Glucose Oxidase Layer Research Articles

Hierarchically decorated magnetic nanoparticles amplify the oxidative stress and promote the chemodynamic/magnetic hyperthermia/immune therapy

Magnetic nanoparticles (MNPs) are promising in tumor treatments due to their capacity for magnetic hyperthermia therapy (MHT), chemodynamic therapy (CDT), and immuno-related therapies, but still suffer from unsatisfactory tumor inhibition in the clinic. Insufficient hydrogen peroxide supply, glutathione-induced resistance, and high-density extracellular matrix (ECM) are the barriers. Herein, we hierarchically decorated MNPs with disulfide bonds (S-S), dendritic L-arginine (R), and glucose oxidase (GOx) to form a nanosystem (MNPs-SS-R-GOx). Its outer GOx layer not only enhanced the H2O2 supply to produce .OH by Fenton reaction, but also generated stronger oxidants (ONOO−) together with the interfaced R layer. The inner S-S layer consumed glutathione to interdict its reaction with oxidants, thus enhancing CDT effects. Importantly, the generated ONOO− tripled the MMP-9 expression to induce ECM degradation, enabling much deeper penetration of MNPs and benefiting CDT, MHT, and immunotherapy. Finally, the MNPs-SS-R-GOx demonstrated a remarkable 91.7% tumor inhibition in vivo. Statement of significanceMagnetic nanoparticles (MNPs) are a promising tumor therapeutic agent but with limited effectiveness. Our hierarchical MNP design features disulfide bonds (S-S), dendritic L-arginine (R), and glucose oxidase (GOx), which boosts H2O2 supply for ·OH generation in Fenton reactions, produces potent ONOO−, and enhances chemodynamic therapy via glutathione consumption. Moreover, the ONOO− facilitates the upregulation of matrix metalloprotein expression beneficial for extracellular matrix degradation, which in turn enhances the penetration of MNPs and benefits the antitumor CDT/MHT/immuno-related therapy. In vivo experiments have demonstrated an impressive 91.7% inhibition of tumor growth. This hierarchical design offers groundbreaking insights for further advancements in MNP-based tumor therapy. Its implications extend to a broader audience, encompassing those interested in material science, biology, oncology, and beyond.

An Amperometric Glucose Biosensor Composed of Prussian Blue, Nafion, and Glucose Oxidase Studied by Flow Injection Analysis

The construction and characterization of a glucose biosensor has been implemented in an instrumental chemical analysis course to provide students experience with chemical sensing, electroanalytical methods, and flow injection analysis (FIA). The glucose biosensor was assembled on a glassy carbon electrode by depositing a layer of Prussian blue (PB) electrocatalyst followed by a layer of glucose oxidase (GOx) immobilized in a Nafion film. GOx catalyzed the oxidation of glucose in the presence of dissolved oxygen to yield gluconic acid and hydrogen peroxide. The hydrogen peroxide produced was then electrocatalytically reduced at the PB film with a mild applied potential. The measured cathodic current provided the analytical signal for the glucose biosensor, which was applied to the quantification of glucose levels in soft drinks using FIA techniques. This experiment exposes students to several electroanalytical methods, including amperometry, cyclic voltammetry, and chronocoulometry, as well as methods that are used to measure biosensor response, deposit the PB film, measure the PB film coverage, and examine its electrocatalytic properties. In addition, students gain experience with FIA and the application of an enzyme-based biosensor.

Design of a bioelectronic tongue for glucose monitoring using zinc oxide nanofibers and graphene derivatives

