A 3D printed dual screen-printed electrode separation device for twin electrochemical mini-cell establishment.

We describe a tiny 3D-printed polymethyl-methacrylate-based plastic sleeve that houses two disposable screen-printed electrodes (SPE) and enables each of the working electrodes (WEs) to work independently, on a different side of a thin barrier, in its own electrochemical (EC) mini-cell, while the SPE counter and reference units are shared for electroanalysis. Optical and EC performance tests proved that the plastic divider between WE1 and WE2 efficiently inhibited solution mixing between the mini-cells. The two neighboring, independently operating mini-cells enabled matched differential measurements in the same sample solution, a tactic designed for elimination of electrochemical interference in complex samples. In a proof-of-principle glucose biosensor trial, a glucose oxidase-modified WE2 and an unmodified WE1 delivered the EC data for the removal of anodic ascorbic acid (AA) interference simply by subtracting the WE1 (background) current from the analyte-specific WE2 current (from buffered sample solution supplemented with glucose/AA), at an anodic H2O2 detection potential of +1 V. The microfabricated SPE accessory is cheap and easy to make and use. For the many dual electrode SPE strips on the market for multiple analytical targets the new device widens the options for their exploitation in assays of biological and environmental samples with complex matrix compositions and significant risks of interference.

Glucose Oxidase-Based Glucose Biosensing with a Simple Dual Ag/AgCl Probe Conductivity Readout.

We describe a conductometric assay of the enzymatic conversion of glucose to gluconic acid by dissolved glucose oxidase (GOx), using the generation of proton and gluconate from the reaction product dissociation for glucose detection. Simple basics of ionic conductivity, a silver/silver chloride wire pair, and a small applied potential translate glucose-dependent GOx activity into a scalable cell current. Enzyme immobilization and complex sensor design, involving extra nanomaterials or microfabrication of electrode structures, are entirely avoided, in contrast to all modern electrochemical glucose biosensors. Assay calibration showed a response linearity up to 500 µM, with a sensitivity of about 1.3 nA/µM. Selectivity tests excluded signals from sugars other than glucose, and glucose quantifications with recovery rates close to 100% were reached with a model sample and a beverage. Easy use of elementary physicochemical phenomena and a satisfactory performance are assets of the proposed non-amperometric glucose biosensing strategy. Assay integration into a planar dual electrode platform, with or without microfluidic application option, is feasible because of the simplicity of the sensor readout and suggests a route to affordable glucose analysis in beverage, food, and body fluid samples.

Photocleavable Protecting Groups Using a Sulfite Self-Immolative Linker for High Uncaging Quantum Yield and Aqueous Solubility.

Using light as an external stimulus to control (bio)chemical processes offers many distinct advantages, most importantly it allows for the spatiotemporal control simply through operating the light source. Photocleavable protecting groups (PPGs) are a cornerstone class of compounds that are used to achieve photocontrol over (bio)chemical processes. PPGs are able to release a payload of interest upon light irradiation. The successful application of PPGs hinges on their efficiency of payload release, captured in the uncaging Quantum Yield (QY). Heterolytic PPGs efficiently release low pKa payloads, but their efficiency drops significantly for payloads with higher pKa values, such as alcohols. For this reason, alcohols are usually attached to PPGs via a carbonate linker. The self-immolative nature of the carbonate linker results in concurrent release of CO2 with the alcohol payload upon irradiation. We introduce herein novel PPGs containing sulfites as self-immolative linkers for photocaged alcohol payloads, for which we discovered that the release of the alcohol proceeds with higher uncaging QY than an identical payload released from a carbonate-linked PPG. Furthermore, we demonstrate that uncaging of the sulfite-linked PPGs results in the release of SO2 and show that the sulfite linker improves water solubility as compared to the carbonate based systems.

Immobilized chitinase as effective biocatalytic platform for producing bioactive di-N-acetyl chitobiose from recycled chitin food waste.

Described is chitinase immobilization on magnetic nanoparticles (MNPs) as biocompatible support for enzymatic production of di-N-acetyl chitobiose from chitin waste. Chitinase immobilization was feasible with an immobilization yield of 88.9 ± 1.6 % with 97.8 ± 1.0 % retention of activity and compared to free enzyme work, immobilization conferred better thermal and storage stability. As practical benefit the attachment to magnetic nanocarriers enabled easy enzyme recovery after repeated application runs and thus sustainable reuse. In fixed state chitinase retained a remarkable 39.7 ± 2.6 % of the starting activity after 16 reaction cycles. Furthermore, immobilized chitinase showed higher catalytic activity than free chitinase in converting shrimp shells and squid-pens chitins into di-N-acetyl chitobiose in a single-step reaction. The final yield of purified compound was 37.0 ± 1.2 % from shrimp shells and 61.1 ± 0.5 % from squid-pens chitin. In conclusion, an efficient MNP-based chitinase immobilization system with the potential for large-scale production was developed.

