Printed Antifouling Electrodes for Biosensing Applications.

Biosensors based on miniaturized, functional electrodes are of high potential for various biosensing applications, especially at the point-of-care setting among others. However, the sensor performance of such electrochemical devices is still strongly limited, especially due to surface fouling in complex sample fluids, such as blood serum. Electrode coatings based on conductive nanomaterials embedded in antifouling matrices offer a promising strategy to overcome this limitation. However, known composite coatings require long (typically >24 h) and complex fabrication processes, which pose a strong barrier for cost-effective mass manufacturing and successful commercialization. Here, we describe a novel polymer/carbon nanotube (CNT) composite coating that can be produced from an ink containing a photoreactive and antifouling copolymer as well as conductive CNTs using fast and highly scalable printing processes. Coatings were prepared on screen-printed electrodes and characterized using cyclic voltammetry (CV) and protein fouling experiments. The coatings offered an electroactive surface area (EASA) comparable to uncoated screen-printed electrodes and retained >90% of initial EASA after 1 h of exposure to concentrated bovine serum albumin solution, while uncoated electrodes decreased to <20% of initial EASA after the same treatment. Utilizing the universal crosslinking reaction of the polymer coating, antibodies against the inflammatory biomarker C-reactive protein (CRP) were photochemically immobilized on the electrodes. Functionalized electrodes were fabricated in <2 h and were successfully used to quantify nanogram-range concentrations of CRP spiked in undiluted human blood serum using a sandwich-immunoassay with electrochemical read-out, demonstrating the high potential of the platform for biosensing applications.

Quality control methods in musculoskeletal tissue engineering: from imaging to biosensors.

Tissue engineering is rapidly progressing toward clinical application. In the musculoskeletal field, there has been an increasing necessity for bone and cartilage replacement. Despite the promising translational potential of tissue engineering approaches, careful attention should be given to the quality of developed constructs to increase the real applicability to patients. After a general introduction to musculoskeletal tissue engineering, this narrative review aims to offer an overview of methods, starting from classical techniques, such as gene expression analysis and histology, to less common methods, such as Raman spectroscopy, microcomputed tomography, and biosensors, that can be employed to assess the quality of constructs in terms of viability, morphology, or matrix deposition. A particular emphasis is given to standards and good practices (GXP), which can be applicable in different sectors. Moreover, a classification of the methods into destructive, noninvasive, or conservative based on the possible further development of a preimplant quality monitoring system is proposed. Biosensors in musculoskeletal tissue engineering have not yet been used but have been proposed as a novel technology that can be exploited with numerous advantages, including minimal invasiveness, making them suitable for the development of preimplant quality control systems.

Nanocellulose aerogel inserts for quantitative lateral flow immunoassays.

The Lateral Flow Immuno Assay (LFIA) is a well-established technique that provides immediate results without high-cost laboratory equipment and technical skills from the users. However, conventional colorimetric LFIA strips suffer from high limits of detection, mainly due to the analysis of a limited sample volume, short reaction time between the target analyte and the conjugation molecules, and a weak optical signal. Thus, LFIAs are mainly employed as a medical diagnostic tool for qualitative and semi/quantitative detection, respectively. We applied a novel cellulose nanofiber (CNF) aerogel material incorporated into LFIA strips to increase the sample flow time, which in turn extends the binding interactions between the analyte of interest and the detection antibody, thus improving the limit of detection (LOD). Compared to a conventional LFIA strip, the longer sample flow time in the aerogel modified LFIA strips improved the LOD for the detection of mouse IgG in a buffer solution by a 1000-fold. The accomplished LOD (0.01 ng/mL) even outperformed specifications of a commercial ELISA kit by a factor of 10, and the CNF aerogel assisted LFIA was successfully applied to detect IgG in human serum with a LOD of 0.72 ng/mL. Next to the improved LOD, the aerogel assisted LFIA could quantify IgG samples in buffer and human serum in the concentration ranges of 0.17 ng/mL - 100 ng/mL (in buffer) and 4.6 ng/mL - 100 ng/mL (in human serum). The presented solution thus poses a unique potential to transform lateral flow assays into highly sensitive, fully quantitative point-of-care diagnostics.

