Electrochemically Stimulated Insulin Release from a Modified Graphene‐functionalized Carbon Fiber Electrode

A graphene‐functionalized carbon fiber electrode was modified with adsorbed polyethylenimine to introduce amino functionalities and then with trigonelline and 4‐carboxyphenylboronic acid covalently bound to the amino groups. The trigonelline species containing quarterized pyridine groups produced positive charge on the electrode surface regardless of the pH value, while the phenylboronic acid species were neutral below pH 8 and negatively charged above pH 9 (note that their pK_a=8.4). The total charge on the monolayer‐modified electrode was positive at the neutral pH and negative at pH > 9. Note that 4‐carboxyphenylboronic acid was attached to the electrode surface in molar excess to trigonelline, thus allowing the negative charge to dominate on the electrode surface at basic pH. Negatively charged fluorescent dye‐labeled insulin (insulin‐FITC) was loaded on the modified electrode surface at pH 7.0 due to its electrostatic attraction to the positively charged interface. The local pH in close vicinity to the electrode surface was increased to ca. 9–10 due to consumption of H⁺ ions upon electrochemical reduction of oxygen proceeding at the potential of −1.0 V (vs. Ag/AgCl) applied on the modified electrode. The process resulted in recharging of the electrode surface to the negative value due to the formation of the negative charge on the phenylboronic acid groups, thus resulting in the electrostatic repulsion of insulin‐FITC and stimulating its release from the electrode surface. The insulin release was characterized by fluorescence spectroscopy (using the FITC‐labeled insulin), by electrochemical measurements on an iridium oxide, IrO_x, electrode and by mass spectrometry. The graphene‐functionalized carbon fiber electrode demonstrated significant advantages in the signal‐stimulated insulin release comparing with the carbon fiber electrode without the graphene species.     

Biomolecular Release Stimulated by Electrochemical Signals at a Very Small Potential Applied

DNA release electrochemically stimulated by applying ?10 mV on the modified electrode was studied. The release process was based on the local (interfacial) pH change produced upon H₂O₂ reduction electrocatalyzed by the immobilized microperoxidase‐11. SiO₂ nanoparticles attached to the electrode surface and functionalized with trigonelline and boronic acid species changed their electrical charge from positive to negative upon the interfacial pH change, thus allowing electrostatic adsorption of negatively charged DNA on the positive interface and then its repulsion/release from the negative interface. The loaded/released DNA molecules were labeled with a fluorescent dye to allow easy detection of the released DNA molecules. The important feature of the developed system is the controlled DNA release upon applying very small electrical potential on the modified electrode.     

DNA Computing Systems Activated by Electrochemically‐triggered DNA Release from a Polymer‐brush‐modified Electrode Array

An array of four independently wired indium tin oxide (ITO) electrodes was used for electrochemically stimulated DNA release and activation of DNA‐based Identity, AND and XOR logic gates. Single‐stranded DNA molecules were loaded on the mixed poly(N ,N ‐dimethylaminoethyl methacrylate) (PDMAEMA)/poly(methacrylic acid) (PMAA) brush covalently attached to the ITO electrodes. The DNA deposition was performed at pH 5.0 when the polymer brush is positively charged due to protonation of tertiary amino groups in PDMAEMA, thus resulting in electrostatic attraction of the negatively charged DNA. By applying electrolysis at −1.0 V(vs. Ag/AgCl reference) electrochemical oxygen reduction resulted in the consumption of hydrogen ions and local pH increase near the electrode surface. The process resulted in recharging the polymer brush to the negative state due to dissociation of carboxylic groups of PMAA, thus repulsing the negatively charged DNA and releasing it from the electrode surface. The DNA release was performed in various combinations from different electrodes in the array assembly. The released DNA operated as input signals for activation of the Boolean logic gates. The developed system represents a step forward in DNA computing, combining for the first time DNA chemical processes with electronic input signals.     