Ten years of laser-induced graphene: impact and future prospect on biomedical, healthcare, and wearable technology.

Since its introduction in 2014, laser-induced graphene (LIG) from commercial polymers has been gaining interests in both academic and industrial sectors. This can be clearly seen from its mass adoption in various fields ranging from energy storage and sensing platforms to biomedical applications. LIG is a 3-dimensional, nanoporous graphene structure with highly tuneable electrical, physical, and chemical properties. LIG can be easily produced by single-step laser scribing at normal room temperature and pressure using easily accessible commercial level laser machines and materials. With the increasing demand for novel wearable devices for biomedical applications, LIG on flexible substrates can readily serve as a technological platform to be further developed for biomedical applications such as point-of-care (POC) testing and wearable devices for healthcare monitoring system. This review will provide a comprehensive grounding on LIG from its inception and fabrication mechanism to the characterization of its key functional properties. The exploration of biomedicals applications in the form of wearable and point-of-care devices will then be presented. Issue of health risk from accidental exposure to LIG will be covered. Then LIG-based wearable devices will be compared to devices of different materials. Finally, we discuss the implementation of Internet of Medical Things (IoMT) to wearable devices and explore and speculate on its potentials and challenges.

The fate of the contact ion pair determines the photochemistry of coumarin-based photocleavable protecting groups.

Photocleavable protecting groups (PPGs) enable the precise spatiotemporal control over the release of a payload of interest, in particular a bioactive substance, through light irradiation. A crucial parameter that determines the practical applicability of PPGs is the efficiency of payload release, largely governed by the quantum yield of photolysis (QY). Understanding which parameters determine the QY will prove crucial for engineering improved PPGs and their effective future applications, especially in the emerging field of photopharmacology. The Contact Ion Pair (CIP) has been recognized as an important intermediate in the uncaging process, but the key influence of its fate on the quantum yield has not been explored yet, limiting our ability to design improved PPGs. Here, we demonstrate that the CIP escape mechanism of PPGs is crucial for determining their payload- and solvent-dependent photolysis QY, and illustrate that an intramolecular type of CIP escape is superior over diffusion-dependent CIP escape. Furthermore, we report a strong correlation of the photolysis QY of a range of coumarin PPGs with the DFT-calculated height of all three energy barriers involved in the photolysis reaction, despite the vastly different mechanisms of CIP escape that these PPGs exhibit. Using the insights obtained through our analysis, we were able to predict the photolysis QY of a newly designed PPG with particularly high accuracy. The level of understanding of the factors determining the QY of PPGs presented here will move the ever-expanding field of PPG applications forward and provides a blueprint for the development of PPGs with QYs that are independent of payload-topology and solvent polarity.

Cation delocalization and photo-isomerization enhance the uncaging quantum yield of a photocleavable protecting group.

Photocleavable protecting groups (PPGs) enable the light-induced, spatiotemporal control over the release of a payload of interest. Two fundamental challenges in the design of new, effective PPGs are increasing the quantum yield (QY) of photolysis and red-shifting the absorption spectrum. Here we describe the combination of two photochemical strategies for PPG optimization in one molecule, resulting in significant improvements in both these crucial parameters. Furthermore, we for the first time identify the process of photo-isomerization to strongly influence the QY of photolysis of a PPG and identify the cis-isomer as the superior PPG.

Rapid and directly interpretable antimicrobial susceptibility profiling by continuous microvolume-electroanalysis of ferricyanide-mediated bacterial respiration.

We present a novel method for the electroanalysis of potassium ferricyanide-mediated bacterial electron transport, to rapidly assess viability and construct interpretable antimicrobial susceptibility profiles. Electrochemical minimum inhibitory concentrations (ecMICs) became determinable with a high correlation to the results from conventional assays.

Dip-coated carbon nanotube surface deposits as stable, effective response enhancers in pencil lead electrode voltammetry.