Cell-Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities.

Pulsed electromagnetic fields (PEMFs) are capable of specifically activating a TRPC1-mitochondrial axis underlying cell expansion and mitohormetic survival adaptations. This study characterizes cell-derived vesicles (CDVs) generated from C2C12 murine myoblasts and shows that they are equipped with the sufficient molecular machinery to confer mitochondrial respiratory capacity and associated proliferative responses upon their fusion with recipient cells. CDVs derived from wild type C2C12 myoblasts include the cation-permeable transient receptor potential (TRP) channels, TRPC1 and TRPA1, and directly respond to PEMF exposure with TRPC1-mediated calcium entry. By contrast, CDVs derived from C2C12 muscle cells in which TRPC1 has been genetically knocked-down using CRISPR/Cas9 genome editing, do not. Wild type C2C12-derived CDVs are also capable of restoring PEMF-induced proliferative and mitochondrial activation in two C2C12-derived TRPC1 knockdown clonal cell lines in accordance to their endogenous degree of TRPC1 suppression. C2C12 wild type CDVs respond to menthol with calcium entry and accumulation, likewise verifying TRPA1 functional gating and further corroborating compartmental integrity. Proteomic and lipidomic analyses confirm the surface membrane origin of the CDVs providing an initial indication of the minimal cellular machinery required to recover mitochondrial function. CDVs hence possess the potential of restoring respiratory and proliferative capacities to senescent cells and tissues.

Screen-Printed Glucose Sensors Modified with Cellulose Nanocrystals (CNCs) for Cell Culture Monitoring.

Glucose sensors are potentially useful tools for monitoring the glucose concentration in cell culture medium. Here, we present a new, low-cost, and reproducible sensor based on a cellulose-based material, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidized-cellulose nanocrystals (CNCs). This novel biocompatible and inert nanomaterial is employed as a polymeric matrix to immobilize and stabilize glucose oxidase in the fabrication of a reproducible, operationally stable, highly selective, cost-effective, screen-printed glucose sensor. The sensors have a linear range of 0.1-2 mM (R2 = 0.999) and a sensitivity of 5.7 ± 0.3 µA cm-2∙mM-1. The limit of detection is 0.004 mM, and the limit of quantification is 0.015 mM. The sensor maintains 92.3 % of the initial current response after 30 consecutive measurements in a 1 mM standard glucose solution, and has a shelf life of 1 month while maintaining high selectivity. We demonstrate the practical application of the sensor by monitoring the glucose consumption of a fibroblast cell culture over the course of several days.

Influence of the Membrane Dye R18 and of DMSO on Cell Penetration of Guanidinium-Rich Peptides.

A quantitative analysis by confocal fluorescence microscopy of the entry into HEK293 and MCF-7 cells by fluorescein-labeled octaarginine (1) and by three octa-Adp derivatives (2 - 4, octamers of the ß-Asp-Arg-dipeptide, derived from the biopolymer cyanophycin) is described, including the effects of the membrane dye R18 and of DMSO on cell penetration.

Cell Penetration, Herbicidal Activity, and in-vivo-Toxicity of Oligo-Arginine Derivatives and of Novel Guanidinium-Rich Compounds Derived from the Biopolymer Cyanophycin.