Imaging Local Proton Fluxes through a Polycarbonate Membrane by Using Scanning Electrochemical Microscopy and Functionalized Alkanethiols

A new application of scanning electrochemical microscopy (SECM) to probe the transport of protons through membranes is described. Herein, a probe ultramicroelectrode (UME) is modified with a self‐assembled monolayer (SAM) of 11‐mercaptoundecanoic acid to qualitatively image areas within different pH regions above a track‐etched membrane. The current response of the modified electrode in the presence of potassium hexacyanoferrate as electroactive component is different in acidic and alkaline solutions. Depending on the pH value of the solution, the SAM‐covered electrode exposes either a neutral or a negatively charged insulating monolayer at pH 3 or 7, respectively, which leads to an increase/decrease in the faradaic current due to electrostatic interactions between the neutral/charged surface and the charged redox mediator. Therefore, local pH changes in the close vicinity of a membrane‐like substrate lead to different current responses recorded at the tip electrode when scanning above the surface.     

Anticancer Drug Modified GCFE,for the Study of Its In‐Vivo Interaction Mechanism

An anticancer drug (Adriamycin) modified Glassy Carbon Fiber Electrode (GCFE) has been prepared to study its interaction with ds‐DNA. The redox reaction of Adriamycin molecules at the chemically modified GCFE helps in understanding the in‐vivo mechanism of action of this anti cancer drug. The modified electrode has been fabricated by the adsorption of Adriamycin on GCFE surface. The results of Differential Pulse Voltammetric (DPV) analysis in acetate buffer of pH 4.5 ± 0.1, showed that the interaction between DNA guanine and adenine bases and electrode surface, is easily detected. A suitable mechanism for the oxidation and reduction of Adriamycin in‐situ intercalated in ds‐DNA immobilized on to the GCFE surface has been explained. The drug‐DNA complex formation at GCFE surface has also been studied. The prepared modified electrode is of utmost relevance because the mechanism of interaction of DNA‐Adriamycin at charged interfaces is parallel to the in‐vivo DNA‐Adriamycin complex reaction, where the nucleic acid is in close contact with charged phospholipid membranes and proteins. The interaction studies of Adriamycin at modified GCFE using DPV method help in understanding the DNA‐Adriamycin reaction mechanism.     

Novel pH‐responsive mixed‐charge copolymer brushes based on carboxylic acid and quaternary amine monomers

In this study, we report on the fabrication of tunable mixed‐charged copolymer brushes consisting of negatively charged carboxylic acid monomer (4‐vinylbenzoic acid, VBA) and positively charged quaternary amine monomer ((ar‐vinylbenzyl)trimethylammonium chloride) via reversible addition–fragmentation chain transfer‐mediated polymerization. The copolymer brushes have negative charge under neutral and basic conditions, and are positively charged under acidic conditions owing to the protonation of the carboxylate groups. The copolymer brushes revealed a unique reversible wetting behavior with pH. The reversible properties of the copolymer brushes can be employed to regulate the adsorption of charged biomacromolecules such as DNA and proteins. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Real Time Electrochemical Monitoring of DNA/PNA Dissociation by Melting Curve Analysis

An immobilization‐free electrochemical method is reported for real‐time monitoring of the DNA hybrid dissociation between a ferrocene labeled peptide nucleic acid (PNA) and a fully‐complementary or single‐base‐mismatched DNA. This method takes advantages of electrostatic charge characteristics and interactions among the neutrally charged PNA, the negatively charged DNA and the negatively charged electrode surface made of indium tin oxide (ITO). When a ferrocene labeled PNA (Fc‐PNA) sequence is hybridized to a complementary DNA strand, electrostatic repulsion between the negatively charged PNA/DNA hybrid and the negative ITO surface retards the diffusion of the electroactive Fc to the electrode, resulting in a much reduced electrochemical signal. On the other hand, when the Fc‐PNA is dissociated from the hybrid at elevated temperatures, the neutrally charged Fc‐PNA easily diffuses to the electrode with an enhanced electrochemical signal. Therefore, an electrochemical melting curve of the Fc‐PNA/DNA hybrid can be obtained by measuring the Fc signal with the increasing temperature. This strategy allows monitoring of the dissociation of the DNA hybrid in real time, which might lead to a simple detection method for single nucleotide polymorphism (SNP) analysis.     