Graphitic pencil leads (PLs) are inexpensive writing accessories, readily available in stationery shops. Because the round filaments have high conductivity, they are excellent candidates for sustainable electroanalytical sensor fabrication. Here, we show that dip-coated carbon nanotube (CNT) surface deposits can stably enhance the faradaic redox response of cylindrical pencil lead electrodes (PLEs), with just ten simple sequential immersions of assembled PLEs in an aqueous suspension of CNTs producing significant improvement in their analytical performance. Cyclic (CV) and differential pulse (DPV) voltammetry of ferricyanide with unmodified and CNT-modified PLEs confirmed the reproducibility of the modification procedure and the reliability of the extent of signal amplification, as well as the stability of the response. A series of DPV tests with drugs, an environmental pollutant, an enzyme-substrate redox label and an industrial chemical proved the practical applicability of the proposed CNT-PLEs. Based on their observed properties, PLEs with dip-coated CNT deposits are suggested as cost-effective tools for advanced electroanalysis and as green platforms for enzyme biosensor construction.

Real-time monitoring of the release of multiple payloads from nanomaterials.

We overcome limitations of conventional methods to monitor the release of two payloads in situ. The concentration of two different corrosion inhibitors are simultaneously determined during their release from nanofibers by square wave voltammetry (SWV). SWV is suitable for direct and simultaneous determination of concentration of two payloads.

Basic Guide to Multilayer Microfluidic Fabrication with Polyimide Tape and Diode Laser.

For normal operations, microfluidic devices typically require an external source of pressure to deliver fluid flow through the microchannel. This requirement limits their use for benchtop research activities in a controlled static environment. To exploit the full potential of the miniaturization and portability of microfluidic platforms, passively driven capillary microfluidic devices have been developed to completely remove the need for an external pressure source. Capillary microfluidics can be designed to perform complex tasks by designing individual components of the device. These components, such as the stop valve and trigger valve, operate through changes in microchannel dimensions and aspect ratios. A direct, maskless fabrication protocol that allows the precise fabrication of microchannels and other microfluidic components is introduced here. A diode laser and polyimide tape on a PMMA substrate are the only components needed to start fabrication. By varying the laser power used and the number of laser repetitions, various depths and widths of the microchannel can be quickly created to meet specific needs. As an example of a functional unit, a trigger valve was fabricated and tested, as proof of the validity of the fabrication protocol.

Diode Laser and Polyimide Tape Enables Cheap and Fast Fabrication of Flexible Microfluidic Sensing Devices.

Wearable devices are a new class of healthcare monitoring devices designed for use in close contact with the patient's body. Such devices must be flexible to follow the contours of human anatomy. With numerous potential applications, a wide variety of flexible wearable devices have been created, taking various forms and functions. Therefore, different fabrication techniques and materials are employed, resulting in fragmentation of the list of equipment and materials needed to make different devices. This study attempted to simplify and streamline the fabrication process of all key components, including microfluidic chip and flexible electrode units. A combination of diode laser CNC machine and polyimide tape is used to fabricate flexible microfluidic chip and laser-induced graphene (LIG) electrodes, to create flexible microfluidic sensing devices. Laser ablation on polyimide tape can directly create microfluidic features on either PDMS substrates or LIG electrodes. The two components can be assembled to form a flexible microfluidic sensing device that can perform basic electrochemical analysis and conform to curved surfaces while undergoing microfluidic flow. This study has shown that simple, commonly available equipment and materials can be used to fabricate flexible microfluidic sensing devices quickly and easily, which is highly suitable for rapid prototyping of wearable devices.

Design and Synthesis of Visible-Light-Responsive Azobenzene Building Blocks for Chemical Biology.

Tetra-ortho-fluoro-azobenzenes are a class of photoswitches useful for the construction of visible-light-controlled molecular systems. They can be used to achieve spatio-temporal control over the properties of a chosen bioactive molecule. However, the introduction of different substituents to the tetra-fluoro-azobenzene core can significantly affect the photochemical properties of the switch and compromise biocompatibility. Herein, we explored the effect of useful substituents, such as functionalization points, attachment handles, and water-solubilizing groups, on the photochemical properties of this photochromic system. In general, all the tested fluorinated azobenzenes exhibited favorable photochemical properties, such as high photostationary state distribution and long half-lives, both in organic solvents and in water. One of the azobenzene building blocks was functionalized with a trehalose group to enable the uptake of the photoswitch into mycobacteria. Following metabolic uptake and incorporation of the trehalose-based azobenzene in the mycobacterial cell wall, we demonstrated photoswitching of the azobenzene in the isolated total lipid extract.

Structural and biochemical evidence for the emergence of a calcium-regulated actin cytoskeleton prior to eukaryogenesis.