Monitoring glucose levels is critical for diabetes management and might be a key step in the development of individualized treatment strategies. In this scenario, tracking salivary glucose has been recognized as a promising strategy due to its merits of ease sampling and non-invasive nature. In this paper, we report on the development of an electrical impedance-based biosensor array to distinguish glucose at different concentrations in saliva. The enzymatic biosensors were made of gold interdigitated electrodes coated with pristine electrospun zinc oxide nanofibers (NFZ) and NFZ combined with graphene-based nanomaterials (i.e., reduced graphene oxide - rGO and graphene quantum dots - GQDs), on which a layer of glucose oxidase (GOx) enzyme was adsorbed. Electrical impedance measurements indicate that the NFZ-GQDs@GOx and NFZ-rGO@GOx platforms presented good linear relationship with glucose concentration in the range of 0.1 to 6 mM. The highest sensitivity was reached for NFZ-rGO@GOx with a detection limit (LOD) of 14 μM, while the LOD was 32 μM for NFZ-GQDs@GOx. Both biosensors were also capable of detecting glucose in artificial saliva using aliquots of 10 μL, with recovery between 87.3 and 106.8%. Furthermore, the three sensing units (NFZ@GOx, NFZ-rGO@GOx and NFZ-GQDs@GOx) were employed to build a bioelectronic tongue. Using Principal Component Analysis (PCA) technique to project the electrical impedance data of all sensing units allowed the discrimination of the different glucose concentrations and interferents. This study reveals the applicability of the developed bioelectronic tongue as non-invasive glucose sensors, which approach could also be pottentially adapted to detect other disease biomarkers present in saliva.

Epidermal Sensor for Potentiometric Analysis of Metabolite and Electrolyte.

Wearable epidermal sensors that can provide noninvasive and continuous analysis of metabolites and electrolytes in sweat have great significance for healthcare monitoring. This study reports an epidermal sensor that can wirelessly, noninvasively, and potentiometrically analyze metabolites and electrolytes. Potentiometry-based ion-selective electrodes (ISE) are most widely used for detecting electrolytes, such as Na+ and K+. We develop an enzyme-based glucose ISE for potentiometric analysis of sweat glucose. The glucose ISE sensor is obtained by modifying a glucose oxidase layer (GOD) on an H+ ISE sensor. GOD catalyzes glucose to generate H+. The generated H+ passes through the H+ selective membrane to change the potential of the electrode. We have fully examined the limit of detection, detecting range, and stability of our epidermal sensor. Meanwhile, using this epidermal sensor, we can easily analyze the relationship between blood glucose and sweat glucose. The concentration curve of sweat glucose can represent blood glucose concentration, significantly contributing to sports and chronic disease monitoring.

Effect of axial ligand on the performance of hemin based catalysts and their use for fuel cells

A new cascade type cathodic catalyst containing hemin and glucose oxidase (GOx) is suggested for enhancing the performances of enzymatic biofuel cells (EBCs). In the cathodic catalyst, the upper GOx layer generates hydrogen peroxide (H2O2) by glucose oxidation reaction (GOR), and then actual cathodic current is determined by H2O2 reduction reaction (HRR) catalyzed by hemin, using the pre-produced H2O2. The reaction potential of hemin is positively shifted by the formation of coordinate bond between its core ions and amine groups, meaning that that of HRR deciding reduction onset potential of the catalyst is positively shifted. As materials providing the ligand containing amine groups, polyethyleneimine (PEI) and imidazole propionic acid (IPA) are considered. According to evaluations, the reaction potential for HRR is favorably moved as the amounts of available ligand and coordinate bond increase. When IPA is applied, the reduction onset potential for HRR is shifted from 0.4 to 0.51V and reduction reaction rate also increases from 55 to 86 μAcm−2. Based on that, the EBC using catalyst containing IPA shows superior performances, such as maximum power density of 66 μWcm−2 and open circuit voltage of 0.65V.

Fabrication of Repeatedly Usable Pt-Electrode Chip Coated With Solidified Glucose Oxidase and Ascorbate Oxidase for the Quantification of Glucose in Urine

A repeated usable electrode sensor for the measurement of glucose in urine has been developed. For improvement of the selectivity in measuring glucose in urine, we fabricated the Pt electrode coated with multilayered composite of glucose oxidase (GOX) and ascorbate oxidase (AOX). This simple layer structure could deliver more electrons from glucose oxidation without interference from ascorbic acids by first oxidizing the ascorbic acids present in urine. Results showed that our newly developed Pt electrode is capable of quantifying the concentrations of glucose in urine in the range between 0 and 1000 mg/dL, as well as maintaining its detection stability during 200 times of repeated measurements. This repeatable use of the sensor is attributable to the solidified structure of AOX and GOX layer.

