Search

Nucleic acid/semiconductor quantum dots (QDs) hybrid systems combine the recognition and catalytic properties of nucleic acids with the unique photophysical features of QDs. These functions of nucleic acid/QDs hybrids are implemented to develop different optical sensing platforms for the detection of DNA, aptamer-substrate complexes, and metal ions. Different photophysical mechanisms including fluorescence, electron transfer quenching, fluorescence resonance energy transfer (FRET), and chemiluminescence resonance energy transfer (CRET) are used to develop the sensor systems. The size-controlled luminescence properties of QDs are further implemented for the multiplexed, parallel analysis of several DNAs, aptamer-substrate complexes, or mixtures of ions. Similarly, methods to amplify the sensing events through the biocatalytic regeneration of the analyte were developed. An additional paradigm in the implementation of nucleic acid/QDs hybrids for sensing applications involves the integration of the systems with electrodes, and the generation of photocurrents as transduction signals for the sensing events. Finally, semiconductor QDs conjugated to functional DNA machines, such as "walker" systems, provide an effective optical label for probing the dynamics and mechanical functions of the molecular devices. The present article addresses the recent advances in the application of nucleic acid/QDs hybrids for sensing applications and DNA nanotechnology, and discusses future perspectives of these hybrid materials.

A hybrid consisting of glucose oxidase-functionalized with hemin/G-quadruplex units is used for the chemiluminescence detection of glucose. The glucose oxidase-mediated oxidation of glucose yields gluconic acid and H(2)O(2). The latter in the presence of luminol acts as substrate for the hemin/G-quadruplex-catalyzed generation of chemiluminescence. The glucose oxidase/hemin G-quadruplex hybrid was immobilized on CdSe/ZnS quantum dots (QDs). The light generated by the hybrid, in the presence of glucose, activated a chemiluminescence resonance energy transfer process to the QDs, resulting in the luminescence of the QDs. The intensities of the luminescence of the QDs at different concentrations of glucose provided an optical means to detect glucose.

Nucleic acid subunits consisting of fragments of the horseradish peroxidase (HRP)-mimicking DNAzyme and aptamer domains against ATP or sequences recognizing Hg(2+) ions self-assemble, in the presence of ATP or Hg(2+), into the active hemin-G-quadruplex DNAzyme structure. The DNAzyme-generated chemiluminescence provides the optical readout for the sensing events. In addition, the DNAzyme-stimulated chemiluminescence resonance energy transfer (CRET) to CdSe/ZnS quantum dots (QDs) is implemented to develop aptamer or DNA sensing platforms. The self-assembly of the ATP-aptamer subunits/hemin-G-quadruplex DNAzyme, where one of the aptamer subunits is functionalized with CdSe/ZnS QDs, leads to the CRET signal. Also, the functionalization of QDs with a hairpin nucleic acid that includes the G-quadruplex sequence in a ''caged'' configuration is used to analyze DNA. The opening of the hairpin structure by the target DNA assembles the hemin-G-quadruplex DNAzyme that stimulates the CRET signal. By the application of three different sized QDs functionalized with different hairpins, the multiplexed analysis of three different DNA targets is demonstrated by the generation of three different CRET luminescence signals.

Three different sensing platforms for the analysis of telomerase activity in human cells are described. One sensing platform involves the label-free analysis of the telomerase activity by a field-effect-transistor (FET) device. The telomerase-induced extension of a primer associated with the gate of the FET device, in the presence of the nucleotide mixture dNTPs, alters the gate potential, and this allows the detection of telomerase extracted from 65 ± 10 293T (transformed human embryonic kidney) cells/μL. The second sensing platform involves the optical detection of telomerase using CdSe/ZnS quantum dots (QDs). The telomerase-stimulated telomerization of the primer-functionalized QDs in the presence of the nucleotide mixture dNTPs results in the synthesis of the G-rich telomeres. The stacking of hemin on the self-organized G-quadruplexes found on the telomers results in the electron transfer quenching of the QDs, thus providing an optical readout signal. This method enables the detection of telomerase originating from 270 ± 20 293T cells/μL. The third sensing method involves the amplified surface plasmon resonance (SPR) detection of telomerase activity. The telomerization of a primer associated with Au film-coated glass slides, in the presence of telomerase and the nucleotide mixture (dNTPs), results in the formation of telomeres on the surface, and these alter the dielectric properties of the surface resulting in a shift in the SPR spectrum. The hybridization of Au NPs functionalized with nucleic acids complementary to the telomere repeat units with the telomeres amplifies the SPR shifts due to the coupling between the local plasmon of the NPs and the surface plasmon wave. This method enables the detection of telomerase extracted from 18 ± 3 293T cells/μL.