Oligo-arginines are thoroughly studied cell-penetrating peptides (CPPs, Figures 1 and 2). Previous in-vitro investigations with the octaarginine salt of the phosphonate fosmidomycin (herbicide and anti-malaria drug) have shown a 40-fold parasitaemia inhibition with P. falciparum, compared to fosmidomycin alone (Figure 3). We have now tested this salt, as well as the corresponding phosphinate salt of the herbicide glufosinate, for herbicidal activity with whole plants by spray application, hoping for increased activities, i.e. decreased doses. However, both salts showed low herbicidal activity, indicating poor foliar uptake (Table 1). Another pronounced difference between in-vitro and in-vivo activity was demonstrated with various cell-penetrating octaarginine salts of fosmidomycin: intravenous injection to mice caused exitus of the animals within minutes, even at doses as low as 1.4 µmol/kg (Table 2). The results show that use of CPPs for drug delivery, for instance to cancer cells and tissues, must be considered with due care. The biopolymer cyanophycin is a poly-aspartic acid containing argininylated side chains (Figure 4); its building block is the dipeptide H-ßAsp-αArg-OH (H-Adp-OH). To test and compare the biological properties with those of octaarginines we synthesized Adp8-derivatives (Figure 5). Intravenouse injection of H-Adp8-NH2 into the tail vein of mice with doses as high as 45 µmol/kg causes no symptoms whatsoever (Table 3), but H-Adp8-NH2 is not cell penetrating (HEK293 and MCF-7 cells, Figure 6). On the other hand, the fluorescently labeled octamers FAM-(Adp(OMe))8-NH2 and FAM-(Adp(NMe2))8-NH2 with ester and amide groups in the side chains exhibit mediocre to high cell-wall permeability (Figure 6), and are toxic (Table 3). Possible reasons for this behavior are discussed (Figure 7) and corresponding NMR spectra are presented (Figure 8).

Transient receptor potential vanilloid 2-mediated shear-stress responses in C2C12 myoblasts are regulated by serum and extracellular matrix.

The developmental sensitivity of skeletal muscle to mechanical forces is unparalleled in other tissues. Calcium entry via reputedly mechanosensitive transient receptor potential (TRP) channel classes has been shown to play an essential role in both the early proliferative stage and subsequent differentiation of skeletal muscle myoblasts, particularly TRP canonical (TRPC) 1 and TRP vanilloid (TRPV) 2. Here we show that C2C12 murine myoblasts respond to fluid flow-induced shear stress with increments in cytosolic calcium that are largely initiated by the mechanosensitive opening of TRPV2 channels. Response to fluid flow was augmented by growth in low extracellular serum concentration (5 vs. 20% fetal bovine serum) by greater than 9-fold and at 18 h in culture, coincident with the greatest TRPV2 channel expression under identical conditions (P < 0.02). Fluid flow responses were also enhanced by substrate functionalization with laminin, rather than with fibronectin, agreeing with previous findings that the gating of TRPV2 is facilitated by laminin. Fluid flow-induced calcium increments were blocked by ruthenium red (27%) and SKF-96365 (38%), whereas they were unaltered by 2-aminoethoxydiphenyl borate, further corroborating that TRPV2 channels play a predominant role in fluid flow mechanosensitivity over that of TRPC1 and TRP melastatin (TRPM) 7.

An adaptable stage perfusion incubator for the controlled cultivation of C2C12 myoblasts.

Here we present a stage perfusion incubation system that allows for the cultivation of mammalian cells within PDMS microfluidic devices for long-term microscopic examination and analysis. The custom-built stage perfusion incubator is adaptable to any x-y microscope stage and is enabled for temperature, gas and humidity control as well as equipped with chip and tubing holder. The applied double-layered microfluidic chip allows the predetermined positioning and concentration of cells while the gas permeable PDMS material facilitates pH control via CO2 levels throughout the chip. We demonstrate the functionality of this system by culturing C2C12 murine myoblasts in buffer free medium within its confines for up to 26 hours. We moreover demonstrated the system's compatibility with various chip configurations, other cells lines (HEK-293 cells) and for longer-term culturing. The cost-efficient system are applicable for any type of PDMS-based cell culture system. Detailed technical drawings and specification to reproduce this perfusion incubation system is provided in the ESI.