Glucose‐Regulated Insulin Release from Acid‐Disintegrable Microgels Covalently Immobilized with Glucose Oxidase and Catalase

The fabrication of a novel type of positively charged acid‐disintegrable microgel loaded with insulin by electrostatic interactions and covalently immobilized with glucose oxidase (GOx) and catalase by inverse emulsion polymerization is reported, aiming for glucose‐regulated insulin release by utilizing GOx/catalase cascade enzymatic reactions to trigger local pH decrease and acid‐cleavage of crosslinking moieties. At the same time, a local pH decrease within the microgels also leads to the diminishment of net surface negative charges of encapsulated insulin. The above two factors both synergistically contribute to the prominently enhanced insulin release at high glucose levels (∼10–20 mM ) compared to that in the absence of glucose.     

A Novel DNA‐Enrichment Technology Based on Amino‐Modified Functionalized Silica Nanoparticles

Abstract

In this paper we report a novel DNA‐enrichment technology based on amino‐modified functionalized silica nanoparticles. The approach takes advantage of the amino‐modified silica nanoparticles that have been prepared in one step by the controlled synchronous hydrolysis of tetraethoxysilane and N‐(β‐amimoethyl)‐γ‐aminopropyltriethoxysilane in water nanodroplets of water‐in‐oil microemulsions. The functionalized silica nanoparticles display a positive surface charge at neutral pH due to the presence of amino groups on the surface of these nanoparticles. DNA‐enrichment has been realized in the form of nanoparticle–DNA complexes that is accomplished through electrostatic binding between the positive charge of the amino group and the negative charge of the phosphate groups of the nucleic acid. These nanoparticles have high affinity to bind DNA. The results show that 1 mg of nanoparticles can bind 97.2 µg of plasmid DNA with 4.3 kb. This novel DNA‐enrichment technology has been used successfully in gene delivery.     

Phenylboronic Acid‐Cross‐Linked Nanoparticles with Improved Stability as Dual Acid‐Responsive Drug Carriers

Herein, a kind of dual acid‐sensitive nanoparticles based on monomethoxy poly(ethylene glycol)‐imine‐β‐cyclodextrin is constructed by a facile phenylboronic acid‐cross‐linked way. The data of dynamic light scattering and transmission electron microscope reveal the cross‐linked nanoparticles have improved stability. The cross‐linked nanoparticles could easily self‐assemble and load the anticancer drug at neutral pH condition. However, when the drug‐loaded nanoparticles are delivered to extracellular tumor sites (pH ≈6.8), the surface of the nanoparticles would be amino positively charged and easily internalized by tumor cell due to the cleavage of the acid‐labile benzoic–imine. Subsequently, with the acidity in subcellular compartments significantly increasing (such as the endosome pH ≈5.3), the loaded drug would fast release from the endocytosis carriers due to the hydrolysis of boronate ester. These features suggest that these dual acid‐sensitive cross‐linked nanoparticles not only possess excellent biocompatibility but also can efficiently load and deliver anticancer drug into tumor cells to enhance the inhibition of cellular proliferation, outlining a favorable platform as drug carriers.

     

Sequence Specific Electrochemical DNA Detection Based on Solution‐Phase Competitive Hybridization