Semiconductor quantum dots (QDs) are used for the optical analysis of casein kinase (CK2) or the hydrolytic activity of alkaline phosphatase (ALP). Two schemes for the analysis of CK2 by a FRET-based mechanism are described. One approach involves the CK2-catalyzed phosphorylation of a serine-containing peptide (1), linked to CdSe/ZnS QDs, with Atto-590-functionalized ATP. The second analytical method involves the specific association of the Atto-590-functionalized antibody to the phosphorylated product. The hydrolytic activity of ALP is followed by the application of phosphotyrosine (4)-modified CdSe/ZnS QDs in the presence of tyrosinase as a secondary reporter biocatalyst. The hydrolysis of (4) yields the tyrosine units that are oxidized by O(2)/tyrosinase to the respective dopaquinone product. The latter quinone units quench the QDs via an electron transfer route, leading to the optical detection of the ALP activity.

Metallic or semiconductor nanoparticles (NPs) are used as labels for the electrochemical, photoelectrochemical, or surface plasmon resonance (SPR) detection of cocaine using a common aptasensor configuration. The aptasensors are based on the use of two anticocaine aptamer subunits, where one subunit is assembled on a Au support, acting as an electrode or a SPR-active surface, and the second aptamer subunit is labeled with Pt-NPs, CdS-NPs, or Au-NPs. In the different aptasensor configurations, the addition of cocaine results in the formation of supramolecular complexes between the NPs-labeled aptamer subunits and cocaine on the metallic surface, allowing the quantitative analysis of cocaine. The supramolecular Pt-NPs-aptamer subunits-cocaine complex allows the detection of cocaine by the electrocatalyzed reduction of H(2)O(2). The photocurrents generated by the CdS-NPs-labeled aptamer subunits-cocaine complex, in the presence of triethanol amine as a hole scavenger, allows the photoelectrochemical detection of cocaine. The supramolecular Au-NPs-aptamer subunits-cocaine complex generated on the Au support allows the SPR detection of cocaine through the reflectance changes stimulated by the electronic coupling between the localized plasmon of the Au-NPs and the surface plasmon wave. All aptasensor configurations enable the analysis of cocaine with a detection limit in the range of 10(-6) to 10(-5) M. The major advantage of the sensing platform is the lack of background interfering signals.

Phenyl boronic acid-functionalized CdSe/ZnS quantum dots (QDs) were synthesized. The modified particles bind nicotinamide adenine dinucleotide (NAD(+)) or 1,4-dihydronicotinamide adenine dinucleotide (NADH). The NAD(+)-functionalized QDs are effectively quenched by an electron transfer process, while the NADH-modified QDs are inefficiently quenched by the reduced cofactor. These properties enable the implementation of the QDs for the fluorescence analysis of ethanol in the presence of alcohol dehydrogenase. The NADH-functionalized QDs were used for the optical analysis of the 1,3,5-trinitrotriazine, RDX explosive, with a detection limit that corresponded to 1 x 10(-10) M. We demonstrate cooperative optical and catalytic functions of the core-shell components of the QDs in the analysis of RDX.

Dopamine (1) and tyrosinase (TR) activities were analyzed by using chemically modified ion-sensitive field-effect transistor (ISFET) devices. In one configuration, a phenylboronic acid functionalized ISFET was used to analyze 1 or TR. The formation of the boronate-1 complex on the surface of the gate altered the electrical potential associated with the gate, and thus enabled 1 to be analyzed with a detection limit of 7x10(-5) M. Similarly, the TR-induced formation of 1, and its association with the boronic acid ligand allowed a quantitative assay of TR to be performed. In another configuration, the surface of the ISFET gate was modified with tyramine or 1 to form functional surfaces for analyzing TR activities. The TR-induced oxidation of the tyramine- or 1-functionalized ISFETs resulted in the formation of the redox-active dopaquinone units. The control of the gate potential by the redox-active dopaquinone units allowed a quantitative assay of TR to be performed. The dopaquinone-functionalized ISFETs could be regenerated to give the 1-modified sensing devices by treatment with ascorbic acid.

