Andrei B. Kharitonov

Enzyme monolayer-functionalized field-effect transistors for biosensor applications

Abstract A gate surface of an ion-selective field-effect transistor was modified with a monolayer enzyme array that stimulates biocatalytic reactions that control the gate potential. Stepwise assemblage of the biocatalytic layer included primary silanization of the Al 2 O 3 -gate with 3-aminopropyltriethoxysilane, subsequent activation of the amino groups with glutaric dialdehyde and the covalent attachment of the enzyme to the functionalized gate surface. Urease, glucose oxidase, acetylcholine esterase and α-chymotrypsin were used to organize the biocatalytic matrices onto the chip gate. The resulting enzyme-based field-effect transistors, ENFETs, demonstrated capability to sense urea, glucose, acetylcholine and N -acetyl- l -tyrosine ethyl ester, respectively. The mechanism of the biosensing involves the alteration of the pH in the sensing layer by the biocatalytic reactions and the detection of the pH change by the ENFET. The major advantage of the enzyme-thin-layered FET devices as biosensors is the fast response-time (several tens of seconds) of these bioelectronic devices. This advantage over traditional thick-polymer-based ENFETs results from the low diffusion barrier for the substrate penetration to the biocatalytic active sites and minute isolation of the pH-sensitive gate surface from the bulk solution.

Probing of bioaffinity interactions at interfaces using impedance spectroscopy and chronopotentiometry

Faradaic impedance spectroscopy and chronopotentiometry are used as electrochemical methods for probing in situ bioaffinity interactions on surfaces between enzymes and their molecular or macromolecular cofactors. Alcohol dehydrogenase (E.C. 1.1.1.1.) and lactate dehydrogenase (E.C. 1.1.1.27) bind to an NAD+-functionalized monolayer electrode with association constants corresponding to 1.6×104 and 1.5×105 M−1, respectively. Cytochrome oxidase binds to an oriented cytochrome c monolayer assembled on an Au electrode with an association constant of Ka=1.2×107 M−1.

Imprinting of specific molecular recognition sites in inorganic and organic thin layer membranes associated with ion-sensitive field-effect transistors

Molecular recognition sites were imprinted in inorganic TiO2 films, and acrylamide–acrylamidephenylboronic acid copolymer membranes, associated with ion-sensitive field-effect transistors, ISFETs, that act as transduction devices for the association of the substrates to the imprinted membranes. Molecular structures of carboxylic acids, e.g. 4-chlorophenoxyacetic acid (1), 2,4-dichlorophenoxyacetic acid (2), fumaric acid (3), and maleic acid (4), are imprinted in TiO2 films. The imprinted sites reveal high specificity, and substrates, structurally-related to the imprinted compounds are fully differentiated by the imprinted membranes. The specificity of the imprinted sites originates from the complementary structural fitting and ligation of the guest carboxylic acid residues to the Ti(IV)–OH sites in the host carboxylic acids to the imprinted cavities. An acrylamide–acrylamidephenylboronic acid copolymer acts as a functional polymer for the imprinting of nucleotides, e.g. adenosine 5′-monophosphate, AMP, (7), guanosine 5′-monophosphate, GMP, (8), or cytosine 5′-monophosphate, CMP, (9). The specificity of the imprinted nucleotide sites originates from the cooperative binding interactions between the nucleotides and the boronic acid ligand and acrylamide H-bonds. The detection regions and sensitivities for sensing of the different substrates by the functional polymers are determined.

Imprinting of Chiral Molecular Recognition Sites in Thin TiO2 Films Associated with Field-Effect Transistors: Novel Functionalized Devices for Chiroselective and Chirospecific Analyses

(R)- or (S)-2-Methylferrocene carboxylic acids, (R)-1 or (S)-1, (R)- or (S)-2-phenylbutanoic acid, (R)-2 or (S)-2, and (R)- or (S)-2-propanoic acid, (R)-3 or (S)-3, can be imprinted in thin TiO2 films on the gate surface of ion-sensitive field-effect transistor (ISFET) devices. The imprinting is performed by hydrolyzing the respective carboxylate TiIV butoxide complex on the gate surface, followed by washing off the acid from the resulting TiO2 film. The imprinted sites reveal chiroselectivity only towards the sensing of the imprinted enantiomer. The chiral recognition sites reveal not only chiroselectivity but also chirospecificity and, for example, the (R)-2-imprinted film is active in the sensing of (R)-2, but insensitive towards the sensing of (R)2-phenylpropanoic acid, (R)-3, which exhibits a similar chirality. Similarly, the (R)-3-imprinted film is inactive in the analysis of (R)-2. The chiroselectivity and chirospecificity of the resulting imprinted films are attributed to the need to align and fit the respective substrates in precise molecular contours generated in the cross-linked TiO2 films upon the imprinting process.

