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.

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.

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 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.

An electrochemical method for detecting the biomarker 4-HPA by allosteric activation of Acinetobacterbaumannii reductase C1 subunit.

The C1 (reductase) subunit of 4-hydroxy-phenylacetate (4-HPA) 3-hydroxylase (HPAH) from the soil-based bacterium Acinetobacterbaumannii catalyzes NADH oxidation by molecular oxygen, with hydrogen peroxide as a by-product. 4-HPA is a potent allosteric modulator of C1, but also a known urinary biomarker for intestinal bacterial imbalance and for some cancers and brain defects. We thus envisioned that C1 could be used to facilitate 4-HPA detection. The proposed test protocol is simple and in situ and involves addition of NADH to C1 in solution, with or without 4-HPA, and direct acquisition of the H2O2 current with an immersed Prussian Blue-coated screen-printed electrode (PB-SPE) assembly. We confirmed that cathodic H2O2 amperometry at PB-SPEs is a reliable electrochemical assay for intrinsic and allosterically modulated redox enzyme activity. We further validated this approach for quantitative NADH electroanalysis and used it to evaluate the activation of NADH oxidation of C1 by 4-HPA and four other phenols. Using 4-HPA, the most potent effector, allosteric activation of C1 was related to effector concentration by a simple saturation function. The use of C1 for cathodic biosensor analysis of 4-HPA is the basis of the development of a simple and affordable clinical routine for assaying 4-HPA in the urine of patients with a related disease risk. Extension of this principle to work with other allosteric redox enzymes and their effectors is feasible.

The C2 entity of chitosugars is crucial in molecular selectivity of the Vibrio campbellii chitoporin.

The marine bacterium Vibrio campbellii expresses a chitooligosaccharide-specific outer-membrane channel (chitoporin) for the efficient uptake of nutritional chitosugars that are externally produced through enzymic degradation of environmental host shell chitin. However, the principles behind the distinct substrate selectivity of chitoporins are unclear. Here, we employed black lipid membrane (BLM) electrophysiology, which handles the measurement of the flow of ionic current through porins in phospholipid bilayers for the assessment of porin conductivities, to investigate the pH dependency of chitosugar-chitoporin interactions for the bacterium's natural substrate chitohexaose and its deacetylated form, chitosan hexaose. We show that efficient passage of the N-acetylated chitohexaose through the chitoporin is facilitated by its strong affinity for the pore. In contrast, the deacetylated chitosan hexaose is impermeant; however, protonation of the C2 amino entities of chitosan hexaose allows it to be pulled through the channel in the presence of a transmembrane electric field. We concluded from this the crucial role of C2-substitution as the determining factor for chitoporin entry. A change from N-acetylamino- to amino-substitution effectively abolished the ability of approaching molecules to enter the chitoporin, with deacetylation leading to loss of the distinctive structural features of nanopore opening and pore access of chitosugars. These findings provide further understanding of the multistep pathway of chitin utilization by marine Vibrio bacteria and may guide the development of solid-state or genetically engineered biological nanopores for relevant technological applications.

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.

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.

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.

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.

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).

Single-channel properties, sugar specificity, and role of chitoporin in adaptive survival of Vibrio cholerae type strain O1.

Vibrio cholerae is a Gram-negative, facultative anaerobic bacterial species that causes serious disease and can grow on various carbon sources, including chitin polysaccharides. In saltwater, its attachment to chitin surfaces not only serves as the initial step of nutrient recruitment but is also a crucial mechanism underlying cholera epidemics. In this study, we report the first characterization of a chitooligosaccharide-specific chitoporin, VcChiP, from the cell envelope of the V. cholerae type strain O1. We modeled the structure of VcChiP, revealing a trimeric cylinder that forms single channels in phospholipid bilayers. The membrane-reconstituted VcChiP channel was highly dynamic and voltage induced. Substate openings O1', O2', and O3', between the fully open states O1, O2, and O3, were polarity selective, with nonohmic conductance profiles. Results of liposome-swelling assays suggested that VcChiP can transport monosaccharides, as well as chitooligosaccharides, but not other oligosaccharides. Of note, an outer-membrane porin (omp)-deficient strain of Escherichia coli expressing heterologous VcChiP could grow on M9 minimal medium supplemented with small chitooligosaccharides. These results support a crucial role of chitoporin in the adaptive survival of bacteria on chitinous nutrients. Our findings also suggest a promising means of vaccine development based on surface-exposed outer-membrane proteins and the design of novel anticholera agents based on chitooligosaccharide-mimicking analogs.