We report a novel electrochemical method for detecting sequence‐specific DNA based on competitive hybridization that occurs in a homogeneous solution phase instead of on a solution‐electrode interface as in previously reported competition‐based electrochemical DNA detection schemes. The method utilizes the competition between the target DNA (t‐DNA) and a ferrocene‐labeled peptide nucleic acid probe (Fc‐PNA) to hybridize with a probe DNA (p‐DNA) in solution. The neutral PNA backbone and the electrostatic repulsion between the negatively‐charged DNA backbone and the negatively‐charged electrode surface are then exploited to determine the result of the competition through measurement of the electrochemical signal of Fc. Upon the introduction of the t‐DNA, the stronger hybridization affinity between the t‐DNA and p‐DNA releases the Fc‐PNA from the Fc‐PNA/p‐DNA hybrid, allowing it to freely diffuse to the negatively charged electrode to produce a significantly enhanced electrochemical signal of Fc. Therefore, the presence of the t‐DNA is indicated by the appearance or enhancement of the electrochemical signal, rendering a signal‐on DNA detection, which is less susceptible to false positive and can produce more reliable results than signal‐off detection methods. All the competitive hybridizations occur in a homogeneous solution phase, resulting in very high hybridization efficiency and therefore extremely short assay time. This simple and fast signal‐on solution‐competition‐based electrochemical DNA detection strategy has promising potential to find application in fields such as nucleic acid‐based point‐of‐care testing.     

Direct Detection of DNA and DNA‐Ligand Interaction by Impedance Spectroscopy

In this work we present an impedimetric detection system for DNA‐ligand interactions. The sensor system consists of thiol‐modified single‐stranded DNA chemisorbed to gold. Impedance measurements in the presence of the redox system ferri‐/ferrocyanide show an increase in charge transfer resistance (R_ct) after hybridisation of a complementary target. Different amounts of capture strands, used for gold electrode modification, result in surface coverages between 3 and 15 pmol/cm² ssDNA. The relative change in R_ct upon hybridisation increases with increasing amount of capture probe on the electrode from 1.5‐ to 4.5‐fold. Impedimetric detection of binding events of a metal‐intercalator ([Ru(phen)₃]²⁺) and a groove binder (spermine) to double‐stranded DNA is demonstrated. Binding of [Ru(phen)₃]²⁺ and spermine exhibits a decrease in charge transfer resistance. Here, the ligand’s interaction leads to electrostatic shielding of the negatively charged DNA backbone. The impedance changes have been evaluated in dependence on the concentration of both DNA binders. Furthermore, the association of a single‐stranded binding protein (SSBP) is found to cause an increase in charge transfer resistance only when incubated with single‐stranded DNA. The specific binding of an anti‐dsDNA antibody to the dsDNA‐modified electrode surface decreases in contrast the interfacial impedance.     

Electrochemical Determination of L‐Lactate Using Phenylboronic Acid Monolayer‐Modified Electrodes

The surface of a gold (Au) disk electrode was modified with a self‐assembled monomolecular layer of dithiobis(4‐butylamino‐m‐phenylboronic acid) (DTBA‐PBA) to prepare L ‐lactate‐sensitive electrodes. The DTBA‐PBA‐modified electrodes exhibited an attenuated cyclic voltammogram (CV) for the Fe(CN)₆^3? ion in the presence of L ‐lactate, as a result of the formation of phenylboronate ester of L ‐lactate accompanied with the addition of OH^? ion to the boron atom. In other words, the negatively charged DTBA‐PBA monolayer blocked the electrode surface from the access of the Fe(CN)₆^3?/4? ions. Thus, the DTBA‐PBA monolayer‐modified Au electrode can be used for determining L ‐lactate on the basis of the change in redox current of Fe(CN)₆^3?/4? ions. The calibration graph useful for determining 1–30 mM L ‐lactate was obtained.     

Poly(L‐lysine)‐Induced Aggregation of Single‐Strand Oligo‐DNA‐Modified Gold Nanoparticles

Single‐strand oligo‐DNA‐modified Au nanoparticles (AuNPs) undergo aggregation in the presence of poly(L ‐lysine) (PLL), which is attributed to the interactions between the oligo‐DNA and PLL. These interactions between the oligo‐DNA and PLL were identified to be electrostatic when the lysine residues of PLL were positively charged and to be hydrogen bonding when the residues were deprotonated. The aggregation was promoted with an increase in the pH value at a pH level lower than the pK_a value of PLL (pK_a≈10.0) due to the gradual deprotonation of the lysine residues and thus suppressed electrostatic interactions between the positively charged lysine residues of PLL and the negatively charged backbone phosphate groups of the oligo‐DNA. At pH levels higher than the pK_a value of PLL, the aggregation was identified to be dominated by the hydrogen bonds between the bases of the oligo‐DNA and the deprotonated lysine residues of PLL. This study prompts the possibility that the spectral, and thus color, change of AuNPs upon aggregation can be used as a probe to follow the interactions between oligo‐DNA and polypeptides.     

