Pyrrolizidine Alkaloids as Hazardous Toxins in Natural Products: Current Analytical Methods and Latest Legal Regulations.

Pyrrolizidine alkaloids (PAs) are toxic compounds that occur naturally in certain plants, however, there are many secondary pathways causing PA contamination of other plants, including medicinal herbs and plant-based food products, which pose a risk of human intoxication. It is proven that chronic exposure to PAs causes serious adverse health consequences resulting from their cytotoxicity and genotoxicity. This review briefly presents PA occurrence, structures, chemistry, and toxicity, as well as a set of analytical methods. Recently developed sensitive electrochemical and chromatographic methods for the determination of PAs in honey, teas, herbs, and spices were summarized. The main strategies for improving the analytical efficiency of PA determination are related to the use of mass spectrometric (MS) detection; therefore, this review focuses on advances in MS-based methods. Raising awareness of the potential health risks associated with the presence of PAs in food and herbal medicines requires ongoing research in this area, including the development of sensitive methods for PA determination and rigorous legal regulations of PA intake from herbal products. The maximum levels of PAs in certain products are regulated by the European Commission; however, the precise knowledge about which products contain trace but significant amounts of these alkaloids is still insufficient.

Bovine Serum Albumin - Hydroxyapatite Nanoflowers as Potential Local Drug Delivery System of Ciprofloxacin.

Introduction: Hybrid nanoflowers are structures consisting of organic (enzymes, proteins, nucleic acids) and inorganic components (mostly metal phosphates) with a flower-like hierarchical structure. Novel hybrid nanoflowers based on bovine serum albumin (BSA) and hydroxyapatite (HA) were obtained and characterized. Study on BSA-HA nanoflowers as potential drug delivery system is reported for the first time. Methods: Embedding ciprofloxacin in the structure of hybrid nanoflowers was confirmed by ATR-FTIR and thermogravimetric analysis. The inorganic phase of the nanoflowers was determined by X-ray diffraction. UV‒Vis spectroscopy was used to evaluate the release profiles of ciprofloxacin from nanoflowers in buffer solutions at pH 7.4 and 5. The agar disk diffusion method was used to study the antibacterial activity of the synthesized nanoflowers against Staphylococcus aureus and Pseudomonas aeruginosa. Results: Bovine serum albumin - hydroxyapatite nanoflowers were obtained with diameters of ca. 1-2 µm. The kinetics of ciprofloxacin release from nanoflowers were described by the Korsmeyer-Peppas model. The antibacterial activity of the synthesized nanoflowers was demonstrated against S. aureus and P. aeruginosa, two main pathogens found in osteomyelitis. Conclusion: The formulated nanoflowers may act as an efficient local antibiotic delivery system. Due to the use of nonhazardous, biodegradable components and benign synthesis, hybrid nanoflowers are very promising drug delivery systems that could be applied in the treatment of skeletal system infections.

Computational Modeling of Gold Nanoparticle Interacting with Molecules of Pharmaceutical Interest in Water.

We derived a theory of biomolecule binding to the surface of Aun clusters and of the Au plane based on the hard soft acid base (HSAB) principle and the free electron metallic surface model. With the use of quantum mechanical calculations, the chemical potential (µ) and the chemical hardness (η) of the biomolecules are estimated. The effect of the gold is introduced via the empirical value of the gold chemical potential (-5.77 eV) as well as by using the expression (modified here) for the chemical hardness (η). The effect of an aqueous environment is introduced by means of the ligand molecular geometry influenced by the PCM field. This theory allows for a fast and low-cost estimation of binding biomolecules to the AuNPs surface. The predicted binding of thiolated genistein and abiraterone to the gold surface is about 20 kcal/mol. The model of the exchange reaction between these biomolecules and citrates on the Au surface corresponds well with the experimental observations for thiolated abiraterone. Moreover, using a model of the place exchange of linear mercaptohydrocarbons on 12-mercaptododecane acid methyl ester bound to the Au surface, the present results reflect the known relation between exchange energy and the size of the reagents.

Thiogenistein-Antioxidant Chemistry, Antitumor Activity, and Structure Elucidation of New Oxidation Products.

