Maya Zayats

Semiconductor Quantum Dots for Bioanalysis

Semiconductor nanoparticles, or quantum dots (QDs), have unique photophysical properties, such as size-controlled fluorescence, have high fluorescence quantum yields, and stability against photobleaching. These properties enable the use of QDs as optical labels for the multiplexed analysis of immunocomplexes or DNA hybridization processes. Semiconductor QDs are also used to probe biocatalytic transformations. The time-dependent replication or telomerization of nucleic acids, the oxidation of phenol derivatives by tyrosinase, or the hydrolytic cleavage of peptides by proteases are probed by using fluorescence resonance energy transfer or photoinduced electron transfer. The photoexcitation of QD-biomolecule hybrids associated with electrodes enables the photoelectrochemical transduction of biorecognition events or biocatalytic transformations. Examples are the generation of photocurrents by duplex DNA assemblies bridging CdS NPs to electrodes, and by the formation of photocurrents as a result of biocatalyzed transformations. Semiconductor nanoparticles are also used as labels for the electrochemical detection of DNA or proteins: Semiconductor NPs functionalized with nucleic acids or proteins bind to biorecognition complexes, and the subsequent dissolution of the NPs allows the voltammetric detection of the related ions, and the tracing of the recognition events.

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.

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.

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.

Controlling the direction of photocurrents by means of CdS nanoparticles and cytochrome c-mediated biocatalytic cascades

Cathodic or anodic photocurrents are generated by a monolayer of CdS nanoparticles in the presence of the oxidized or reduced states of cytochrome c, respectively, and the photocurrents are amplified by enzyme-generated biocatalytic cascades mediated by cytochrome c.

Electronic Transduction of HIV-1 Drug Resistance in AIDS Patients

A drug composition consisting of nucleoside reverse transcriptase inhibitors (NRTIs), non‐nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) is commonly used in AIDS therapy. A major difficulty encountered with the therapeutic composite involves the emergence of drug‐resistant viruses, especially to the PIs, regarded as the most effective drugs in the composition. We present a novel bioelectronic means to detect the appearance of mutated HIV‐1 exhibiting drug resistance to the PI saquinavir. The method is based on the translation of viral RNA, the association of cleaved or uncleaved Gag polyproteins at an electrode surface functionalized with the respective antibodies, and the bioelectronic detection of the Gag polyproteins associated with the surface. The bioelectronic process includes the association of anti‐MA or anti‐CA antibodies, the secondary binding of an antibody–horseradish peroxidase (HRP) conjugate, and the biocatalyzed precipitation of an insoluble product on the electronic transducers. Faradaic impedance measurements and quartz crystal microbalance analyses are employed to follow the autoprocessing of the Gag polyproteins. The method was applied to determine drug resistance in infected cultured cells and also in blood samples of consenting AIDS patients. The method described here is also applicable to the determination of drug effectiveness in AIDS patients and to screening of the efficiency of newly developed drugs.

Photoelectrochemical and Optical Applications of Semiconductor Quantum Dots for Bioanalysis

Semiconductor nanoparticles (NPs) or quantum dots (QDs) exhibit unique photophysical properties reflected by size-controlled fluorescence, high fluorescence quantum yields, and stability against photobleaching. These properties are utilized by applying the QDs as optical labels for the multiplexed analysis of immunocomplexes and DNA hybridization. Also, semiconductor QDs are used to probe biocatalytic transformations. The time-dependent replication or telomerization of nucleic acids, the oxidation of phenol derivatives by tyrosinase, and the hydrolytic cleavage of peptides by proteases are probed by using fluorescence resonance energy transfer or photoinduced electron transfer. The photoexcitation of semiconductor NP-biomolecule hybrids associated with electrodes enables the photoelectrochemical transduction of biorecognition events or biocatalytic transformations. This is exemplified with the generation of photocurrents by duplex DNA assemblies bridging CdS NPs to electrodes, and by the formation of photocurrents as a result of biocatalyzed transformations, or redox protein-mediated electron transfer in the presence of the NPs.

Amplified DNA Biosensors

Amplified detection of DNA is a central research topic in modern bioanalytical science. Electronic or optical transduction of DNA recognition events provides readout signals for DNA biosensors. Amplification of the DNA analysis is accomplished by the coupling of nucleic acid-functionalized enzymes or nucleic acid-functionalized nanoparticles (NP) as labels for the DNA duplex formation. This chapter discusses the amplified amperometric analysis of DNA by redox enzymes, the amplified optical sensing of DNA by enzymes or DNAzymes, and the amplified voltammetric, optical, or microgravimetric analysis of DNA using metallic or semiconductor nanoparticles. Further approaches to amplify DNA detection involve the use of micro-carriers of redox compounds as labels for DNA complex formation on electrodes, or the use of micro-objects such as liposomes, that label the resulting DNA complexes on electrodes and alter the interfacial properties of the electrodes. Finally, DNA machines are used for the optical detection of DNA, and the systems are suggested as future analytical procedures that could substitute the polymerase chain reaction (PCR) process.