Electropolymerization of aniline and (N-phenyl-o-phenylenediamine) for glucose biosensor application

Fabrication of conductive polyaniline nanofibers (PAnNFs) and poly(aniline-co-N-phenyl-o-phenylenediamine) nanoflakes [P(An-co-PoPD)] can promote a high surface area which influences rapid ion exchange and hence improved sensitivity in biosensor applications. Here, fabrication of two conductive films of PAnNFs from aniline monomer and a mixture of aniline/N-phenyl-o-phenylenediamine (oPD) monomers through cyclic voltammetry (potentiodynamic electropolymerization) technique was performed. The electrochemical features of aniline monomer generate polyaniline nanofibers compared to aniline with addition of oPD monomer that generate nano-flakes. Electro-fabricated electrodes were characterized by SEM, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). A molecular recognition layer of glucose oxidase within chitosan (GOx:CHI) was applied to the electrode-supported polymer and produced chronoamperometry dose–responses to different glucose molar concentrations with sensitivities that were larger for P(An-co-PoPD) compared to PAnNFs electrode.

Development of a Glucose Sensor Using Palladium Electrode

An amperometric glucose sensor was introduced where glucose oxidase was immobilized on palladium electrode. This sensor based on the glucose oxidase catalyzed oxidation of glucose and electrochemical oxidation of hydrogen peroxide which was produced by enzyme reaction. Basic concept of the glucose sensor is composed of inner and outer membrane systems, immobilized glucose oxidase layer, and palladium electrode. Inner membrane prevents the access of electrochemical interfering substrates. Outer membrane works as a diffusion barrier of glucose. The glucose sensor can achieve a wide dynamic range with this outer membrane. In this research, to verify this concept especially the role of outer membrane, the glucose sensor which was made with palladium electrode, glucose oxidase layer, and outer membrane was tested on different glucose concentrations. The sensor showed a good correlation between sensor responses and glucose concentrations. In addition, the dynamic range of this sensor was enough for glucose monitoring of diabetic patients. This sensor also demonstrated stable sensor responses during several days. These results show that this sensor has a strong potential for application to continuous glucose monitoring, or CGM. Disclosure T. Han: Employee; Self; Panasonic Healthcare Co., Ltd. S. Noda: Employee; Self; Panasonic Healthcare Co., Ltd. K. Haneda: Employee; Self; Panasonic Healthcare Co., Ltd. N. Shigetou: Employee; Self; Panasonic Healthcare Co., Ltd.

Development and Optimization of a Glucose Biosensor Based on Tris [5-amino-1,10-Phenanthroline]Iron(II) Polymer Films

A bilayered glucose biosensor consisting of tris[5-amino-1, 10-phenanthroline]iron(II) polymer film redox mediator and a glucose oxidase layer was prepared on glassy carbon surfaces. The polymer film of the iron complex was immobilized onto the electrode using cyclic voltammetry via electropolymerization reactions, while the enzyme layer was formed using a BSA and glutaraldehyde crosslinking reaction. The biosensors gave the largest response in the pH range of 7-8 and were evaluated with respect to storage conditions of room temperature and 4oC. There was no significant difference between the detection of glucose using the biosensor stored at room temperature versus one stored at 4°C and both bilayered films remained active for 20 days. The detection limit of the biosensors was found to be 0.30 mM which corresponds to a signal to noise ratio of 3:1.

Highly selective FET-type glucose sensor based on shape-controlled palladium nanoflower-decorated graphene

The glucose concentration in human body fluids is main marker for diagnose diabetes mellitus, which is a disease caused by insufficient insulin. Although diagnostic devices based on glucose level in the blood have been commercialized, a non-invasive detection methodology is required. Here, selectivity enhanced field-effect-transistor (FET)-type glucose sensor based on shape-controlled palladium nanoflower-decorated CVD graphene (SPNFG) with glucose oxidase (GOx) and nafion was fabricated using surface functionalization, electrodeposition and spin coating techniques. The surface-functionalized CVD graphene is used as substrate for electrodeposition and a p-type channel for FET-type sensor. The palladium nanoflower (PNF) shape was controlled by adjusting the sulfuric acid concentration in the electrolyte during the electrodeposition. Nafion and GOx layer were deposited onto SPNFG using spincoating method. The GOx/Nafion/SPNFG electrode within FET system was applied to high sensitive and selective glucose sensor. The GOx/Nafion/SPNFG based sensors exhibit high selectiviy for uric acid and ascorbic acid with low minimum detection limit for glucose (1 nM). and The high durability can be expected for the FET-type sensor by virtue of direct decoration of palladium on the CVD graphene surface.