Isoflavonoids such as genistein (GE) are well known antioxidants. The predictive biological activity of structurally new compounds such as thiogenistein (TGE)-a new analogue of GE-becomes an interesting way to design new drug candidates with promising properties. Two oxidation strategies were used to characterize TGE oxidation products: the first in solution and the second on the 2D surface of the Au electrode as a self-assembling TGE monolayer. The structure elucidation of products generated by different oxidation strategies was performed. The electrospray ionization mass spectrometry (ESI-MS) was used for identifying the product of electrochemical and hydrogen peroxide oxidation in the solution. Fourier transform infrared spectroscopy (FT-IR) with the ATR mode was used to identify a product after hydrogen peroxide treatment of TGE on the 2D surface. The density functional theory was used to support the experimental results for the estimation of antioxidant activity of TGE as well as for the molecular modeling of oxidation products. The biological studies were performed simultaneously to assess the suitability of TGE for antioxidant and antitumor properties. It was found that TGE was characterized by a high cytotoxic activity toward human breast cancer cells. The research was also carried out on mice macrophages, disclosing that TGE neutralized the production of the LPS-induced reactive oxygen species (ROS) and exhibits ABTS (2,2'-azino-bis-3-(ethylbenzothiazoline-6-sulphonic acid) radical scavenging ability. In the presented study, we identified the main oxidation products of TGE generated under different environmental conditions. The electroactive centers of TGE were identified and its oxidation mechanisms were proposed. TGE redox properties can be related to its various pharmacological activities. Our new thiolated analogue of genistein neutralizes the LPS-induced ROS production better than GE. Additionally, TGE shows a high cytotoxic activity against human breast cancer cells. The viability of MCF-7 (estrogen-positive cells) drops two times after a 72-h incubation with 12.5 µM TGE (viability 53.86%) compared to genistein (viability 94.46%).

Anti-Cancer and Electrochemical Properties of Thiogenistein-New Biologically Active Compound.

Pharmacological and nutraceutical effects of isoflavones, which include genistein (GE), are attributed to their antioxidant activity protecting cells against carcinogenesis. The knowledge of the oxidation mechanisms of an active substance is crucial to determine its pharmacological properties. The aim of the present work was to explain complex oxidation processes that have been simulated during voltammetric experiments for our new thiolated genistein analog (TGE) that formed the self-assembled monolayer (SAM) on the gold electrode. The thiol linker assured a strong interaction of sulfur nucleophiles with the gold surface. The research comprised of the study of TGE oxidative properties, IR-ATR, and MALDI-TOF measurements of SAM before and after electrochemical oxidation. TGE has been shown to be electrochemically active. It undergoes one irreversible oxidation reaction and one quasi-reversible oxidation reaction in PBS buffer at pH 7.4. The oxidation of TGE results in electroactive products composed likely from TGE conjugates (e.g., trimers) as part of polymer. The electroactive centers of TGE and its oxidation mechanism were discussed using IR supported by quantum chemical and molecular mechanics calculations. Preliminary in-vitro studies indicate that TGE exhibits higher cytotoxic activity towards DU145 human prostate cancer cells and is safer for normal prostate epithelial cells (PNT2) than genistein itself.

Biosupercapacitor with an enzymatic cascade at the anode working in a sucrose solution.

In this report, we demonstrate the advantages of the dual-mode operation of an enzymatic biosupercapacitor with nanostructured polypyrrole/nanocellulose, gold nanoparticle-based paper electrodes, sucrose as the anode fuel and molecular oxygen as the oxidant. The device allowed conversion of the sucrose biofuel, and offered storage of the generated power in the same, small-scale device. The external and internal biosupercapacitor re-charging modes were compared. The specific capacitance of the device was 1.8 F cm-2 at a discharge current density of 1 mA cm-2. The cell used in the charge/discharge mode of operation allowed retention of 49% of the initial capacitance after eight days of exhaustive discharging under external load. The discontinuous capacitive mode, preserved the biocatalysts activity for much longer time. The use of such enzyme-based electrical energy sources in the capacitive mode i.e. under discontinuous charging was demonstrated as a solution for preserving high specific capacitance and long-term operational stability.

Cysteamine assay for the evaluation of bioactive electrophiles.

