Electrochemical and Surface-Plasmon Correlation of a Serum-Autoantibody Immunoassay with Binding Insights: Graphenyl Surface versus Mercapto-Monolayer Surface.

We present here the correlation of picomolar affinities between surface-plasmon and electrochemical immunoassays for the binding of serum glutamic acid decarboxylase 65 autoantibody (GADA), a biomarker of type 1 diabetes (T1D), to its antigen GAD-65. Carboxylated (∼5.0%)-graphene-modified immunoassembly on a gold surface-plasmon chip or on an electrochemical array provided significantly larger binding affinity, higher sensitivity, and lower detection limits than a self-assembled monolayer surface of mercaptopropionic acid (MPA). Estimation of the relative surface -COOH groups by covalent tagging of an electroactive aminoferrocene showed that the graphenyl surface displayed a greater number of -COOH groups (9-fold) than the MPA surface. X-ray-photoelectron-spectroscopy analysis showed more C-O and C═O functionalities on the graphene-COOH surface than on the MPA surface. The graphene-COOH coating on gold exhibited ∼5.5-fold enhancement of plasmon signals compared with a similar coating on a plain glass surface. In summary, this article provides a quantitative comparison of carboxylated graphene with a mercapto-monolayer immunoassembly. Additionally, we propose that the binding-constant value can be useful as a quality-control checkpoint for reproducible and reliable production of large-scale biosensors for clinical bioassays.

Electrochemical and surface plasmon insulin assays on clinical samples.

Diabetes is a complex immune disorder that requires extensive medical care beyond glycemic control. Recently, the prevalence of diabetes, particularly type 1 diabetes (T1D), has significantly increased from 5% to 10%, and this has affected the health-associated complication incidences in children and adults. The 2012 statistics by the American Diabetes Association reported that 29.1 million Americans (9.3% of the population) had diabetes, and 86 million Americans (age ≥20 years, an increase from 79 million in 2010) had prediabetes. Personalized glucometers allow diabetes management by easy monitoring of the high millimolar blood glucose levels. In contrast, non-glucose diabetes biomarkers, which have gained considerable attention for early prediction and provide insights about diabetes metabolic pathways, are difficult to measure because of their ultra-low levels in blood. Similarly, insulin pumps, sensors, and insulin monitoring systems are of considerable biomedical significance due to their ever-increasing need for managing diabetic, prediabetic, and pancreatic disorders. Our laboratory focuses on developing electrochemical immunosensors and surface plasmon microarrays for minimally invasive insulin measurements in clinical sample matrices. By utilizing antibodies or aptamers as the insulin-selective biorecognition elements in combination with nanomaterials, we demonstrated a series of selective and clinically sensitive electrochemical and surface plasmon immunoassays. This review provides an overview of different electrochemical and surface plasmon immunoassays for insulin. Considering the paramount importance of diabetes diagnosis, treatment, and management and insulin pumps and monitoring devices with focus on both T1D (insulin-deficient condition) and type 2 diabetes (insulin-resistant condition), this review on insulin bioassays is timely and significant.

Pyrenyl-carbon nanostructures for scalable enzyme electrocatalysis and biological fuel cells.

The objective of this article is to demonstrate the electrode geometric area-based scalability of pyrenyl-carbon nanostructure modification for enzyme electrocatalysis and fuel cell power output using hydrogenase anode and bilirubin oxidase cathode as the model system.

Measuring Ultra-low Levels of Nucleotide Biomarkers Using Quartz Crystal Microbalance and SPR Microarray Imaging Methods: A Comparative Analysis.

