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Human liver subcellular fractions, including liver microsomes (HLM), liver cytosol fractions, and S9 fractions, are extensively utilized in in vitro assays to predict liver metabolism. The S9 fractions are supernatants of human liver homogenates that contain both microsomes and cytosol, which include most cytochrome P450 (CYP) enzymes and soluble phase II enzymes such as glucuronosyltransferases and sulfotransferases. This study reports on the direct electrochemistry and biocatalytic features of redox-active enzymes in S9 fractions for the first time. We investigated the electrochemical properties of S9 films by immobilizing them onto a high-purity graphite (HPG) electrode and performing cyclic voltammetry under anaerobic (Ar-saturated) and aerobic (O 2 -saturated) conditions. The heterogeneous electron transfer rate between the S9 film and the HPG electrode was found to be 14 ± 3 s -1 , with a formal potential of -0.451 V vs. Ag/AgCl reference electrode, which confirmed the electrochemical activation of the FAD/FMN cofactor containing CYP450-reductase (CPR) as the electron receiver from the electrode. The S9 films have also demonstrated catalytic oxygen reduction under aerobic conditions, identical to HLM films attached to similar electrodes. Additionally, we investigated CYP activity in the S9 biofilm for phase I metabolism using diclofenac hydroxylation as a probe reaction and identified metabolic products using liquid chromatography-mass spectrometry (LC-MS). Investigating the feasibility of utilizing liver S9 fractions in such electrochemical assays offers significant advantages for pharmacological and toxicological evaluations of new drugs in development while providing valuable insights for the development of efficient biosensor and bioreactor platforms.

Human exposure to acute and chronic levels of heavy metal ions are linked with various health issues, including reduced children's intelligence quotients, developmental challenges, cancers, hypertension, immune system compromises, cytotoxicity, oxidative cellular damage, and neurological disorders, among other health challenges. The potential environmental HMI contaminations, the biomagnification of heavy metal ions along food chains, and the associated risk factors of heavy metal ions on public health safety are a global concern of top priority. Hence, developing low-cost analytical protocols capable of rapid, selective, sensitive, and accurate detection of heavy metal ions in environmental samples and consumable products is of global public health interest. Conventional flame atomic absorption spectroscopy, graphite furnace atomic absorption spectroscopy, atomic emission spectroscopy, inductively coupled plasma-optical emission spectroscopy, inductively coupled plasma-mass spectroscopy, X-ray diffractometry, and X-ray fluorescence have been well-developed for HMIs and trace element analysis with excellent but varying degrees of sensitivity, selectivity, and accuracy. In addition to high instrumental running and maintenance costs and specialized personnel training, these instruments are not portable, limiting their practicality for on-demand, in situ, field study, or point-of-need HMI detection. Increases in the use of electrochemical and colorimetric techniques for heavy metal ion detections arise because of portable instrumentation, high sensitivity and selectivity, cost-effectiveness, small size requirements, rapidity, and visual detection of colorimetric nanosensors that facilitate on-demand, in situ, and field heavy metal ion detections. This review highlights the new approach to low-cost, rapid, selective, sensitive, and accurate detection of heavy metal ions in ecosystems (soil, water, air) and consumable products. Specifically, the review highlights low-cost, portable, and recent advances in smartphone-operated screen-printed electrodes (SPEs), plastic chip SPES, and carbon fiber paper-based nanosensors for environmental heavy metal ion detection. In addition, the review highlights recent advances in colorimetric nanosensors for heavy metal ion detection requirements. The review provides the advantages of electrochemical and optical nanosensors over the conventional methods of HMI analyses. The review further provides in-depth coverage of the detection of arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), manganese (Mn), nickel (Ni), lead (Pb), and zinc (Zn) ions in the ecosystem, with emphasis on environmental and biological samples. In addition, the review discusses the advantages and challenges of the current electrochemical and colorimetric nanosensors protocol for heavy metal ion detection. It provides insight into the future directions in the use of the electrochemical and colorimetric nanosensors protocol for heavy metal ion detection., Competing Interests: The authors declare no conflict of interest.