Covalent modifications of thiol and amine groups may control the function of proteins involved in the regulatory and signaling pathways of the cell. In this study, we developed a simple cysteamine assay which can be used to study the reactivity of electrophilic compounds towards primary amine and thiol groups in an aqueous environment. The detection principle is based on the electrochemical, photometrical and mass spectrometric analyses of cysteamine (2-aminoethanethiol) as the molecular probe. This technique is useful for studying the reaction kinetics of electrophiles with thiol (SH) and amino (NH2) groups. The decrease in analytical responses of cysteamine was monitored to evaluate the reactivity of three electrophilic activators of the Nrf2 pathway, which mediates the cellular stress response. The SH-reactivity under cell-free conditions of the tested electrophiles decreased in the following order: 4-hydroxy-2-nonenal ≥ nitro-oleic acid > sulforaphane. However, as shown in RAW264.7 cells, the tested compounds activated Nrf2-dependent gene expression in the opposite order: sulforaphane > nitro-oleic acid ≥ 4-hydroxy-2-nonenal. Although other factors in addition to chemical reactivity play a role in biological systems, we conclude that this cysteamine assay is a useful tool for screening potentially bioactive electrophiles and for studying their reactivity at a molecular level.

Multi-Substrate Biofuel Cell Utilizing Glucose, Fructose and Sucrose as the Anode Fuels.

A significant problem still exists with the low power output and durability of the bioelectrochemical fuel cells. We constructed a fuel cell with an enzymatic cascade at the anode for efficient energy conversion. The construction involved fabrication of the flow-through cell by three-dimensional printing. Gold nanoparticles with covalently bound naphthoquinone moieties deposited on cellulose/polypyrrole (CPPy) paper allowed us to significantly improve the catalysis rate, both at the anode and cathode of the fuel cell. The enzymatic cascade on the anode consisted of invertase, mutarotase, Flavine Adenine Dinucleotide (FAD)-dependent glucose dehydrogenase and fructose dehydrogenase. The multi-substrate anode utilized glucose, fructose, sucrose, or a combination of them, as the anode fuel and molecular oxygen were the oxidant at the laccase-based cathode. Laccase was adsorbed on the same type of naphthoquinone modified gold nanoparticles. Interestingly, the naphthoquinone modified gold nanoparticles acted as the enzyme orienting units and not as mediators since the catalyzed oxygen reduction occurred at the potential where direct electron transfer takes place. Thanks to the good catalytic and capacitive properties of the modified electrodes, the power density of the sucrose/oxygen enzymatic fuel cells (EFC) reached 0.81 mW cm-2, which is beneficial for a cell composed of a single cathode and anode.

NAD(P)-dependent glucose dehydrogenase: Applications for biosensors, bioelectrodes, and biofuel cells.

This review discusses the physical and chemical properties of nicotinamide redox cofactor dependent glucose dehydrogenase (NAD(P) dependent GDH) and its extensive application in biosensors and bio-fuel cells. GDHs from different organisms show diverse biochemical properties (e.g., activity and stability) and preferences towards cofactors, such as nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+). The (NAD(P)+) play important roles in biological electron transfer, however, there are some difficulties related to their application in devices that originate from their chemical properties and labile binding to the GDH enzyme. This review discusses the electrode modifications aimed at immobilising NAD+ or NADP+ cofactors and GDH at electrodes. Binding of the enzyme was achieved by appropriate protein engineering techniques, including polymerisation, hydrophobisation or hydrophilisation processes. Various enzyme-modified electrodes applied in biosensors, enzymatic fuel cells, and biobatteries are compared. Importantly, GDH can operate alone or as part of an enzymatic cascade, which often improves the functional parameters of the biofuel cell or simply allows use of cheaper fuels. Overall, this review explores how NAD(P)-dependent GDH has recently demonstrated high potential for use in various systems to generate electricity from biological sources for applications in implantable biomedical devices, wireless sensors, and portable electronic devices.

The ligand exchange of citrates to thioabiraterone on gold nanoparticles for prostate cancer therapy.

