-cost mold materials in UV nanoimprint lithography (UV-NIL). Here, we demonstrate a nanoimprint process with molds made of rigid polymers novel for this application. These polymer molds are found to show high performance in the patterning with UV-NIL. Sub-50nm structures were fabricated with this process.

In reported low-field nuclear magnetic resonance (NMR) measurements using Superconducting Quantum Interference Device (SQUID) detection, the pre-polarizing magnetic field has been usually oriented orthogonal to the measuring field, B(p) perpendicular B(m). Melton et al. systematically analyzed the consequences of B(p) decay in time after turnoff and showed that this decay should be nonadiabatic. We evaluated our measuring procedure in the light of that analysis, and found good quantitative agreement. It was showed that, when the decay time constant is comparable to the precession period of the magnetization of the sample, M, the optimum procedure is to orient B(p) parallel to B(m) and to apply a pi/2 pulse to flip M, similar as in the case of conventional NMR.

Three-dimensional surface reconstructions from high resolution image stacks of biological specimens, observed by confocal microscopy, have changed the perspective of morphological understanding. In the field of cell-cell or cell-substrate interfaces, combining these two techniques leads to new insights yet also creates a tremendous amount of data. In this article, we present a technique to reduce large, multidimensional data sets from confocal microscopy into one single curve: a membrane allocation profile. Reconstructed cells are represented in a three-dimensional surface from image sections of individual cells. We virtually cut segments of the reconstructed cell membrane parallel to the substrate and calculate the surface areas of each segment. The obtained membrane allocation profiles lead to morphological insights and yield an in vivo ratio of attached and free membrane areas without cell fixation. As an example, glass substrates were modified with different proteins (fibronectin, laminin, concavalin A, extracellular matrix gel, and both isomers of poly-lysine) and presented to HEK293 cells to examine differences in cell morphology and adhesion. We proved that proteins on a substrate could increase the attached portion of a cell membrane, facing the modified substrate, from an average of 32% (glass) to 45% (poly-lysine) of the total membrane surface area.

Nanostructured surfaces have recently gained in importance for electrochemical applications because of an enhanced surface area compared to planar substrates. Due to this property, structured substrates are well suited for electrochemical (bio-)sensors, capacitive coupling with electrogenic cells, and other bioelectronic applications. However, the relationship between electrolytically exposed and redox-active surface areas of nanostructured electrodes compared to planar electrodes is still under discussion. Here, we performed a series of comparative studies to elucidate processes taking place at the electrochemically active surface of gold nanopillars. The pillars, approximately 200 nm in height and 50 nm in diameter, were fabricated using template-assisted nanostructuring. The surface area increase compared to planar electrodes was determined by scanning electron microscopy (SEM), and the redox-active surface area of the same sample was derived from cyclovoltammetric studies. We found consistency between the SEM results and the electrochemically active surfaces determined by cyclic voltammetry of immobilized ferrocenylhexanethiol, immobilized cytochrome c, and oxidation/reduction of gold for small scan rates. Similar values were derived from the capacitance measured by cyclic voltammetry, whereas impedimetric measurements revealed values twice as high. Commonly used diffusion-controlled systems, such as hexacyanoferrate, showed a smaller increase of the electroactive surface area. Finally, we conclude that the sterically restricted diffusion of redox-active species leads to an inaccurate determination of the electroactive surface area of nanosized electrodes.

We present a complementary metal oxide semiconductor (CMOS) process with floating gate field-effect transistors (FG FETs) as sensor inputs for bioelectronic applications. In order to achieve a stable passivation of the devices against electrolyte solutions, we included additional fabrication steps and materials into our advanced process. Main changes to the standard CMOS process were the application of titanium silicide for all interconnects, a passivation layer formed by LPCVD deposition of silicon nitride and oxide, and an oxidation step for the sensing areas exposed to the electrolyte at the end of the fabrication process. Different arrays of FG FETs were fabricated. We present extracellular recordings from rat cardiac myocytes and from genetically modified human embryonic kidney (HEK) cells. The extracellular recordings showed a good signal-to-noise ratio. Due to the design, the chips were robust and showed an excellent long-term stability. They were able to withstand several cleaning procedures and subsequent cell cultures. The CMOS process allowed the implementation of simple integrated circuits. With two 5-bit decoders it was possible to address an array of 32 × 32 FG FETs. Our study paves the way for future fabrication of higher integrated chips with a stable and robust operation when used in bioelectronic applications.