Nanoparticle-based electrochemical immunosensors demonstrate high sensitivity toward biomarker detection due to the large surface area of the nanoparticles and their ability to amplify the signal of the target molecule. Additionally, they have a fast response time, relatively lower cost, and can be easily miniaturized for point-of-care applications. Among noble metals, silver nanoparticles (AgNPs) have been extensively used in electrochemical sensors due to their unique properties, such as catalytic activity and excellent electrical conductivity. This Feature Article describes six approaches for incorporating AgNPs in electrochemical platforms, featuring the most recent developments in the silver-gold galvanic exchange-based detection strategy. With a few exceptions, many of these detection methods use AgNP oxidation into Ag + ions, followed by electrodeposition of Ag + ions onto the working electrode as zero-valent Ag metal and a final stripping step using a voltammetric technique. Combining these steps provides desirable low detection limits and good sensitivity for various biomarkers. A few other methods involved the reduction of Ag + ions and depositing them as Ag metal onto the electrode using a reagent mixture so that the striping analysis could be performed. Typically, this reagent mixture includes Ag + ions, a reducing agent, or an enzyme substrate. Besides, AgNPs have also been directly used to modify the surface of electrodes to facilitate kinetically favored redox-mediated electrochemical reactions. In addition to Ag detection methods, this report will also provide recent examples to illustrate how the size and shape of AgNPs impact the detection limits and sensitivity of an electrochemical assay. Finally, we discuss recent developments in lab-on-a-chip type immunosensors designed explicitly for Ag-based metalloimmunoassay detection, and we envision that this article will provide a comprehensive summary of the operational principles and new insights into such immunoassay systems.

In this paper we demonstrate the use of dual-shaped silver nanoparticles (AgNPs) as detection labels for electrochemical bioassays. The key finding is that by using AgNP labels having two different shapes simultaneously, the limit of detection (LOD) for the assays is lowered compared to using either of the two shapes separately. The two shapes were silver nanocubes (AgNCs) having edge lengths of 40 ± 4 nm and spherical AgNPs (sAgNPs) having diameters of 20 ± 3 nm. Two different bioassays were examined. In both cases the Ag labels were functionalized with antibodies. In the one assay, the labels are directly linked to a second antibody immobilized on magnetic beads. In the second assay, the antibodies on the AgNP labels and the antibodies on the magnetic beads are linked via a peptide. The peptide is N-terminal prohormone brain natriuretic peptide (NT-proBNP), which is a heart failure marker. The efficacy of the two electrochemical assays as a function of the ratio of the two labels was investigated using a galvanic exchange/anodic stripping voltammetry method. The key finding is that by optimizing the ratio of the two types of AgNP labels, it is possible to decrease the LOD of the assays without compromising the dynamic range compared to using either of the two labels independently. This made it possible to achieve the clinically relevant range for NT-proBNP analysis used by physicians for heart failure risk stratification., Competing Interests: Conflict of Interest The technology reported in this manuscript could eventually be used by Galvanyx, LLC. The corresponding author has an interest in this company.

Quality checks, assessments, and the assurance of food products, raw materials, and food ingredients is critically important to ensure the safeguard of foods of high quality for safety and public health. Nevertheless, quality checks, assessments, and the assurance of food products along distribution and supply chains is impacted by various challenges. For instance, the development of portable, sensitive, low-cost, and robust instrumentation that is capable of real-time, accurate, and sensitive analysis, quality checks, assessments, and the assurance of food products in the field and/or in the production line in a food manufacturing industry is a major technological and analytical challenge. Other significant challenges include analytical method development, method validation strategies, and the non-availability of reference materials and/or standards for emerging food contaminants. The simplicity, portability, non-invasive, non-destructive properties, and low-cost of NIR spectrometers, make them appealing and desirable instruments of choice for rapid quality checks, assessments and assurances of food products, raw materials, and ingredients. This review article surveys literature and examines current challenges and breakthroughs in quality checks and the assessment of a variety of food products, raw materials, and ingredients. Specifically, recent technological innovations and notable advances in quartz crystal microbalances (QCM), electroanalytical techniques, and near infrared (NIR) spectroscopic instrument development in the quality assessment of selected food products, and the analysis of food raw materials and ingredients for foodborne pathogen detection between January 2019 and July 2020 are highlighted. In addition, chemometric approaches and multivariate analyses of spectral data for NIR instrumental calibration and sample analyses for quality assessments and assurances of selected food products and electrochemical methods for foodborne pathogen detection are discussed. Moreover, this review provides insight into the future trajectory of innovative technological developments in QCM, electroanalytical techniques, NIR spectroscopy, and multivariate analyses relating to general applications for the quality assessment of food products.

In the present article we report a new hybrid microfluidic device (hyFlow) comprising a disposable paper electrode and a three-dimensional (3D) printed plastic chip for the electrochemical detection of a magnetic bead-silver nanoparticle (MB-AgNP) bioconjugate. This hybrid device evolved due to the difficulty of incorporating micron-scale MBs into paper-only fluidic devices. Specifically, paper fluidic devices can entrap MB-containing conjugates within their cellulose or nitrocellulose fiber matrix. The hyFlow system was designed to minimize such issues and transport MB conjugates more efficiently to the electrochemical detection zone of the device. The hyFlow system retains the benefit of fluid transport by pressure-driven flow, however, no pump is required for its operation. The hyFlow device is capable of detecting either pre-formed MB-AgNP conjugates or conjugates formed in situ. The detection limit of AgNPs using this device is 12 pM, which represents just 22 AgNPs per MB.