Co-Detection of Dopamine and Glucose with High Temporal Resolution

Neuronal activity and brain glucose metabolism are tightly coupled, where triggered neurotransmission leads to a higher demand for glucose. To better understand the regulation of neuronal activity and its relation to high-speed metabolism, development of analytical tools that can temporally resolve the transients of vesicular neurotransmitter release and fluctuations of metabolites such as glucose in the local vicinity of the activated neurons is needed. Here we present an amperometric biosensor design for rapid co-detection of glucose and the neurotransmitter dopamine. The sensor is based on the immobilization of an ultra-thin layer of glucose oxidase on to a gold-nanoparticle-covered carbon fiber microelectrode. Our electrode, by altering the potential applied at the sensor surface, allows for the high-speed recording of both glucose and dopamine. We demonstrate that, even though glucose is electrochemically detected indirectly through the enzymatic product and the electroactive dopamine is sensed directly, when exposing the sensor surface to a mixture of the two analytes, fluctuations in glucose and dopamine concentrations can be visualized with similar speed and at a millisecond time scale. Hence, by minimizing the enzyme coating thickness at the sensor surface, dual detection of glucose and dopamine can be realized at the same sensor surface and at time scales necessary for monitoring fast metabolic alterations during neurotransmission.

Surface plasmon resonance biosensor for enzymatic detection of small analytes

Surface plasmon resonance (SPR) biosensing is based on the detection of small changes in the refractive index on a gold surface modified with molecular recognition materials, thus being mostly limited to detecting large molecules. In this paper, we report on a SPR biosensing platform suitable to detect small molecules by making use of the mediator-type enzyme microperoxidase-11 (MP11) in layer-by-layer films. By depositing a top layer of glucose oxidase or uricase, we were able to detect glucose or uric acid with limits of detection of 3.4 and 0.27 μmol l−1, respectively. Measurable SPR signals could be achieved because of the changes in polarizability of MP11, as it is oxidized upon interaction with the analyte. Confirmation of this hypothesis was obtained with finite difference time domain simulations, which also allowed us to discard the possible effects from film roughness changes observed in atomic force microscopy images. The main advantage of this mediator-type enzyme approach is in the simplicity of the experimental method that does not require an external potential, unlike similar approaches for SPR biosensing of small molecules. The detection limits reported here were achieved without optimizing the film architecture, and therefore the performance can in principle be further enhanced, while the proposed SPR platform may be extended to any system where hydrogen peroxide is generated in enzymatic reactions.

Ratiometric glucose sensing based on fluorescent oxygen films and glucose oxidase

A new two-layer sensor film was constructed for sensing glucose based on glucose oxidase and oxygen sensing material. The first layer of film containing the oxygen sensor and intra-reference material was polymerized, then the second layer of glucose oxidase and glutaraldehyde was formed on the oxygen sensor layer. The two-layer sensor film has a resolution up to 0.05mM and a detection range from 0 to 5mM to glucose. The effects of pH and temperature on the sensing performance were systematically investigated. The selective detection of glucose among other monosaccharides, such as fructose, mannose and galactose indicated that the sensing film has excellent selectivity. The prepared sensor was successfully applied for glucose sample detection of glucose concentration in artificial tears.

Mathematical Models for the Impedance Response of Subcutaneous Glucose Sensors

Since 1974, the artificial pancreas has been acknowledged by the greater scientific community to be a possible way to manage type 1 diabetes [1]. In 2008, a case study showed that a subcutaneous continuous glucose monitor sensor could be implanted for 28 days in a patient with type 1 diabetes [2]. However, commercially available subcutaneous continuous glucose monitors are generally changed every seven days to avoid unreliable results. The object of this work is to explore the potential of impedance spectroscopy to monitor the state of health of subcutaneous continuous glucose monitor sensors. A mathematical model has been developed for the impedance response of a glucose oxidase enzyme-based electrochemical biosensor. The model accounts for a glucose limiting membrane GLM, which controls the amount of glucose participating in the enzymatic reaction. The glucose oxidase was assumed to be immobilized within a thin film adjacent to the electrode. In the glucose oxidase layer, a process of enzymatic catalysis transforms the glucose into peroxide, which can be detected electrochemically. This system may be considered to be a special case of the coupled homogeneous and heterogeneous reactions addressed by Levich [3]. The model development required two steps. The nonlinear coupled differential equations governing this system were solved under the assumption of a steady state. The steady-state concentrations resulting from the steady-state simulation were used in the solution of the linearized set of differential equations describing the sinusoidal steady state. The enzymatic catalysis was treated in terms of two homogeneous reactions, one consuming the glucose oxidase and forming gluconic acid, and the other regenerating the glucose oxidase and forming the peroxide. References A. M. Albisser, B. S. Leibel, T. G. Ewart, Z. Davidovac, C. K. Botz, and W. Zingg, Diabetes 23 (1974), 389–396.J. J. Chamberlain and D. Small, Clin. Diabetes, 26 (2008), 138–139.V. G. Levich, Physicochemical Hydrodynamics, Prentice Hall, Englewood Cliffs, NJ, (1962), p. 345-357.