For magnetic immunoassay applications, a novel magnetic reader for quantification of magnetic particle concentrations has been developed. Upon magnetic excitation at two distinct frequencies incident on the sample, the response signal generated at a sum frequency is detected. The low-frequency field component periodically drives the magnetic particles into saturation, which is probed by the high-frequency field. The appearance of frequency mixing lines is highly specific to the nonlinearity of the superparamagnetic particles’ magnetization. The optimization of field coils, differential pickup coil, preamplifier, demodulators and filters are discussed. The output signal is linear for four orders of magnitude in iron concentration.

Field-effect-based capacitive electrolyte-insulator-semiconductor (EIS) sensors have been utilised for the deoxyribonucleic acid (DNA) immobilisation and hybridisation detection as well as for monitoring the layer-by-layer adsorption of polyelectrolytes (anionic poly(sodium 4-styrene sulfonate) (PSS) and cationic poly(allylamine hydrochloride) (PAH)). The EIS sensors with charged macromolecules have been systematically characterised by capacitance-voltage, constant-capacitance, impedance spectroscopy and atomic-force microscopy methods. The effect of the number and polarity of the polyelectrolyte layers on the shift of the capacitance-voltage curves has been investigated. Alternating potential shifts of about 30-90 mV have been observed after the adsorption of each polyanion and polycation layer, respectively. The DNA immobilisation and hybridisation signals were 35-55 and 24-33 mV, respectively. The possible mechanisms for the sensor responses are discussed.

Recording of extracellular signals with planar metal microelectrodes (ME) has already been presented more than 30 years ago. To date, microelectrode array (MEA) systems are able to measure extracellular signals at about 64 sites, simultaneously. This enables monitoring of electrical activity of many cells in a large area. The extracellular recording technique has become a widely used method for neurological, toxicological or pharmacological studies. It already proved its potential to supplement the classical methods in electrophysiology. The interpretation of the recorded signal shapes in order to extract electrophysiological meaningful data--however--is still under discussion. In this article, we analyse the preamplifier circuit for extracellular recording of cardiac myocyte signals. We use a circuit model for the cell-electrode contact including the first amplification stage. In test experiments, we observe different signal shapes, when different shunt resistors are introduced at the input of the preamplifier. According to the frequency spectra of the recordings, we evaluate the transfer function between the source signal and the readout signal. As a result of our studies, an optimum readout electronics for originally, preserved extracellular signal shapes is proposed. Our amplifier design will be most valuable, if the use of small microelectrodes with high input impedances for in vitro as well as for in vivo experiments is desired.

Capacitive EIS (electrolyte–insulator–semiconductor) sensors and ISFETs (ion-sensitive field-effect transistor) have been utilised for the detection of the layer-by-layer adsorption of charged macromolecules. The field-effect-based sensors with the adsorbed multilayers have been characterised by capacitance–voltage, constant-capacitance and constant-voltage-mode methods. The effect of the number and polarity of the polyelectrolyte layers on the shift of the capacitance–voltage curves along the capacitance and voltage axis has been investigated. Alternating potential shifts of about 30–50 mV have been observed after the adsorption of each polyanion and polycation layer, respectively. The possible mechanisms for the sensor response are discussed.