Charting the emergence of eukaryotic traits is important for understanding the characteristics of organisms that contributed to eukaryogenesis. Asgard archaea and eukaryotes are the only organisms known to possess regulated actin cytoskeletons. Here, we determined that gelsolins (2DGels) from Lokiarchaeota (Loki) and Heimdallarchaeota (Heim) are capable of regulating eukaryotic actin dynamics in vitro and when expressed in eukaryotic cells. The actin filament severing and capping, and actin monomer sequestering, functionalities of 2DGels are strictly calcium controlled. We determined the X-ray structures of Heim and Loki 2DGels bound actin monomers. Each structure possesses common and distinct calcium-binding sites. Loki2DGel has an unusual WH2-like motif (LVDV) between its two gelsolin domains, in which the aspartic acid coordinates a calcium ion at the interface with actin. We conclude that the calcium-regulated actin cytoskeleton predates eukaryogenesis and emerged in the predecessors of the last common ancestor of Loki, Heim and Thorarchaeota.

Strategy for Engineering High Photolysis Efficiency of Photocleavable Protecting Groups through Cation Stabilization.

Photolabile protecting groups (PPGs) enable the precise activation of molecular function with light in many research areas, such as photopharmacology, where remote spatiotemporal control over the release of a molecule is needed. The design and application of PPGs in recent years have particularly focused on the development of molecules with high molar absorptivity at long irradiation wavelengths. However, a crucial parameter, which is pivotal to the efficiency of uncaging and which has until now proven highly challenging to improve, is the photolysis quantum yield (QY). Here, we describe a novel and general approach to greatly increase the photolysis QY of heterolytic PPGs through stabilization of an intermediate chromophore cation. When applied to coumarin PPGs, our strategy resulted in systems possessing an up to a 35-fold increase in QY and a convenient fluorescent readout during their uncaging, all while requiring the same number of synthetic steps for their preparation as the usual coumarin systems. We demonstrate that the same QY engineering strategy applies to different photolysis payloads and even different classes of PPGs. Furthermore, analysis of the DFT-calculated energy barriers in the first singlet excited state reveals valuable insights into the important factors that determine photolysis efficiency. The strategy reported herein will enable the development of efficient PPGs tailored for many applications.

Cheap and Sustainable Biosensor Fabrication by Enzyme Immobilization in Commercial Polyacrylic Acid/Carbon Nanotube Films.

Novel glucose biosensors were constructed by loading glucose oxidase (GOx) into the nanopores of homogenous carbon nanotube (CNT) films on the surface of Pt disk electrodes and trapping the enzyme by subsequent deposition of polyacrylic acid (PAA), forming PAA/GOx-CNT-modified Pt disks. In amperometric biosensing with anodic hydrogen peroxide (H2O2) detection at a potential of +600 mV, increasing electrolyte glucose concentrations produced instantaneous steps in the H2O2 oxidation current. Glucose biosensor amperometry was feasible down to 10 µM, with a sensitivity of about 34 µA mM-1 cm-2 and linear current response up to 5 mM. The biosensors reliably determined glucose concentrations in human serum and a beverage. Successful trials with PAA/GOx-CNT-modified screen-printed Pt electrode disks demonstrated the potential of this means of enzyme fixation in biosensor mass fabrication, which offers a unique combination of cheap availability of the two matrix constituents and sensor layer formation through simple drop-and-dry steps. PAA/GOx-CNT/Pt biosensors are green and user-friendly bioanalytical tools that do not need large budgets, special skills, or laboratory amenities for their production. Any user, from industrial, university, or school laboratories, even if inexperienced in biosensor construction, can prepare functional biosensors with GOx, as in these proof-of-principle studies, or with other redox enzymes, for clinical, environmental, pharmaceutical, or food sample analysis.

Light-Control over Casein Kinase 1δ Activity with Photopharmacology: A Clear Case for Arylazopyrazole-Based Inhibitors.

Protein kinases are responsible for healthy cellular processes and signalling pathways, and their dysfunction is the basis of many pathologies. There are numerous small molecule inhibitors of protein kinases that systemically regulate dysfunctional signalling processes. However, attaining selectivity in kinase inhibition within the complex human kinome is still a challenge that inspires unconventional approaches. One of those approaches is photopharmacology, which uses light-controlled bioactive molecules to selectively activate drugs only at the intended space and time, thereby avoiding side effects outside of the irradiated area. Still, in the context of kinase inhibition, photopharmacology has thus far been rather unsuccessful in providing light-controlled drugs. Here, we present the discovery and optimisation of a photoswitchable inhibitor of casein kinase 1δ (CK1δ), important for the control of cell differentiation, circadian rhythm, DNA repair, apoptosis, and numerous other signalling processes. Varying the position at which the light-responsive azobenzene moiety has been introduced into a known CK1δ inhibitor, LH846, revealed the preferred regioisomer for efficient photo-modulation of inhibitory activity, but the photoswitchable inhibitor suffered from sub-optimal (photo)chemical properties. Replacement of the bis-phenyl azobenzene group with the arylazopyrazole moiety yielded a superior photoswitch with very high photostationary state distributions, increased solubility and a 10-fold difference in activity between irradiated and thermally adapted samples. The reasons behind those findings are explored with molecular docking and molecular dynamics simulations. Results described here show how the evaluation of privileged molecular architecture, followed by the optimisation of the photoswitchable unit, is a valuable strategy for the challenging design of the photoswitchable kinase inhibitors.