Circulating serum nucleotide biomarkers are useful indicators for early diagnosis of cancer, respiratory illnesses, and other deadly diseases. In this work, we compared detection performances of a quartz crystal microbalance (QCM), which is a mass sensor, with that of a surface plasmon resonance (SPR) microarray for an oligonucleotide mimic of a microRNA-21 biomarker. A surface immobilized capture oligonucleotide probe was used to hybridize with the target oligonucleotide (i.e., the microRNA-21 mimic) to facilitate selective detection. To obtain ultra-low femtomolar (fM) detection sensitivity, gold nanoparticles (50 nm) were conjugated with the target oligonucleotide. We achieved detection limits of 28and 47 fM for the target oligonucleotide by the QCM and SPRi microarray, respectively. We also conducted sample recovery studies and performed matrix effect analysis. Although the QCM had a lower detection limit, the microarray approach offered better throughput for analysis of up to 16 samples. We confirmed that the designed assay was selective for the target oligonucleotide and did not show signals for the control oligonucleotide with five mismatch sites relative to the target sequence. Combination of the QCM and microarray methods that utilize the same assay chemistry on gold are useful for overcoming clinical sample matrix effects and achieving ultra-low detection of small nucleotide biomarkers with quantitative insights.

Label-Free Real-Time Microarray Imaging of Cancer Protein-Protein Interactions and Their Inhibition by Small Molecules.

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.

Voltammetric immunosensor assembled on carbon-pyrenyl nanostructures for clinical diagnosis of type of diabetes.

Herein we report the first serum insulin voltammetric immunosensor for diagnosis of type 1 and type 2 diabetic disorders. The sensor is composed of multiwalled carbon nanotube-pyrenebutyric acid frameworks on edge plane pyrolytic graphite electrodes (PGE/MWNT/Py) to which an anti-insulin antibody was covalently attached. The detection of picomolar levels of serum insulin binding to the surface antibody was achieved by monitoring the decrease in voltammetric current signals of a redox probe taken in the electrolyte solution. This method offered a detection limit of 15 pM for free insulin present in serum. This detection limit was further lowered to 5 pM by designing serum insulin conjugates with poly(acrylic acid)-functionalized magnetite nanoparticles (100 nm hydrodynamic diameter) and detecting the binding of MNP-serum insulin conjugate to the surface insulin-antibody on PGE/MWNT/Py electrodes. When tested on real patient serum samples, the sensor accurately measured insulin levels. To our knowledge, this is the first report of a voltammetric immunosensor capable of both diagnosing and distinguishing the type of diabetes based on serum insulin levels in diabetic patients.

A simple construction of electrochemical liver microsomal bioreactor for rapid drug metabolism and inhibition assays.

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 6ß-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.

Edge-to-edge interaction between carbon nanotube-pyrene complexes and electrodes for biosensing and electrocatalytic applications.

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.

An electrochemical mass sensor for diagnosing diabetes in human serum.

An electrochemical mass sensor for clinically relevant detection of insulin in human serum conjugated to magnetic nanoparticles and captured onto antibody immobilized gold coated quartz resonators is reported for the first time.

Nanobioelectrocatalysis Using Human Liver Microsomes and Cytochrome P450 Bactosomes: Pyrenyl-Nanocarbon Electrodes.

Human liver microsomes containing various drug-metabolizing cytochrome P450 (P450) enzymes, along with their NADPH-reductase bound to phospholipid membranes, were absorbed onto 1-pyrene butylamine pi-pi stacked with amine-functionalized multiwalled carbon nanotube-modified graphite electrodes. The interfaced microsomal biofilm demonstrated direct electrochemical communication with the underlying electrode surface and enhanced oxygen reduction electrocatalytic activity typical of heme enzymes such as P450s over the unmodified electrodes and nonenzymatic currents. Similar enhancements in currents were observed when the bioelectrodes were constructed with recombinant P450 2C9 (single isoform) expressed bactosomes. The designed liver microsomal and 2C9 bactosomal bioelectrodes successfully facilitated the electrocatalytic conversion of diclofenac, a drug candidate, into 4'-hydroxydiclofenac. The enzymatic electrocatalytic metabolite yield was several-fold greater on the modified electrodes than on the unmodified bulk graphite electrodes adsorbed with a microsomal or bactosomal film. The nonenzymatic metabolite production was less than the enzymatically catalyzed metabolite yield in the designed microsomal and bactosomal biofilm electrodes. To test the throughput potential of the designed biofilms, eight-electrode array configurations were tested with the microsomal and bactosomal biofilms toward electrochemical 4'-hydroxydiclofenac metabolite production from diclofenac. The stability of the designed microsomal bioelectrode was assessed using nonfaradaic impedance spectroscopy over 40 h, which indicated good stability.