Chemically modified CdSe/ZnS quantum dots (QDs) are used as fluorescent probes for the analysis of explosives, and specifically, the detection of trinitrotoluene (TNT) or trinitrotriazine (RDX). The QDs are functionalized with electron-donating ligands that bind nitro-containing explosives, exhibiting electron-acceptor properties, to the QD surface, via supramolecular donor-acceptor interactions leading to the quenching of the luminescence of the QDs.

The vascular endothelial growth factor, VEGF, is an important biomarker for different diseases and clinical disorders. We present a series of optical aptasensor-based sensing platforms for VEGF that include the following: (i) A FRET-based sensor that involves the VEGF-induced separation of aptamer-functionalized quantum dots blocked by a quencher nucleic acid (detection limit 1 nM). (ii) A FRET-based sensor based on the VEGF-induced assembly of the aptamer subunits functionalized with QDs and a dye acceptor (Cy5), respectively (detection limit 12 nM). (iii) A chemiluminescence aptasensor based on VEGF-induced assembly of a hemin/G-quadruplex catalyst (detection limit 18 nM). (iv) A chemiluminescence aptasensor based on the VEGF-stimulated assembly of two aptamer subunits into the hemin/G-quadruplex catalyst (detection limit 2.6 nM). (v) A chemiluminescence resonance energy transfer (CRET) aptasensor based on the VEGF-induced assembly of a semiconductor QDs-hemin/G-quadruplex supramolecular structure (detection limit 875 pM). Furthermore, an amplified optical aptasensor system based on the Exonuclease III (Exo III) recycling of the VEGF analyte was developed. In this system, one aptamer subunit is modified at its 5' and 3' ends with QDs and a black hole quencher, respectively. The VEGF-induced self-assembly of the aptamer subunits result in the digestion of the quencher units and the autonomous recycling of the analyte, while triggering-on the luminescence of the QDs (detection limit 5 pM). The system was implemented to analyze VEGF in human sera samples.

Stimuli-triggered wettability of surfaces and controlled uptake and release of substrates by "smart" materials are essential for drug delivery and microfluidic control. A composite "sponge" consisting of bis-aniline-bridged Au nanoparticles (NPs), functionalized with photoisomerizable nitrospiropyran/nitromerocyanine that includes selective imprinted molecular recognition sites for N,N'-bis(3-sulfonatopropyl)-4,4'-bipyridinium (PVS) was electropolymerized on a Au electrode. The system is triggered by photonic and/or electrical signals to yield four different states exhibiting variable binding/release capacities for PVS and controlled wettability of the surface. The electrical/optical uptake and release of PVS to and from the Au NPs "sponge", respectively, is followed by CdSe/ZnS quantum dots, acting as an auxiliary photonic label.

The optical detection of DNA or the sensing of low-molecular-weight substrates or proteins by aptamer nucleic acids is a long term challenge in the design of biosensors. Similarly, the detection of the telomerase activity, a versatile biomarker of cancer cells, is important for rapid cancer diagnostics. We implement the luminescence quenching of the CdSe/ZnS quantum dots (QDs) as a versatile process to develop DNA sensors and aptasensors, and to design an analytical platform for the detection of telomerase activity. The formation of nucleic acid duplexes on QDs, or the assembly of aptamer-substrate complexes on the QDs (substrate=cocaine or thrombin) is accompanied by the intercalation of doxorubicin (DB) into the duplex domains of the resulting recognition complexes. The intercalated DB quenches the luminescence of the QDs, thus leading to the detection readout signal. Similarly, the telomerase-induced formation of the telomere chains on the QDs is followed by the hybridization of nucleic-acid units complementary to the telomere repeat units, and the intercalation of DB into the resulting duplex structure. The resulting luminescence quenching of the QDs provides an indicating signal for the activity of telomerase.