Modeling Multi-Scale Carbon Fiber Supports for Thin Film Bioelectrodes

Carbon electrodes with their super electro-catalytic properties are commonly used in electrochemistry, especially in biomolecular redox reactions1. Electrospinning as a low cost, facile technique is applied to produce uniform carbon nanofibers between 150 nm and 1µm fiber diameter by pyrolyzing electrospun Polyacrylonitrile (PAN) fiber at 250°C and 1100°C. Varying the concentration of PAN in N,N-Dimethylformamide precursor solution changes the fiber diameter and leads to diversity in materials properties, such as surface area, void fraction and conductivity. These electrospun carbon nano fiber (ECNF) properties are characterized by SEM, BET, and four-point probe resistivity measurements. Thin film Os+/2+ mediated electron in hydrogel immobilized glucose oxidase layer is coated on either single micro fiber 2 or free - standing carbon nanofiber mats (Figure 1) to study the effect of carbon fiber physical properties on electron transfer in mediated redox hydrogel bioanode. Electron transport via redox polymer Os+/2+ is enhanced by reducing the average hydrogel film thickness with uniform distribution of small fiber of high surface area (<1.6E3 cm-1 surface area per volume). A transport model is developed to give more insight into these correlations and yield more understanding of electron transfer mechanism in thin film porous electrode. Figure 1

Printable organic thin film transistors for glucose detection incorporating inkjet-printing of the enzyme recognition element

The effect of device architecture upon the response of printable enzymatic glucose sensors based on poly(3-hexythiophene) (P3HT) organic thin film transistors is presented. The change in drain current is used as the basis for glucose detection and we show that significant improvements in drain current response time can be achieved by modifying the design of the sensor structure. In particular, we show that eliminating the dielectric layer and reducing the thickness of the active layer reduce the device response time considerably. The results are in good agreement with a diffusion based model of device operation, where an initial rapid dedoping process is followed by a slower doping of the P3HT layer from protons that are enzymatically generated by glucose oxidase (GOX) at the Nafion gate electrode. The fitted diffusion data are consistent with a P3HT doping region that is close to the source-drain electrodes rather than located at the P3HT:[Nafion:GOX] interface. Finally, we demonstrate that further improvements in sensor structure and morphology can be achieved by inkjet-printing the GOX layer, offering a pathway to low-cost printed biosensors for the detection of glucose in saliva.

Fabrication of biofuel cell containing enzyme catalyst immobilized by layer-by-layer method

Enzymatic biofuel cell (EBC) employing a layer-by-layer (LbL) structure consisting of multiple layers of glucose oxidase (GOx) and poly(ethyleneimine) (PEI) at carbon nanotube (CNT) ([GOx/PEI]n/CNT) is fabricated. The [GOx/PEI]n/CNT serves as anode catalyst for promoting glucose reaction, while Pt is employed as cathode catalyst. To evaluate effect of [GOx/PEI]n/CNT on EBC performance and stability, several characterizations are conducted. The optimal GOx/PEI layer is determined electrochemically, and it turns out that [GOx/PEI]2/CNT is the best. Electron transfer rate constant of the optimal layer is 11.3s−1, its glucose sensitivity is 83 μAmM−1cm−2, and maximum power density of EBC adopting [GOx/PEI]2/CNT is 1.34 mWcm−2. The values are superior to those of other reference structures, indicating that the [GOx/PEI]2/CNT can produce excellent reactivity, followed by improved EBC performance. In terms of redox reaction mechanism of flavin adenine dinucleotide (FAD) within [GOx/PEI]2/CNT, glucose does not affect the redox reaction of FAD, while oxygen serves as mediator in transferring electrons and protons produced by glucose oxidation into those for reduction reaction of FAD. It is also found that the [GOx/PEI]2/CNT is confined by surface reaction and the reaction is quasi-reversible. Regarding long-term stability, [GOx/PEI]2/CNT maintains ∼83% of initial activity even after two weeks.