Low-cost, voltage-driven biocatalytic designs for rapid drug metabolism assay, chemical toxicity screening, and pollutant biosensing represent considerable significance for pharmaceutical, biomedical, and environmental applications. In this study, we have designed biointerfaces of human liver microsomes with various roughened, high-purity graphite disk electrodes to study electrochemical and electrocatalytic properties. Successful spectral and microscopic characterizations, direct bioelectronic communication, direct electron-transfer rates from the electrode to liver microsomal enzymes, microsomal heme-enzyme specific oxygen reduction currents, and voltage-driven diclofenac hydroxylation (chosen as the probe reaction) are presented., Competing Interests: Declaration of Competing Interest There are no conflicts to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Electrode materials play an important role on the electrocatalytic properties of immobilized biocatalysts. In this regard, achieving direct electronic communication between the electrode and redox sites of biocatalysts eliminates the need for additional electron transfer mediators for biocatalytic applications in fuel cells and other electrochemical energy devices. In order to increase electrocatalytic currents and power in fuel cells and metal-air batteries, conductive carbon-nanostructure-modified large surface area electrodes are quite useful. Among various electrode materials, freestanding buckypapers made from carbon nanotubes have gained significance as they do not require a solid support material and thus facilitate miniaturization. In this article, we present the effect of buckypaper (BP) thickness on the electrocatalytic properties of a bilirubin oxidase (BOD) enzyme. In this study, we prepared BPs of varying thicknesses ranging from 87 μm, the minimum thickness for suitable handling with a good stability in aqueous experiments, to 380 μm. BOD was adsorbed overnight onto the BPs, mostly via hydrophobic and π-π interactions since the nanotubes used were not chemically functionalized. Furthermore, intercalation of the BOD molecules onto the nanotubes' multicylindrical network is feasible. We determined that the lower range BP thickness (<220 μm) exhibited better sigmoidal shaped electrocatalytic currents than the higher BP-thickness-based BOD biofilms with larger capacitive currents. An oxygen reduction current density of up to 3 mA cm -2 is achieved without the use of any redox mediators or tedious electrode modifications. Using the 87 μm thick BP as the representative case, we were able to obtain distinguishable peaks for all Cu sites of BOD and assign their types, T1, T2, and T3, based on the peak-width at half-maximum in anaerobic cyclic voltammograms. Our peak assignment is further supported by the appearance of dual electrocatalytic oxygen reduction waves at a higher scan rate region (>10 mV s -1 ) in oxygen-saturated buffer, which is identified to be driven by an ∼3.5 times faster electron transfer rate from the buckypaper to the T2/T3 center than the T1 Cu site. Findings from this study are significant for designing enzyme electrocatalytic systems and biosensors in general and fuel cells and aerobic energy storage devices in particular, where the cathodic oxygen reduction current is often inadequate.

We report single drop electroanalytical measurements of pharmaceutically and biologically relevant compounds using screen printed electrodes (SPEs) modified with carboxylated multiwalled carbon nanotubes (MWCNT-COOH) as the sensor surface. Acetaminophen, nicotine, ascorbic acid, and nicotinamide adenine dinucleotide reduced form (NADH) were detected in a single drop of solution. We show that combined polar and nonpolar interactions of analytes with -COOH functional groups and large surface area of MWCNT, respectively, allow highly sensitive analyte detection with wide dynamic range. Smaller analytes can bind to a significantly greater number of sensor sites than the bulkier analytes and offer better detection sensitivity. Results suggest that sensitivity is controlled by predominant nonpolar interactions that an analyte can undergo with the MWCNT-COOH SPE sensor surface, whereas limit of detection is controlled by the extent of polar interactions between an analyte and the sensor surface, facilitating interfacial charge transport and an electrochemical signal output. Furthermore, a combination of polar and nonpolar analyte interactions with the sensor surface shows a synergistic effect on sensitivity and detection limit. This could be a likely reason for why sensitivity does not need to always correlate with lower detection limits as variations in the interfacial interactions are critical. Application of the designed single drop method to real samples was validated by estimating the amounts of acetaminophen, nicotine, ascorbic acid, and NADH in commercially available pharmaceuticals with excellent recovery.