Cancer is one of the leading causes of morbidity and mortality worldwide and nanotechnology has a significant potential to enhance the therapeutic and diagnostic performance of anti-cancer agents. Our work offers a simple and feasible strategy for thiocompound nanomedicines to be used in cancer therapy. Novel gold nanoparticles conjugated with thioabiraterone (AuNP-S-AB) were synthesized and significant new analytical methodologies were developed for their characterization by UV-Vis, TEM, IR, NMR and TGA. Our synthetic approach was based on the ligand exchange of citrates to thioabiraterone on gold nanoparticles. The average particle size of AuNP-S-AB was 14.5 nm with a spherical shape. The identity of thioabiraterone on the gold nanoparticles was proved by NMR and IR spectroscopy. The coverage of the gold nanoparticles with 40.9% (m/m) thioabiraterone was calculated from a TGA analysis. Molecular interactions between the thiol group of thioabiraterone and gold nanoparticles were evaluated through a combined experimental and theoretical study using the density functional theory (DFT). Additionally, an experiment conducted on hepatocytes or human prostate epithelial cells proved that newly synthesized thiol forms of abiraterone, as well as AuNP-S-AB, are more biocompatible than abiraterone. Our proposed idea of delivering abiraterone with our newly designed AuNP-S-AB may constitute a promising and novel prospect in cancer therapy.

Design of Therapeutic Self-Assembled Monolayers of Thiolated Abiraterone.

The aim of our work was to synthetize of a new analogue of abiraterone-thiolated abiraterone (HS-AB) and design a gold surface monolayer, bearing in mind recent advances in tuning monolayer structures and using them as efficient drug delivery systems. Therapeutic self-assembled monolayers (TSAMs) were prepared by chemically attaching HS-AB to gold surfaces. Their properties were studied by voltammetry and atomic force microscopy (AFM). A gold electrode with immobilized thioglycolic acid (HS-GA) was used for comparison. The surface concentration of HS-AB on the gold surface was 0.572 nmol/cm², determined from the area of the voltammetric reduction peaks (desorption process). The area per one molecule estimated from the voltammetry experiments was 0.291 nmol/cm². The capacity of thus prepared electrode was also tested. The calculated capacity for the HS-AB modified electrode is 2.90 µF/cm². The obtained value indicates that the monolayer on the gold electrode is quite well ordered and well-packed. AFM images show the formation of gold nanoparticles as a result of immersing the HS-AB modified gold electrode in an aqueous solution containing 1 mM HAuCl4·3H2O. These structures arise as a result of the interaction between the HS-AB compound adsorbed on the electrode and the AuCl4- ions. The voltammetric experiments also confirm the formation of gold structures with specific catalytic properties in the process of oxygen reduction.

Design and Molecular Modeling of Abiraterone-Functionalized Gold Nanoparticles.

The aim of our work was the synthesis and physicochemical characterization of a unique conjugate consisting of gold nanoparticles (AuNPs) and a pharmacologically active anticancer substance abiraterone (AB). The direct coupling of AB with gold constitutes an essential feature of the unique AuNPs⁻AB conjugate that creates a promising platform for applications in nanomedicine. In this work, we present a multidisciplinary, basic study of the obtained AuNPs⁻AB conjugate. Theoretical modeling based on the density functional theory (DFT) predicted that the Aun clusters would interact with abiraterone preferably at the N-side. A sharp, intense band at 1028 cm-1 was observed in the Raman spectra of the nanoparticles. The shift of this band in comparison to AB itself agrees well with the theoretical model. AB in the nanoparticles was identified by means of electrochemistry and NMR spectroscopy. The sizes of the Au crystallites measured by XRPD were about 9 and 17 nm for the nanoparticles obtained in pH 7.4 and 3.6, respectively. The size of the particles as measured by TEM was 24 and 30 nm for the nanoparticles obtained in pH 7.4 and pH 3.6, respectively. The DLS measurements revealed stable, negatively charged nanoparticles.

Pemetrexed conjugated with gold nanoparticles - Synthesis, characterization and a study of noncovalent interactions.

Gold nanoparticles (AuNPs) have been widely used as nanocarriers in drug delivery application. However, the binding mechanism between AuNPs and drug bases still remains a puzzle. Our study included: (i) optimization of three synthesis of the AuNPs-pemetrexed (PE) nanocomposites formation which was monitored by UV-Vis spectroscopy, (ii) identification of PE in gold nanocomposites and mechanism of PE interaction with gold nanoparticles by electrochemistry, NMR and Raman measurements, (iii) characterization of the three nanocomposites by TEM, DSL, ESL, zeta potential, XRPD and TGA analysis. The obtained nanocomposites are homogeneously shaped and have a maximum diameter of around 14nm and 88nm, as measured by the TEM and DSL techniques, respectively. The zeta potential of the nanocomposites is -43mV and suggests a high stability of the nanoparticles and lower toxicity for the normal cells. Quantum chemical calculations were also performed on model systems to estimate the strength of the AuNPs-PE interaction. Taking into account the experimental and theoretical data a mechanism of the nanocomposites' formation has been proposed in which PE interacts with the gold surface by the COOH/COO- group.