On-chip enzyme quantification of single Escherichia coli bacteria by immunoassay-based analysis.

Individual bacteria of an isogenic population can differ significantly in their phenotypic characteristics. This cellular heterogeneity is thought to increase the adaptivity to environmental changes on a population level. Analytical methods for single-bacteria analyses are essential to reveal the different factors that may contribute to this cellular heterogeneity, among them the stochastic gene expression, cell cycle stages and cell aging. Although promising concepts for the analysis of single mammalian cells based on microsystems technology were recently developed, platforms suitable for proteomic analyses of microbial cells are by far more challenging. Here, we present a microfluidic device optimized for the analysis of single Escherichia coli bacteria. Individual bacteria are captured in a trap and isolated in a volume of only 155 pL. In combination with an immunoassay-based analysis of the cell lysate, the platform allowed the selective and sensitive analysis of intracellular enzymes. The limit of detection of the developed protocol was found to be 200 enzymes. Using this platform, we could investigate the levels of ß-galactosidase in cells grown under different nutrient conditions. We successfully determined the enzyme copy numbers in cells cultured in defined medium (3517 ± 1578) and in complex medium (4710 ± 2643), and verified the down-regulation of expression in medium that contained only glucose as carbon source. The strong variations we found for individual bacteria confirm the phenotype heterogeneity. The capability to quantify proteins and other molecules in single bacterial lysates is encouraging to use the new analysis platform in future proteomics studies of isogenic bacteria populations.

A new mechanobiological era: microfluidic pathways to apply and sense forces at the cellular level.

Fueled by technological advances in micromanipulation methodologies, the field of mechanobiology has boomed in the last decade. Increasing needs for clinical solutions to better maintain our major mechanosensitive tissues (muscle, bone, and cartilage) with increasing age and new insights into cellular adaptations to mechanical stresses beckon for novel approaches to meet the needs of the future. In particular, the emergence of microfluidics has inspired new interdisciplinary strategies to decipher cellular mechanotransduction on the biochemical as well as macromolecular level. Cellular actuation by locally varying fluid shear can serve to accurately alter membrane surface tension as well as produce direct compressive and strain forces onto cells. Moreover, incorporating microelectronic technologies into microfluidic platforms has led to further advances in actuation and readout possibilities. In this review, we discuss the application of microfluidics to mechanobiological research with particular focus on microfluidic platforms that are able to simultaneously monitor cellular adaptation to mechanical forces and interpret biochemical mechanotransduction.

Response of Pseudomonas putida KT2440 to increased NADH and ATP demand.

Adenosine phosphate and NAD cofactors play a vital role in the operation of cell metabolism, and their levels and ratios are carefully regulated in tight ranges. Perturbations of the consumption of these metabolites might have a great impact on cell metabolism and physiology. Here, we investigated the impact of increased ATP hydrolysis and NADH oxidation rates on the metabolism of Pseudomonas putida KT2440 by titration of 2,4-dinitrophenol (DNP) and overproduction of a water-forming NADH oxidase, respectively. Both perturbations resulted in a reduction of the biomass yield and, as a consequence of the uncoupling of catabolic and anabolic activities, in an amplification of the net NADH regeneration rate. However, a stimulation of the specific carbon uptake rate was observed only when P. putida was challenged with very high 2,4-dinitrophenol concentrations and was comparatively unaffected by recombinant NADH oxidase activity. This behavior contrasts with the comparably sensitive performance described, for example, for Escherichia coli or Saccharomyces cerevisiae. The apparent robustness of P. putida metabolism indicates that it possesses a certain buffering capacity and a high flexibility to adapt to and counteract different stresses without showing a distinct phenotype. These findings are important, e.g., for the development of whole-cell redox biocatalytic processes that impose equivalent burdens on the cell metabolism: stoichiometric consumption of (reduced) redox cofactors and increased energy expenditures, due to the toxicity of the biocatalytic compounds.