Sustainable and Efficient: A Reusable DIY Three-Electrode Base Plate for Microfluidic Electroanalysis and Biosensing.

A sustainable three-electrode platform for affordable microfluidic electroanalysis is described. The device can be handmade using common tools and, facilitating broad applicability, is indefinitely reusable through simple surface polishing. Compact prototypes with Pt counter, Pt working, and Ag/AgCl reference electrode disks were combined with silicone lid plates containing a microchannel for electrolyte flow. Redox voltammetry/amperometry of excellent quality was achieved in static and flowing ferricyanide solutions, respectively. Modified with a glucose oxidase surface layer, base plate Pt WEs performed very well as amperometric biosensors for microfluidic blood glucose testing. The electrode system is recyclable, compatible with matching lid plate microchannels, and functionally adaptable regarding the constituent metal and electrode surface modifications. This asset combination makes the device a sustainable detection tool for microfluidic electroanalysis, with applications ranging from direct detection of redox-active analytes to bioreceptor-assisted biosensing. It avoids costly microfabrication with clean-room use, and the accessibility of microfluidic EC (bio)sensing is thus greatly increased, especially for users with restricted budgets.

Reductive stability evaluation of 6-azopurine photoswitches for the regulation of CKIα activity and circadian rhythms.

Photopharmacology develops bioactive compounds whose pharmacological potency can be regulated by light. The concept relies on the introduction of molecular photoswitches, such as azobenzenes, into the structure of bioactive compounds, such as known enzyme inhibitors. Until now, the development of photocontrolled protein kinase inhibitors proved to be challenging for photopharmacology. Here, we describe a new class of heterocyclic azobenzenes based on the longdaysin scaffold, which were designed to photo-modulate the activity of casein kinase Iα (CKIα) in the context of photo-regulation of circadian rhythms. Evaluation of a set of photoswitchable longdaysin derivatives allowed for better insight into the relationship between substituents and thermal stability of the cis-isomer. Furthermore, our studies on the chemical stability of the azo group in this type of heterocyclic azobenzenes showed that they undergo a fast reduction to the corresponding hydrazines in the presence of different reducing agents. Finally, we attempted light-dependent modulation of CKIα activity together with the accompanying modulation of cellular circadian rhythms in which CKIα is directly involved. Detailed structure-activity relationship (SAR) analysis revealed a new potent reduced azopurine with a circadian period lengthening effect more pronounced than that of its parent molecule, longdaysin. Altogether, the results presented here highlight the challenges in the development of light-controlled kinase inhibitors for the photomodulation of circadian rhythms and reveal key stability issues for using the emerging class of heteroaryl azobenzenes in biological applications.

Controlling the Circadian Clock with High Temporal Resolution through Photodosing.

Circadian clocks, biological timekeepers that are present in almost every cell of our body, are complex systems whose disruption is connected to various diseases. Controlling cellular clock function with high temporal resolution in an inducible manner would yield an innovative approach for the circadian rhythm regulation. In the present study, we present structure-guided incorporation of photoremovable protecting groups into a circadian clock modifier, longdaysin, which inhibits casein kinase I (CKI). Using photodeprotection by UV or visible light (400 nm) as the external stimulus, we have achieved quantitative and light-inducible control over the CKI activity accompanied by an accurate regulation of circadian period in cultured human cells and mouse tissues, as well as in living zebrafish. This research paves the way for the application of photodosing in achieving precise temporal control over the biological timing and opens the door for chronophotopharmacology to deeper understand the circadian clock system.