Electrocatalytic features of a heme protein attached to polymer-functionalized magnetic nanoparticles.

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.

Efficient bioelectronic actuation of the natural catalytic pathway of human metabolic cytochrome P450s.

Cytochrome (cyt) P450s comprise the enzyme superfamily responsible for human oxidative metabolism of a majority of drugs and xenobiotics. Electronic delivery of electrons to cyt P450s could be used to drive the natural catalytic cycle for fundamental investigations, stereo- and regioselective synthesis, and biosensors. We describe herein 30 nm nanometer-thick films on electrodes featuring excess human cyt P450s and cyt P450 reductase (CPR) microsomes that efficiently mimic the natural catalytic pathway for the first time. Redox potentials, electron-transfer rates, CO-binding, and substrate conversion rates confirmed that electrons are delivered from the electrode to CPR, which transfers them to cyt P450. The film system enabled electrochemical probing of the interaction between cyt P450 and CPR for the first time. Agreement of film voltammetry data with theoretical simulations supports a pathway featuring a key equilibrium redox reaction in the natural catalytic pathway between reduced CPR and cyt P450 occurring within a CPR-cyt P450 complex uniquely poised for substrate conversion.

Plasmonic nucleotide hybridization chip for attomolar detection: localized gold and tagged core/shell nanomaterials.

We report a large amplification of surface plasmon signals for a double hybridization microarray chip assembly that bridges localized gold and detection probe-carrying-core/shell Fe3O4@Au nanoparticles for detection of as low as 80 aM miRNA-155 marker in solution. The plasmonic wavelength match of the gold shell with surface localized gold nanoparticles and the additional scattering band of the core/shell material in resonance with the incident 800 nm light source are the underlying factors for the observed remarkable analyte signal at ultra-low (10-18 order) concentrations.

Simultaneous cortisol/insulin microchip detection using dual enzyme tagging.

Here we describe the development of a dual electrochemical immunosensor microchip for simultaneous detection of insulin (I) and cortisol (C) biomarkers that can enhance the ability to improve glucose regulation using automated insulin delivery. The successful realization of the simultaneous I and C measurements has been realized by integrating different enzymatically-tagged competitive and sandwich immunoassay formats on a single chip platform. The insulin detection is based on a peroxidase (HRP)-labeled sandwich assay whereas the cortisol detection relies on an alkaline phosphatase (ALP)-labeled competitive immunoassay. The attractive analytical performance of the dual marker immunosensor, with no apparent cross-talk, was achieved through systematic optimization of the incubation and amperometric detection of the different captured enzyme tags. Evaluation of dual biosensor chip in untreated serum samples indicated favorable simultaneous detection of picomolar (pM) insulin and nanomolar (nM) cortisol concentrations in a single microliter sample droplet within less than 25min. The new dual immunosensor chip offers considerable promise for frequent decentralized testing of I and C towards a tighter glycemic control and improved management of diabetes.

Roughened graphite biointerfaced with P450 liver microsomes: Surface and electrochemical characterizations.

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.

Control of electrochemical and ferryloxy formation kinetics of cyt P450s in polyion films by heme iron spin state and secondary structure.

Voltammetry of cytochrome P450 (cyt P450) enzymes in ultrathin films with polyions was related for the first time to electronic and secondary structure. Heterogeneous electron transfer (hET) rate constants for reduction of the cyt P450s depended on heme iron spin state, with low spin cyt P450cam giving a value 40-fold larger than high spin human cyt P450 1A2, with mixed spin human P450 cyt 2E1 at an intermediate value. Asymmetric reduction-oxidation peak separations with increasing scan rates were explained by simulations featuring faster oxidation than reduction. Results are consistent with a square scheme in which oxidized and reduced forms of cyt P450s each participate in rapid conformational equilibria. Rate constants for oxidation of ferric cyt P450s in films by t-butyl hydroperoxide to active ferryloxy cyt P450s from rotating disk voltammetry suggested a weaker dependence on spin state, but in the reverse order of the observed hET reduction rates. Oxidation and reduction rates of cyt P450s in the films are also likely to depend on protein secondary structure around the heme iron.