Reticulated vitreous carbon as a scaffold for enzymatic fuel cell designing.

Three - dimensional (3D) electrodes are successfully used to overcome the limitations of the low space - time yield and low normalized space velocity obtained in electrochemical processes with two - dimensional electrodes. In this study, we developed a three - dimensional reticulated vitreous carbon - gold (RVC-Au) sponge as a scaffold for enzymatic fuel cells (EFC). The structure of gold and the real electrode surface area can be controlled by the parameters of metal electrodeposition. In particular, a 3D RVC-Au sponge provides a large accessible surface area for immobilization of enzyme and electron mediators, moreover, effective mass diffusion can also take place through the uniform macro - porous scaffold. To efficiently bind the enzyme to the electrode and enhance electron transfer parameters the gold surface was modified with ultrasmall gold nanoparticles stabilized with glutathione. These quantum sized nanoparticles exhibit specific electronic properties and also expand the working surface of the electrode. Significantly, at the steady state of power generation, the EFC device with RVC-Au electrodes provided high volumetric power density of 1.18±0.14mWcm-3 (41.3±3.8µWcm-2) calculated based on the volume of electrode material with OCV 0.741±0.021V. These new 3D RVC-Au electrodes showed great promise for improving the power generation of EFC devices.

CE method for the in-process control of the synthesis of active substances conjugated with gold nanoparticles.

A fast capillary electrophoresis method was developed and validated for the in-process control (IPC) of the synthesis of active substances (APIs) with gold nanoparticles (AuNPs). The capillary electrophoresis method was key to ensure that the reaction step conducted in order to obtain AuNP and API conjugates will produce the expected product without the presence of free APIs, which is a critical parameter determining the quality of the synthetic material. Capillary electrophoresis was performed using uncoated fused-silica capillaries with the effective length of 40cm, 50µm i.d. and the background electrolyte consisted of 20mM borate buffer (pH 8.5) with the application of hydrodynamic injection 50mbar/5s, voltage 20kV, temperature of the capillary cassette 25°C and UV detection at 261nm for GE, 541nm for AuNP-GE, 227nm for PE and 535nm for AuNP-PE. During validation the specificity, linearity, accuracy, precision, range, and stability of the sample solution were confirmed. The linear regression (R2=0.999) between the corrected peak areas of the analytes and their amount was fulfilled in the range from 2.4µg/mL to 0.3mg/mL for genistein and from 4.6µg/mL to 0.6mg/mL for pemetrexed. Within this range the method was proved to be accurate (99.0% for genistein and 99.9% for pemetrexed) and precise for both analytes with the intra-day RSD values of 0.77% and 0.97% for the migration time of genistein and pemetrexed, respectively. The inter-day RSD values were 1.90% and 2.27% for the migration time of genistein and pemetrexed, respectively. The LOD and LOQ values for pemetrexed were 1.4µg/mL and 4.6µg/mL, respectively, and for genistein 0.72µg/mL and 2.4µg/mL, respectively. The results obtained during the validation indicate that the method is sufficient to be applied for the IPC of the synthesis of APIs with gold nanoparticles.

Synthesis and characterization of genistein conjugated with gold nanoparticles and the study of their cytotoxic properties.