The luminescence of CdSe/ZnS QDs is quenched via electron transfer by hemin/G-quadruplex associated with the particles. This phenomenon is implemented to develop DNA sensors or aptasensors by tailoring hairpin-functionalized QDs that generate the hemin/G-quadruplex quenchers upon sensing of the respective analytes.

Beta-cyclodextrin (beta-CD)-functionalized CdSe/ZnS quantum dots (QDs) are used for optical sensing and chiroselective sensing of different substrates using a fluorescence resonance energy transfer (FRET) or an electron transfer (ET) mechanisms. The FRET between the QDs and Rhodamine B incorporated in the beta-CD receptor sites is used for the competitive analysis of adamantanecarboxylic acid and of p-hydroxytoluene. Also, the dye-incorporated beta-CD-modified QDs are used for the chiroselective optical discrimination between D,L-phenylalanine and D,L-tyrosine. The receptor-functionalized QDs are also implemented for the optical detection of p-nitrophenol using an ET quenching route.

The fluorescence resonance energy transfer (FRET) between CdSe-ZnS QDs and fluorophore-labeled galactose or fluorophore-labeled dopamine linked to phenyl boronic acid-functionalized QDs is used to develop competitive assays for the optical detection of galactose, glucose, or dopamine.

CdS nanoparticles linked through a duplex DNA to a Au electrode do not lead to a noticeable photocurrent upon their illumination in the presence of triethanolamine, TEOA, 20 mM, pH = 7.2. The intercalation of doxorubicin into the duplex DNA stimulates, however, the generation of a photocurrent. This is attributed to the trapping of photoexcited conduction-band electrons by the intercalator units that facilitates, by a hopping mechanism, the electron transport to the electrode. The oxidation of TEOA by valence band holes allows the formation of a steady state photocurrent. This basic phenomenon is used to probe the operation of a DNA-based machine through the assembly of CdS nanoparticles on a Au electrode. The machine includes a nucleic acid “track”, (1), that binds a primer, (2), through hybridization to a predefined domain. In the presence of polymerase, the nucleotide mixture, dNTPs, and the nicking enzyme, the autonomous replication, nicking and displacement of the “waste product”, (3), are activated. The “waste product” bridges the (4)-functionalized CdS nanoparticles and the nucleic acid (5)-functionalized Au electrode, resulting in the assembly of the nanoparticles on the electrode. The intercalation of doxorubicin into the DNA–CdS nanostructures results in the generation of photocurrents upon illumination in the presence of TEOA, pH = 7.2. The photocurrents are controlled by the time intervals used to operate the DNA machine.

CdSe/ZnS QDs enable the optical probing of the biocatalytic oxidation of tyrosine derivatives and of the scission of peptides by thrombin. CdSe/ZnS QDs were modified with tyrosine methyl ester or with a tyrosine-containing peptide. The tyrosine units were reacted with tyrosinase/O2 to yield the respective l-DOPA and quinone derivatives. The luminescence of QDs modified by the enzyme-generated quinone units is quenched. The quinone-functionalized peptide associated with the QDs was cleaved by thrombin, a process that restored the luminescence of the QDs.

The self-assembly of labeled aptamer sub-units in the presence of their substrates provides a method for the optical (fluorescence) or electrochemical detection of the substrate. One of the sub-units is linked to CdSe/ZnS quantum dots (QDs), and the self-assembly of the dye-functionalized second sub-unit with the modified QDs, in the presence of cocaine, stimulates fluorescence resonance energy transfer (FRET). This enables the detection of cocaine with a detection limit corresponding to 1 x 10(-6) M. Alternatively, the aptamer fragments are modified with pyrene units. The formation of a supramolecular aptamer-substrate complex allosterically stabilizes the formation of excimer supramolecular structure, and its characteristic emission is observed. In addition, the thiolated aptamer sub-unit is assembled on an Au electrode. The Methylene Blue-labeled sub-unit binds to the surface-confined fragment in the presence of cocaine. The amperometric response of the system allows the detection of cocaine with a detection limit of 1 x 10(-5) M. The approach is generic and can be applied to other substrates, e.g. adenosine triphosphate.