A spatially resolved delivery of substances integrated with cell culture substrates shows promise for application in pharmacological assays, bioanalytical studies on cell signaling pathways and cell-based biosensors, where control over the extracellular biochemical environment with a cellular resolution is desirable. In this work, we studied a biohybrid system where rat embryonic cortical neuronal networks are reconstructed on microstructured silicon chips and interfaced to microfluidics. The design of cell–cell and cell–medium interactions in confined geometries is presented. We developed an aligned microcontact printing technique (AμCP) for poly(lysine)-extracellular matrix proteins on microstructured chips, which allows a high degree of geometrical control over the network architecture and alignment of the neuronal network with the microfluidic features of a substrate. Spatially resolved on-chip delivery of compounds with a cellular resolution is demonstrated by chemical stimulation of patterned rat cortical neurons within a network with a number of solutions of excitatory neurotransmitter glutamate delivered via microfluidics. The combination of the system described with a patch-clamp technique allowed both modulation of the biochemical environment on a cellular level and the monitoring of electrophysiological properties in the reconstructed rat embryonic cortical networks changed by this microenvironment.

Silanization protocols for glass slides and silicon oxide substrates usually include acid rinsing steps to activate the surfaces prior to silanization. In our group, field-effect transistor devices and electrolyte–insulator–semiconductor structures are used to electronically record signals from cells or to detect biomolecular interactions at the solid–liquid interface. A miniaturized, high sensitive, field-effect-based semiconductor device should expose at its input stage just a thin oxide (

A critical evaluation of the possibilities and limitations of the label-free detection of deoxyribonucleic acid (DNA) hybridization by means of field-effect-based devices is discussed. A new DNA-detection method is introduced, which utilizes an ion-sensitive field-effect device as transducer. The upon the DNA hybridization induced redistribution of the ion concentration within the intermolecular spaces and/or the alteration of the ion sensitivity of the device is proposed as detection mechanism. The theoretical calculations predict a substantial change in the average ion concentration within the intermolecular spaces induced upon hybridization that are enough to obtain a detectable sensor signal.

The labelfree detection of nucleic acid sequences is one of the modern attempts to develop quick, cheap and miniaturised hand-held devices for the future genetic testing in biotechnology and medical diagnostics. We present an approach to detect the hybridisation of DNA sequences using electrolyte-oxide-semiconductor field-effect transistors (EOSFETs) with micrometer dimensions. These semiconductor devices are sensitive to electrical charge variations that occur at the surface/electrolyte interface, i.e. upon hybridisation of oligonucleotides with complementary single-stranded (ss) oligonucleotides, which are immobilised on the oxide surface of the transistor gate. This method allows direct, time-resolved and in situ detection of specific nucleic acid binding events without any labelling. We focus on the detection mechanism of our sensors by using oppositely charged polyelectrolytes (PAH and PSS) subsequently attached to the transistor structures. Our results indicate that the sensor output is charge sensitive and distance dependent from the gate surface, which pinpoints the need for very defined surface chemistry at the device surface. The hybridisation of natural 19 base-pair sequences has been successfully detected with the sensors. In combination with nano-transistors a PCR free detection system might be feasible in future.

Experimental control over cellular polarity in a neuronal network is a promising tool to study synapse formation and network behavior. We aimed to exploit a mechanism described by Stenger et al. [J. Neurosci. Methods 82 (1998) 167] to manipulate the direction of axonal versus dendritic outgrowth on a micropattern. The group had used laser ablation to create patterns of aminated silanes for cell attachment on a background of repellent fluorinated silanes. The pattern offered continuous adhesive pathways for axonal and interrupted pathways for dendritic outgrowth. By microcontact printing, we created similar patterns containing continuous and interrupted pathways consisting of extracellular matrix proteins on a background of polystyrene. Neuronal polarity was determined on the functional level through double patch clamp measurements, detecting synapses and their orientation. Although our pattern reproduced the properties that were assumed to be critical for the described effect, namely contrasting pathways of different adhesiveness, we failed to reproduce the above results. It is indicated that other qualities of alternative pathways than mere differences in adhesiveness are required to orient neuronal polarity in vitro. We suggest that the effect observed by Stenger et al. has to be attributed to less universal characteristics of the micropattern, e.g. to the specific chemical groups that were utilized.