Gold nanoparticles conjugated with drug substances are used in diagnostics and therapies. Apart from the combinations involving gold nanoparticles conjugated with drug substances through linkers, a direct bonding is also known. In our paper the example of such a direct bonding between gold nanoparticles and genistein (AuNPs-GE) is presented. This conjugate was obtained in a one-pot synthesis and the formation of AuNPs-GE was monitored in terms of color change and UV-Vis spectroscopy. It has been shown that genistein reduces Au3+ ions to spherical Au0 nanocrystallites and acts as a stabilizing agent. The efficiency of the purification of the conjugate from free genistein was controlled by the capillary electrophoresis. Gold nanoparticles are homogeneously shaped and have a narrow range of size from 14 to 33nm and the size of the nanoparticles modified with genistein is around 64.64±0.41nm, as measured by the TEM and DSL techniques, respectively. The zeta potential of the gold nanoparticles modified with genistein is -19.32±0.82mV and suggests a high stability of the nanoparticles and lower toxicity for the normal cells. The identity of genistein on the gold nanoparticles was proved by the electrochemistry, NMR and Raman spectroscopy. The mechanism of the conjugate forming has been proposed. The coverage of gold nanoparticles with genistein 5.09% (m/m) has been calculated from the TGA analysis. Moreover, it has been proved that the obtained conjugate is characterized by a high cytotoxic activity towards cancer cells, as observed in the cell line test.

Bioelectrodes based on pseudocapacitive cellulose/polypyrrole composite improve performance of biofuel cell.

Enzymatic electrodes with high internal capacitance, based on cellulose/polypyrrole composite were optimized and utilized to design improved enzymatic fuel cell. Fructose dehydrogenase Gluconobacter sp. specifically adsorbed on the cellulose/polypyrrole matrix and electrophoretically immobilized and electrochemically entrapped Laccase Trametes versicolor, were used as the anode and cathode bioelectrocatalysts, respectively. The cellulose/polypyrrole composite film exhibited pseudocapacitive properties under mild pH conditions. Following modification with carboxylic groups the composite material enabled highly efficient adsorption of enzyme and provided good electrical contact between the enzymatic active sites and the electrode surface. The modified cellulose/polypyrrole composite based electrode was used for the anode leading to mediatorless fructose oxidation giving large catalytic current density, 12.8mAcm(-2). Laccase and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as the mediator entrapped in the cellulose/polypyrrole composite film generated dioxygen reduction current density of 2mAcm(-2). Application of pseudocapacitive matrix and decreasing the distance between electrodes to 1mm lead to improvement of the biofuel cell power output and its regeneration ability. The power of the cell was found to increase by introduction of a preconditioning step during which the cell was kept at open circuit voltage under fuel flow. After 24h of preconditioning the matrix was recharged and the device output reached the power, 2.1mWcm(-2) and OCV, 0.59V.

Biosupercapacitors for powering oxygen sensing devices.

A biofuel cell comprising electrodes based on supercapacitive materials - carbon nanotubes and nanocellulose/polypyrrole composite was utilized to power an oxygen biosensor. Laccase Trametes versicolor, immobilized on naphthylated multi walled carbon nanotubes, and fructose dehydrogenase, adsorbed on a porous polypyrrole matrix, were used as the cathode and anode bioelectrocatalysts, respectively. The nanomaterials employed as the supports for the enzymes increased the surface area of the electrodes and provide direct contact with the active sites of the enzymes. The anode modified with the conducting polymer layer exhibited significant pseudocapacitive properties providing superior performance also in the high energy mode, e.g., when switching on/off the powered device. Three air-fructose biofuel cells connected in a series converted chemical energy into electrical giving 2 mW power and open circuit potential of 2V. The biofuel cell system was tested under various externally applied resistances and used as a powering unit for a laboratory designed two-electrode minipotentiostat and a laccase based sensor for oxygen sensing. Best results in terms of long time measurement of oxygen levels were obtained in the pulse mode -45 s for measurement and 15 min for self-recharging of the powering unit.

Hybrid biobattery based on arylated carbon nanotubes and laccase.

Single-walled carbon nanotubes (SWCNT) were covalently modified with anthracene and anthraquinone and used for the construction of cathodes for biocatalytic reduction of dioxygen. The nanotubes with aromatic groups casted onto the electrode increased the working surface of the electrode and enabled efficient direct electron transfer (DET) between the enzyme and the electrode. The aryl groups enter the hydrophobic pocket of the T1 center of laccase responsible for exchanging electrons with the substrate. Glassy carbon electrode covered with arylated SWCNT and coated with a layer of neutralized Nafion containing laccase was found to be a very efficient cathode in the hybrid battery. Zn wire covered with a Nafion film served as the anode. The cell parameters were determined: power density was 2 mW/cm(2) and the open circuit potential was 1.5 V.