Sensitively Electrochemical Sensing for Sequence‐Specific Detection of Phosphinothricin Acetyltransferase Gene: Layer‐by‐Layer Films of Poly‐L‐Lysine and Au‐Carbon Nanotube Hybrid

An electrochemical DNA sensing film was constructed based on the multilayers comprising of poly‐L ‐lysine (pLys) and Au‐carbon nanotube (Au‐CNT) hybrid. A precursor film of mercaptopropionic acid (MPA) was firstly self‐assembled on the Au electrode surface. pLys and Au‐CNT hybrid layer‐by‐layer assembly films were fabricated by alternately immersing the MPA‐modified electrode into the pLys solution and Au‐CNT hybrid solution. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)₆]^3?/4? and [Co(phen)₃]^3+/2+ as the redox indicators. The outer layer of the multilayer film was the positively charged pLys, on which the DNA probe was easily linked due to the strong electrostatic affinity. The hybridization detection of DNA was accomplished by using methylene blue (MB) as the indicator, which possesses different affinities to dsDNA and ssDNA. Differential pulse voltammetry was employed to record the signal response of MB and determine the amount of the target DNA sequence. The established biosensor has high sensitivity, a relatively wide linear range from 1.0×10^?10 mol/L to 1.0×10^?6 mol/L and the ability to discriminate the fully complementary target DNA from single or double base‐mismatched DNA. The sequence‐specific DNA related to phosphinothricin acetyltransferase gene from the transgenically modified plants was successfully detected.     

表面等离子体子共振技术实时研究蛋白质在修饰表面的静电吸附行为

研究蛋白质在固相表面的静电吸附特性,进而控制蛋白质在修饰表面的静电吸附尤为重要,表面等离子体子共振可以检测金属表面吸附物质厚度和折射率的变化^[1]。这种技术已在研究生物分子相互作用^[2]和考察自组装单层的形成^[3]及蛋白质在固体表面吸附行为^[9-11]等方面得到广泛的应用。对蛋白质在固体表面吸附行为的研究多为考察不同的蛋白质在不同的修饰表面的吸附行为。然而,对蛋白质在修饰表面静电吸附的本质影响因素的研究却少有报道^[4]。本文使用表面等离子体子共振技术实时研究了蛋白质在甲羧基化葡聚糖修饰表面的静电吸附与溶液pH值及离子强度的依赖关系。     

342 - Reorientation of the Cytosine Derivatives at the Electrode surface

The alternating current polarograms of cytosine, cytidine and deoxycytidine at pH 5 and cytidine at pH 9 were measured. With increasing concentration of the cytosine derivatives a sort of a pit appears on the a.c. polarograms indicating the region of potentials at which the bases and/or nucleosides are reoriented at the electrode surface from the planar position to the perpendicular one, associate and form a compact surface film. Cytidine at pH 5 begins to form a pit starting at a concentration of about 15 mM. At pH 9 cytidine begins associate already at a concentration of 7 mM. Deoxycytidine at pH 5 associate on the electrode surface at still lower concentrations than cytidine.The potential U_pit of the centre of the pit of the investigated derivatives shifts to more negative values in the order deoxycytidine pH 5 (−0.45) - cytidine pH 5 (−0.70) - cytidine pH 9 (−0.85) cytosine pH 5 (−1.20 V). This order seems to correlate with the increasing value of the electrostatic attraction of the cytosine derivatives on the negatively charged electrode via their positive charge on N-3 and/or dipole moment. According to this conception the greatest electrostatic adsorption on the negatively charged electrode should be exhibited by cytosine at pH 5, which is attracted to the electrode via the protonated N-3, the next in the series being cytidine at pH 9, which is attracted to the electrode via the dipole moment. The smallest electrostatic adsorption exhibits deoxycytidine at pH 5, by which the effective value of the dipole moment is reduced due to the positive charge on N-3.The more negative is the potential U_pit of the pit of nucleosides, the lower is the capacitance of the minimum of the pit indicating the changes in the orientation of the adsorbed nucleosides and/or changes of the compactness of the surface film. The possible orientations of the cytosine derivatives in the compact layer on the electrode surface are presented. At more negative electrode potentials the positive charge (cytosine at pH 5) or positive end of dipole moment (cytidine at pH 9) points to the electrode surface.With cytidine at pH 9 the pit disappears at concentrations higher than about 100 mM, probably due to the self-association of the neutral cytidine molecules in the bulk of solution. At pH 5 the pit is observed with all of the investigated derivatives up to the highest concentrations. The self-association is at pH 5 possibly prevented due to the repulsion of charges at N-3.     