Selective sensing of triazine herbicides in imprinted membranes using ion-sensitive field-effect transistors and microgravimetric quartz crystal microbalance measurements

A series of triazine herbicides consisting of the chlorotriazine atranex (atrazine), (1), prozinex, (2), tyllanex, (3), simanex, (4) and the methylthiolated triazines ametrex, (5), prometrex, (6) and terbutex, (7), were imprinted in an acrylamide-methacrylate copolymer. The polymer was deposited on the gate surface of ion-sensitive field-effect transistors (ISFETs) and piezoelectric Au-quartz crystals. Selective sensing of the imprinted substrates was accomplished by the imprinted polymer membrane associated with the ISFETs and Au-quartz crystals. Binding of the substrates onto the imprinted polymer associated with the gate of the ISFET alters the electrical charge and potential of the gate interface, thus allowing the potentiometric transduction of the binding events. The association of the substrates with the imprinted membrane linked to the Au-quartz crystal results in the membrane swelling, thus enabling the microgravimetric quartz crystal microbalance assay of the substrate binding events. The specificity of the imprinted recognition sites is attributed to complementary H-bond and electrostatic interactions between the substrates and the acrylamide-methacrylic acid copolymer.

An integrated NAD+-dependent enzyme-functionalized field-effect transistor (ENFET) system : development of a lactate biosensor

An integrated NAD+-dependent enzyme field-effect transistor (ENFET) device for the biosensing of lactate is described. The aminosiloxane-functionalized gate interface is modified with pyrroloquinoline quinone (PQQ) that acts as a catalyst for the oxidation of NADH. Synthetic amino-derivative of NAD+ is covalently linked to the PQQ monolayer. An affinity complex formed between the NAD+/PQQ-assembly and the NAD+-cofactor-dependent lactate dehydrogenase (LDH) is crosslinked and yields an integrated biosensor ENFET-device for the analysis of lactate. Biocatalyzed oxidation of lactate generates NADH that is oxidized by PQQ in the presence of Ca2+-ions. The reduced catalyst, PQQH2, is oxidized by O2 in a process that constantly regenerates PQQ at the gate interface. The biocatalyzed formation of NADH and the O2-stimulated regeneration of PQQ yield a steady-state pH gradient between the gate interface and the bulk solution. The changes in the pH of the solution near the gate interface and, consequently, the gate potential are controlled by the substrate (lactate) concentration in the solution. The device reveals the detection limit of 1 x 10(-4) M for lactate and the sensitivity of 24+/-2 mV dec(-1). The response time of the device is as low as 15 s.

Electrocatalytic intercalator-induced winding of double-stranded DNA with polyaniline.

The intercalation of doxorubicin into double-stranded DNA stimulates the electocatalyzed oxidation of aniline to polyaniline and its winding on the DNA template.

Potential-controlled molecular machinery of bipyridinium monolayer-functionalized surfaces: an electrochemical and contact angle analysis

A non-dense long-chain tethered bipyridinium monolayer linked to an electrode support is reversibly retracted and attracted from and to the electrode respectively by the applied potential. The “molecular machinery” functions of the monolayer are probed by chronoamperometry and contact angle measurements.

Probing photoinduced electron transfer reactions by in situ electrochemical contact angle measurements

In situ electrochemical static contact angle measurements are employed to probe photoinduced electron transfer processes at electrode surfaces. Photosystems consisting of tris(2,2′-bipyridyl)ruthenium(II), (Ru(bpy)32+), N,N′-dimethyl-4,4′-bipyridinium (MV2+), Na2EDTA or CdS nanoparticles, MV2+, triethanolamine (TEOA) were included in aqueous droplets that were coupled with a ferrocene monolayer-functionalized electrode. Upon the oxidation of the interface (0.5 V vs. Ag quasi-reference electrode), and the illumination of the systems, photoinduced electron transfer to the modified electrode proceeds. The electron transfer processes yield photocurrents, and the photoelectrochemical transformations are followed by contact angle measurements. Similarly, a CdS/bipyridinium monolayer is assembled on an Au electrode support. The redox transformations of the bipyridinium units control the hydrophilic/hydrophobic properties of the interface, and are followed by contact angle analyses. Irradiation of the CdS/bipyridinium layer associated with the electrode biased at −0.3 V yields a photocurrent. The changes in the wetting properties of the irradiated interface are investigated by contact angle measurements.

The effect of lipophilic anionic additives on detection limits of ion-selective electrodes based on ionophores with phosphoryl complexing groups

Abstract The dependence of detection limits of ion-selective electrodes (ISEs) based on ionophores with phosphoryl-containing terminal groups on the nature and concentration of lipophilic anionic compounds was investigated. It was shown that by including superstoichiometric amounts of lipophilic anionic compounds into the membrane, the lower detection limit can be decreased significantly. A model describing the mechanism of the dependence of the lower detection limits on both the amount and nature of the lipophilic anionic additives used is presented.