Amperometric glucose biosensor based on layer-by-layer films of microperoxidase-11 and liposome-encapsulated glucose oxidase

An important step in several bioanalytical applications is the immobilization of biomolecules. Accordingly, this procedure must be carefully chosen to preserve their biological structure and fully explore their properties. For this purpose, we combined the versatility of the layer-by-layer (LbL) method for the immobilization of biomolecules with the protective behavior of liposome-encapsulated systems to fabricate a novel amperometric glucose biosensor. To obtain the biosensing unit, an LbL film of the H2O2 catalyst polypeptide microperoxidase-11 (MP-11) was assembled onto an indium-tin oxide (ITO) electrode followed by the deposition of a liposome-encapsulated glucose oxidase (GOx) layer. The biosensor response toward glucose detection showed a sensitivity of 0.91±0.09 (μA/cm2)/mM and a limit of detection (LOD) of 8.6±1.1μM, demonstrating an improved performance compared to similar biosensors with a single phospholipid-liposome or even containing a non-encapsulated GOx layer. Finally, glucose detection was also performed in a zero-lactose milk sample to demonstrate the potential of the biosensor for food analysis.

Amperometric glucose biosensor based on glucose oxidase dispersed in multiwalled carbon nanotubes/graphene oxide hybrid biocomposite

An amperometric glucose biosensor based on enhanced and fast direct electron transfer (DET) of glucose oxidase (GOx) at enzyme dispersed multiwalled carbon nanotubes/graphene oxide (MWCNT/GO) hybrid biocomposite was developed. The fabricated hybrid biocomposite was characterized by transmission electron microscopy (TEM), Raman and infrared spectroscopy (IR). The TEM image of hybrid biocomposite reveals that a thin layer of GOx was covered on the surface of MWCNT/GO hybrid composite. IR results validate that the hybrid biocomposite was formed through the electrostatic interactions between GOx and MWCNT/GO hybrid composite. Further, MWCNT/GO hybrid composite has also been characterized by TEM and UV–visible spectroscopy. A pair of well-defined redox peak was observed for GOx immobilized at the hybrid biocomposite electrode than that immobilized at the MWCNT modified electrode. The electron transfer rate constant (Ks) of GOx at the hybrid biocomposite was calculated to be 11.22s−1. The higher Ks value revealed that fast DET of GOx occurred at the electrode surface. Moreover, fabricated biosensor showed a good sensitivity towards glucose oxidation over a linear range 0.05–23.2mM. The limit of detection (LOD) was estimated to be 28μM. The good features of the proposed biosensor could be used for the accurate detection of glucose in the biological samples.

Sol–gel immobilized enzymatic glucose biosensor on gold interdigitated array (IDA) microelectrode

An enzymatic glucose biosensor with good sensitivity, selectivity and stability employing interdigitated array microelectrode (IDA μ-electrode) was reported. IDA μ-electrode was prepared by photolithography method with its surface immobilized with a layer of glucose oxidase (GOx), entrapped in a three-dimensional network composed of chitosan and tetraethyl orthosilicate sol–gel. The surface of the as-prepared IDA μ-electrode was characterized by scanning electron microscope, electron spectroscopy for chemical analysis, and atomic force microscopy. The experimental parameters for the best glucose sensing performance were optimized according to the loading of GOx, the applied voltages, the concentration of mediator, and the pH for glucose detection. The resulted biosensor exhibited a good response to glucose with a wide linear range from 0 to 35 mM and a low detection limit of 1 mM. The glucose sensor also showed a short response time (within 5 s) that the fast response was reflected by the small Michaelis–Menten constant (KM app) with a value of 2.94 mM. The reported glucose biosensor exhibited good sensitivity (8.74 μA/mM.cm2), reproducibility, and stability.