Amperometric enzymatic sensing of glucose using porous carbon nanotube films soaked with glucose oxidase.

Glucose oxidase was soaked into a porous carbon nanotube film coating on a platinum disk electrode, then trapped beneath a topcoat of electrodeposition paint. The resulting sensors, operated at a potential of +0.6 V (vs. Ag/AgCl), produced a glucose signal that was linear up to 40 mM, with a 50 µM detection limit. Signal stability over >100 h of continuous operation in a flow cell showed the remarkable functional durability of the biosensor, and confirmed that the electropaint coating effectively prevented loss of the enzyme. This performance is deemed to derive from the minimalistic immobilization layer design and the prevention of protein leakage. The immobilization method has a potentially wide scope, in that it may also be applicable in other types of enzymatic biosensor. Graphical abstract Illustration of an enzyme biosensor design that uses glucose oxidase in bare carbon nanotube electrode modifications with electropaint topcoat for amperometric glucose quantification. Immobilization matrix supplementation with extra functional (nano-) materials was unnecessary for high-quality and stable analysis performance.

Functional analysis of an unusual porin-like channel that imports chitin for alternative carbon metabolism in Escherichia coli.

Escherichia coli have the genetic potential to use chitin as a carbon source in the absence of glucose, importing it via the chitin-uptake channel EcChiP for processing by the glucosamine catabolic pathway. The chip gene is usually not expressed when E. coli are grown on glucose-enriched nutrients, providing a general regulatory mechanism for the pathway. EcChiP is unusual in that it is homologous to porins and monomeric instead of trimeric, the typical form of sugar-specific channels, making it unclear how this channel operates. We recently reported that EcChiP could form a stable channel in lipid membranes and that the channel is specific for chitooligosaccharides. This report describes the biophysical nature of sugar-channel interactions and the kinetics of sugar association and dissociation. Titrating EcChiP with chitohexaose resulted in protein fluorescence enhancement in a concentration-dependent manner, yielding a binding constant of 2.9 × 105 m-1, consistent with the value of 2.5 × 105 m-1 obtained from isothermal titration microcalorimetry. Analysis of the integrated heat change suggested that the binding process was endothermic and driven by entropy. Single-channel recordings confirmed the voltage dependence of the penetration of chitohexaose molecules into and their release from EcChiP. Once inside the pore, the sugar release rate (koff) from the affinity site increased with elevated voltage, regardless of the side of sugar addition. Our findings revealed distinct thermodynamic and kinetic features of the activity of sugar-specific EcChiP and advance our knowledge of the physiological possibility of chitin utilization by non-chitinolytic bacteria.

Simple and Economical Analytical Voltammetry in 15 µL Volumes: Paracetamol Voltammetry in Blood Serum as a Working Example.

Reported is a three-electrode mini-cell for voltammetry in 15 µL solutions. The key device component is a rolled platinum foil of an inverted omega-shaped cross section, which functions as both the electrolyte container and the counter-electrode. The analytical assembly was completed with properly sized working and reference electrodes in the two terminals of the quasi-tubular Pt trough. Its applicability in electrochemical assays of 15 µL solutions was verified by redox mediator voltammetry at graphite and noble metal sensors and by trace lead stripping voltammetry. Real sample analysis was adequate for drug detection in a volunteer's blood, drawn before and 1 or 4 h after ingestion of paracetamol. In line with its known pharmacokinetics, lack of drug as well as drug presence and clearance were proven correctly in the three samples. The mini-cell here is easy to assemble and operate, indefinitely reusable, and offers valuable economy in chemical usage and minimal waste. This is primarily a versatile device for electrochemical laboratory analysis of samples that are available only in small quantities, and cost-effective quantitative screens for expensive high-molecular-weight compounds, products of microsynthesis, physiological microdialysis collections, and finger-prick blood sampling are seen as feasible targets.

Tuned Amperometric Detection of Reduced ß-Nicotinamide Adenine Dinucleotide by Allosteric Modulation of the Reductase Component of the p-Hydroxyphenylacetate Hydroxylase Immobilized within a Redox Polymer.