Prussian Blue/Carbon Nanotube Sensor Spread with Gelatin/Zein Glaze: A User-Friendly Modification for Stable Interference-Free H2O2 Amperometry.

We report the production and characterization of effective amperometric sensors for cathodic hydrogen peroxide (H2O2) detection. The proposed electrodes involve a combination of a H2O2-signaling Prussian Blue (PB)/carbon nanotube (CNT) layer with a glaze of the biopolymers gelatin (top) and zein (beneath) for protection against PB leakage. The sandwich-type sensor was constructed through simple "drop and dry" steps with (1) suspensions of the CNTs in a soluble PB solution, (2) zein in ethanol, and (3) gelatin in water, applied sequentially to the carbon working electrode disk of a screen-printed carbon electrode (SPCE) platform. The PB in the signaling layer acted as the electrocatalyst for H2O2 reduction at -150 mV vs Ag/AgCl/3 M KCl, enabling cathodic H2O2 amperometry with good target proportionality. Calibration trials confirmed the linearity of the response up to 700 µM (R2 > 0.998), with a sensitivity of 0.425 µA µM-1 cm-2 and a practical detection limit of 1 µM. Quantification of H2O2 in model and real samples with gelatin-zein-PB/CNT-SPCEs had a recovery of close to 100% of the true value. Since they are easily and cheaply made and yield accurate target assessments, gelatin-zein-PB/CNT-SPCEs are an ideal tool for electrochemical H2O2 analyses in human body fluids, health care products, and samples from industries that use H2O2 as a bleach and germicide. Workers with little experience in sensor fabrication and limited funding will particularly benefit from utilization of the proposed H2O2 probes, which as well as being used in H2O2 testing also have a potential application as the transducer unit of oxidase-based biosensors with amperometric H2O2 readout.

Hypothesis-Driven, Structure-Based Design in Photopharmacology: The Case of eDHFR Inhibitors.

Photopharmacology uses light to regulate the biological activity of drugs. This precise control is obtained through the incorporation of molecular photoswitches into bioactive molecules. A major challenge for photopharmacology is the rational design of photoswitchable drugs that show light-induced activation. Computer-aided drug design is an attractive approach toward more effective, targeted design. Herein, we critically evaluated different structure-based approaches for photopharmacology with Escherichia coli dihydrofolate reductase (eDHFR) as a case study. Through the iterative examination of our hypotheses, we progressively tuned the design of azobenzene-based, photoswitchable eDHFR inhibitors in five design-make-switch-test-analyze cycles. Targeting a hydrophobic subpocket of the enzyme and a specific salt bridge only with the thermally metastable cis-isomer emerged as the most promising design strategy. We identified three inhibitors that could be activated upon irradiation and reached potencies in the low-nanomolar range. Above all, this systematic study provided valuable insights for future endeavors toward rational photopharmacology.

Computational Design, Synthesis, and Photochemistry of Cy7-PPG, an Efficient NIR-Activated Photolabile Protecting Group for Therapeutic Applications.

Photolabile Protecting Groups (PPGs) are molecular tools used, for example, in photopharmacology for the activation of drugs with light, enabling spatiotemporal control over their potency. Yet, red-shifting of PPG activation wavelengths into the NIR range, which penetrates the deepest in tissue, has often yielded inefficient or insoluble molecules, hindering the use of PPGs in the clinic. To solve this problem, we report herein a novel concept in PPG design, by transforming clinically-applied NIR-dyes with suitable molecular orbital configurations into new NIR-PPGs using computational approaches. Using this method, we demonstrate how Cy7, a class of NIR dyes possessing ideal properties (NIR-absorption, high molecular absorptivity, excellent aqueous solubility) can be successfully converted into Cy7-PPG. We report a facile synthesis towards Cy7-PPG from accessible precursors and confirm its excellent properties as the most redshifted oxygen-independent NIR-PPG to date (λmax =746 nm).