A rapid optical microarray imaging approach for anticancer drug screening at specific cancer protein-protein interface targets with binding kinetics and validation by a mass sensor is reported for the first time. Surface plasmon resonance imager (SPRi) demonstrated a 3.5-fold greater specificity for interactions between murine double minute 2 protein (MDM2) and wild-type p53 over a nonspecific p53 mutant in a real-time microfluidic analysis. Significant percentage reflectivity changes (Δ%R) in the SPRi signals and molecular-level mass changes were detected for both the MDM2-p53 interaction and its inhibition by a small-molecule Nutlin-3 drug analogue known for its anticancer property. We additionally demonstrate that synthetic, inexpensive binding domains of interacting cancer proteins are sufficient to screen anticancer drugs by an array-based SPRi technique with excellent specificity and sensitivity. This imaging array, combined with a mass sensor, can be used to study quantitatively any protein-protein interaction and screen for small molecules with binding and potency evaluations.

We demonstrate here that the edge-to-edge interaction between carbon nanotubes (CNTs) and edge plane electrodes plays an important role in exposing a large proportion of the basal planes of the CNTs to allow enhanced π-π stacking of a pyrenyl compound and subsequent high density protein immobilization yielding large electrocatalytic currents.

In order to design a green microsomal bioreactor on suitably identified carbon electrodes, it is important to understand the direct electrochemical properties at the interfaces between various carbon electrode materials and human liver microsomes (HLM). The novelty of this work is on the investigation of directly adsorbed HLM on different carbon electrodes with the goal to develop a simple, rapid, and new bioanalytical platform of HLM useful for drug metabolism and inhibition assays. These novel biointerfaces are designed in this study by a one step adsorption of HLM directly onto polished basal plane pyrolytic graphite (BPG), edge plane pyrolytic graphite (EPG), glassy carbon (GC), or high-purity graphite (HPG) electrodes. The estimated direct electron transfer (ET) rate constant of HLM on the smooth GC surface was significantly greater than that of the other electrodes. On the other hand, the electroactive surface coverage and stability of microsomal films were greater on highly surface defective, rough EPG and HPG electrodes compared to the smooth GC and less defective hydrophobic BPG surfaces. The presence of significantly higher oxygen functionalities and flatness of the GC surface is attributed to favoring faster ET rates of the coated layer of thin HLM film compared to other electrodes. The cytochrome P450 (CYP)-specific bioactivity of the liver microsomal film on the catalytically superior, stable HPG surface was confirmed by monitoring the electrocatalytic conversion of testosterone to 6β-hydroxytestosterone and its inhibition by the CYP-specific ketoconazole inhibitor. The identification of optimal HPG and EPG electrodes to design biologically active interfaces with liver microsomes is suggested to have immense significance in the design of one-step, green bioreactors for stereoselective drug metabolite synthesis and drug metabolism and inhibition assays.

Direct electron-transfer and electrocatalytic kinetics of covalently attached myoglobin (MB) films on magnetic nanoparticles (MB-MNP(covalent)), in comparison to the corresponding physisorbed films and individual components, are reported for the first time. MB-MNP(covalent) ("-" denotes a covalent linkage) was adsorbed onto a cationic poly(ethyleneimine) layer (PEI) coated high-purity graphite (HPG) electrode. Similarly, films of myoglobin physisorbed on magnetic nanoparticles (MB/MNP(adsorbed), "/" denotes a noncovalent nature), only MB, or only MNP were constructed on HPG/PEI electrodes for comparison. The observed electron-transfer rate constants (k(s), s(-1)) were in the following order: MB-MNP(covalent) (69 ± 6 s(-1)), MB/MNP(adsorbed) (37 ± 2 s(-1)), only MB (27 ± 2 s(-1)), and only MNP (16 ± 3 s(-1)). The electrocatalytic properties of these films were investigated with the aid of tert-butylhydroperoxide as a model reactant, and its reduction kinetics were examined. We observed the following order of catalytic current density: MB-MNP(covalent) > MB/MNP(adsorbed) > only MNP > only MB, in agreement with the electron-transfer (ET) rates of MB-MNP(covalent) and MB/MNP(adsorbed) films. The crucial function of MNP in favorably altering the direct ET and electrocatalytic properties of both covalently bound MB and physisorbed MB molecules are discussed. In addition, the occurrence of a highly enhanced electron-hopping mechanism in the designed covalent MB-MNP(covalent) films over the corresponding physisorbed MB/MNP(adsorbed) film is proposed. The enhanced electron-transfer rates and catalytic current density suggest the advantages of using metalloenzymes covalently attached to polymer-functionalized magnetic nanoparticles for the development of modern highly efficient miniature biosensors and bioreactors.