A 64-channel amplifier system for the recording of extracellular signals with planar metal microelectrodes is presented. Gold metal microelectrodes on glass wafers were fabricated using standard photolithographic techniques. The measurement system was divided into a headstage preamplifier and a main amplifier. The inherent noise of the extracellular recording system was minimized by using an independent battery supply. The metal electrodes were directly connected to the gates of low noise junction field effect transistors (JFETs) using a specially designed electronic circuit. With this set-up, it was possible to record extracellular signals with planar metal microelectrodes without any surface modification for impedance reduction. A feedback circuit in the first amplification stage compensated slow drifts of the gold microelectrodes, which made online sampling of all 64 channels with a sampling rate of 10 kHz possible. Recordings were taken from rat cardiac myocytes cultured on fibronectin coated sensor chips. The system exhibited a good signal-to-noise ratio. It was able to detect the signal propagation within the cardiac cell layer and it could be used for pharmacological investigations involving the heart.

Dissociated neuronal cultures on substrates patterned with extracellular matrix (ECM) proteins have yielded much information in the past. However, although the culture of brain slices has many advantages over dissociated neuronal cultures, its feasibility on patterned substrates has not been demonstrated to date. In the present study, neuronal outgrowth from brain stem slices onto homogeneous control substrates, and onto laminin structures of grid- and line-shape was achieved. Cultures were evaluated by means of phase contrast microscopy, antibody staining, and patch-clamp measurements. Only patterns with line sizes of more than 4 microm yielded satisfactory neuronal outgrowth. The size of the nodes in the pattern influenced the nodal compliance of the spreading cells and the amount of unstructured overgrowth. Best grid patterns were 4 microm lines and 10 microm nodes, best line patterns were 4 microm lines and 20 microm nodes. On patterned substrates, average sodium and potassium currents were reduced by approximately 50% compared to controls, whereas area-normalized ion-currents were in the same order of magnitude. This indicates that as a consequence of the pattern-enforced geometrical confinement, neurons tend to have a smaller surface. In addition, neurons on patterned substrates were rapidly covered with glial overgrowth. This was shown by antibody staining.

An extracellular recording system has been designed for the detection of electrical cell signals using p-channel or n-channel field-effect transistor (FET) arrays with non-metallized gates. Signals from rat heart muscle cell were recorded by these devices and the results described on the basis of an equivalent circuit. This technique is sensitive enough to detect minute changes of the extracellular membrane voltage and has potential applications in drug screening. We show that known cardiac stimulants (isoproterenol, norepinephrine) and relaxants (verapamil, carbamylcholine) have characteristic effects on the heart cells in terms of the changes of beat frequencies in the absence or presence of corresponding agents.

Neuronal cell growth in vitro can be controlled with micropatterned structures of extracellular matrix proteins such as laminin. This technique is a powerful tool for studying neuronal cell function in order to increase experimental reproducibility and to specifically design innovative experimental setups. In this paper the correlation between the structural dimensions of the ECM pattern and the shape of the resulting cellular network is analyzed. The aim of the present study was to position neuronal cell bodies as precisely as possible and to induce directed cell differentiation. PCC7-MzN cells were cultured on laminin patterns. The line width, node size and gap size in-between cell adhesion sites was varied systematically. Micrographs of the samples were taken and statistically analyzed using Student's t-test and linear correlation methods. Precise cell positioning has successfully been performed and evidence for controlled neuronal polarization has been found. With a structure geometry of 4 microm line width, 20 microm node size and 10 microm gap size a nodal compliance of 86% (+/- 10%) has been achieved.

Electrogenic cells fire spontaneous or triggered action potentials (transient changes of their membrane potentials) and can be electronically coupled to external electrodes (arrays). Signals from rat heart-muscle cells were recorded by a field-effect transistor and the results described on the basis of an equivalent circuit. This technique has potential applications in drug screening, such as measuring the dose-response curve of isoproterenol, a β-adrenergic agonist with a positive chronotropic effect.