We report the fabrication of an amperometric NADH biosensor system that employs an allosterically modulated bacterial reductase in an adapted osmium(III)-complex-modified redox polymer film for analyte quantification. Chains of complexed Os(III) centers along matrix polymer strings make electrical connection between the immobilized redox protein and a graphite electrode disc, transducing enzymatic oxidation of NADH into a biosensor current. Sustainable anodic signaling required (1) a redox polymer with a formal potential that matched the redox switch of the embedded reductase and avoided interfering redox interactions and (2) formation of a cross-linked enzyme/polymer film for stable biocatalyst entrapment. The activity of the chosen reductase is enhanced upon binding of an effector, i.e. p-hydroxy-phenylacetic acid ( p-HPA), allowing the acceleration of the substrate conversion rate on the sensor surface by in situ addition or preincubation with p-HPA. Acceleration of NADH oxidation amplified the response of the biosensor, with a 1.5-fold increase in the sensitivity of analyte detection, compared to operation without the allosteric modulator. Repetitive quantitative testing of solutions of known NADH concentration verified the performance in terms of reliability and analyte recovery. We herewith established the use of allosteric enzyme modulation and redox polymer-based enzyme electrode wiring for substrate biosensing, a concept that may be applicable to other allosteric enzymes.

Automated Quantitative Enzyme Biosensing in 24-Well Microplates.

We report a novel system for glucose estimation in model and real samples, utilizing enzyme-modified pencil leads (PL) as effective electrochemical biosensors for robotic substrate quantification in 24-well microplates. Electrochemically formed carboxyl groups on the surface of the graphite were cross-linked to amino groups in the enzyme so as to attach glucose oxidase to the PL surface. Automated amperometric sensing of glucose solutions in microtiter-plate wells used computer-controlled stepper motors to move the biosensor/counter/reference electrode assemblies sequentially between the samples. This setup achieved stable analyte response and, in calibration trials, a linear response range and detection limit of 0.1-8 mM and 0.05 ± 0.01 mM, respectively. The biosensor microplate assay offered accurate "hands-off" evaluation of 4 or 20 samples per plate run, in the standard addition or calibration curve mode, respectively. Mode-independent glucose assays in standard solutions and human serum samples worked reproducibly with close to 100% recovery. The choice of cheap and practical PL enzyme biosensors and simple nonmicrofluidic measurement automation offers a convenient, labor- and cost-efficient form of quantitative biosensing, with a reduced risk of operator errors. The robotic approach is best suited to repetitive measurements of sample series, with academic research and clinical, environmental, pharmaceutical, or biotechnological analysis being potential areas for future exploitations of the methodology.

Chitoporin from the Marine Bacterium Vibrio harveyi: PROBING THE ESSENTIAL ROLES OF TRP136 AT THE SURFACE OF THE CONSTRICTION ZONE.

VhChiP is a sugar-specific porin present in the outer membrane of the marine bacterium Vibrio harveyi. VhChiP is responsible for the uptake of chitin oligosaccharides, with particular selectivity for chitohexaose. In this study, we employed electrophysiological and biochemical approaches to demonstrate that Trp(136), located at the mouth of the VhChiP pore, plays an essential role in controlling the channel's ion conductivity, chitin affinity, and permeability. Kinetic analysis of sugar translocation obtained from single channel recordings indicated that the Trp(136) mutations W136A, W136D, W136R, and W136F considerably reduce the binding affinity of the protein channel for its best substrate, chitohexaose. Liposome swelling assays confirmed that the Trp(136) mutations decreased the rate of bulk chitohexaose permeation through the VhChiP channel. Notably, all of the mutants show increases in the off-rate for chitohexaose of up to 20-fold compared with that of the native channel. Furthermore, the cation/anion permeability ratio Pc/Pa is decreased in the W136R mutant and increased in the W136D mutant. This demonstrates that the negatively charged surface at the interior of the protein lumen preferentially attracts cationic species, leading to the cation selectivity of this trimeric channel.