Reflectometric study of the adsorption of poly(vinyl imidazole) on a gold electrode, effects of pH, and applied potential

The adsorption-desorption behavior of poly(vinyl imidazole), a weak polybase (pH-dependent positive charge), on a gold electrode was investigated using optical fixed-angle reflectometry. Using an instrument comprising an impinging-jet system, the hydrodynamic conditions were well defined, making it possible to study the adsorption rate. Comparison between the actual adsorption rate and that of a purely diffusion-controlled process revealed the occurrence and the change of an electrostatic barrier in the adsorption process. The surface charge of the gold electrode was varied by means of an externally applied potential. The surface charge density was evaluated by separate electrochemical impedance spectroscopy. The uptake and the adsorption rate were very sensitive to pH and electrode polarization. At pH 3, the adsorption of the fully charged polymer increased fairly regularly with cathodic polarization, whereas it remained at about 0.4 mg m(-2) in the anodic zone At pH 8, the adsorption of the uncharged polymer decreased with the negative charge of the electrode due to the more favorable adsorption of potassium ions on the charged electrode. Discrepancies in adsorption-desorption measurements taken while cycling the pH were due to an electrostatic adsorption barrier.     

Electrografting of 4‐tert‐Butylcatechol on GC Electrode. Selective Electrochemical Determination of Homocysteine

A new sensor based on the grafting of 4‐tert‐butylcatechol on the surface of a glassy carbon electrode (GC) was developed for the catalytic oxidation of homocysteine ( Hcy ). The GC‐modified electrode exhibited a reversible redox response at neutral pH. Under the optimum conditions cyclic voltammetric results indicated the excellent electrocatalytic activity of modified electrode toward the oxidation of Hcy at reduced over‐potential about 350 mV. A linear dynamic range of 0.01–3.0 mM and a detection limit of 1.0 µM were obtained for Hcy . The modified electrode was used as an electrochemical sensor for selective determination of Hcy in human blood.     

Effect of Electrostatic Interaction on Electrodeposition of Nickel Hexacyanoferrate with Functional MWCNTs and Their Application for the Determination of Persulfate and Tannic Acid

A nanocomposite film of NiHCF electrodeposited onto AFCNT was modified on GCE by the electrostatic interaction of multiple scan cyclic voltammetry. Amino groups have been introduced with a positive charge onto the surface of MWCNTs. The presence of NiHCF‐AFCNT/GCE was characterized by using FE‐SEM, EDS, EIS, and XRD studies. The nanocomposite film of NiHCF‐AFCNT was further utilized to determine persulfate and tannic acid, respectively. The nanocomposite film modified electrode displays linear response from 0.1 to 19 mM and 10 to 170 µM for persulfate and tannic acid with a correlation coefficient of 0.9921 and 0.9869. The detection limits were found to be 0.1 mM and 1 µM, respectively. As results, the electrostatic interaction between positively charged metal ions, amino functionalized CNT and negatively charged hexacyanoferrate can be a new subject for many interesting applications.