Differences in metabolite-mediated toxicity of tamoxifen in rodents versus humans elucidated with DNA/microsome electro-optical arrays and nanoreactors.

Tamoxifen, a therapeutic and chemopreventive breast cancer drug, was chosen as a model compound because of acknowledged species specific toxicity differences. Emerging approaches utilizing electro-optical arrays and nanoreactors based on DNA/microsome films were used to compare metabolite-mediated toxicity differences of tamoxifen in rodents versus humans. Hits triggered by liver enzyme metabolism were first provided by arrays utilizing a DNA damage end point. The arrays feature thin-film spots containing an electrochemiluminescent (ECL) ruthenium polymer ([Ru(bpy)(2)PVP(10)](2+); PVP, polyvinylpyridine), DNA, and liver microsomes. When DNA damage resulted from reactions with tamoxifen metabolites, it was detected by an increase in light from the oxidation of the damaged DNA by the ECL metallopolymer. The slope of ECL generation versus enzyme reaction time correlated with the rate of DNA damage. An approximate 2-fold greater ECL turnover rate was observed for spots with rat liver microsomes compared to that with human liver microsomes. These results were supported by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of reaction products using nanoreactors featuring analogous films on silica nanoparticles, allowing the direct measurement of the relative formation rate for alpha-(N(2)-deoxyguanosinyl)tamoxifen. We observed 2-5-fold more rapid formation rates for three major metabolites, i.e., alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and tamoxifen N-oxide, catalyzed by rat liver microsomes compared to human liver microsomes. Comparable formation rates were observed for N-desmethyl tamoxifen with rat and human liver microsomes. A better detoxifying capacity for human liver microsomes than rat liver microsomes was confirmed utilizing glucuronyltransferase in microsomes together with UDP-glucuronic acid. Taken together, lower genotoxicity and higher detoxication rates presented by human liver microsomes correlate with the lower risk of tamoxifen in causing liver carcinoma in humans, provided the glucuronidation pathway is active.

Buckypaper-Bilirubin Oxidase Biointerface for Electrocatalytic Applications: Buckypaper Thickness.

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.

Synergistic metabolic toxicity screening using microsome/DNA electrochemiluminescent arrays and nanoreactors.

Platforms based on thin enzyme/DNA films were used in two-tier screening of chemicals for reactive metabolites capable of producing toxicity. Microsomes were used for the first time as sources of cytochrome (cyt) P450 enzymes in these devices. Initial rapid screening involved electrochemiluminescent (ECL) arrays featuring spots containing ruthenium poly(vinylpyridine), DNA, and rat liver microsomes or bicistronically expressed human cyt P450 2E1 (h2E1). Cyt P450 enzymes were activated via the NADPH/reductase cycle. When bioactivation of substrates in the films gives reactive metabolites, they are trapped by covalent attachment to DNA bases. The rate of increase in ECL with enzyme reaction time reflects relative DNA damage rates. "Toxic hits" uncovered by the array were studied in structural detail by using enzyme/DNA films on silica nanospheres as "nanoreactors" to provide nucleobase adducts from reactive metabolites. The utility of this synergistic approach was demonstrated by estimating relative DNA damage rates of three mutagenic N-nitroso compounds and styrene. Relative enzyme turnover rates for these compounds using ECL arrays and LC-UV-MS correlated well with TD 50 values for liver tumor formation in rats. Combining ECL array and nanoreactor/LC-MS technologies has the potential for rapid, high-throughput, cost-effective screening for reactive metabolites and provides chemical structure information that is complementary to conventional toxicity bioassays.