Gossypol-Capped Mitoxantrone-Loaded Mesoporous SiO2 NPs for the Cooperative Controlled Release of Two Anti-Cancer Drugs.

Mesoporous SiO2 nanoparticles, MP-SiO2 NPs, are functionalized with the boronic acid ligand units. The pores of the MP-SiO2 NPs are loaded with the anticancer drug mitoxantrone, and the pores are capped with the anticancer drug gossypol. The resulting two-drug-functionalized MP-SiO2 NPs provide a potential stimuli-responsive anticancer drug carrier for cooperative chemotherapeutic treatment. In vitro experiments reveal that the MP-SiO2 NPs are unlocked under environmental conditions present in cancer cells, e.g., acidic pH and lactic acid overexpressed in cancer cells. The effective unlocking of the capping units under these conditions is attributed to the acidic hydrolysis of the boronate ester capping units and to the cooperative separation of the boronate ester bridges by the lactate ligand. The gossypol-capped mitoxantrone-loaded MP-SiO2 NPs reveals preferential cytotoxicity toward cancer cells and cooperative chemotherapeutic activities toward the cancer cells. The MCF-10A epithelial breast cells and the malignant MDA-MB-231 breast cancer cells treated with the gossypol-capped mitoxantrone-loaded MP-SiO2 NPs revealed after a time-interval of 5 days a cell death of ca. 8% and 60%, respectively. Also, the gossypol-capped mitoxantrone-loaded MP-SiO2 NPs revealed superior cancer-cell death (ca. 60%) as compared to control carriers consisting of ß-cyclodextrin-capped mitoxantrone-loaded (ca. 40%) under similar loading of the mitoxantrone drug. The drugs-loaded MP-SiO2 NPs reveal impressive long-term stabilities.

Aptasensors based on supramolecular structures of nucleic acid-stabilized Ag nanoclusters.

Two-sized luminescent nucleic acid-functionalized Ag nanoclusters (NCs) are implemented for the analysis and multiplexed detection of adenosine monophosphate, AMP, and of cocaine using aptamer-ligand complexes.

Multiplexed analysis of genes using nucleic acid-stabilized silver-nanocluster quantum dots.

Luminescent nucleic acid-stabilized Ag nanoclusters (Ag NCs) are applied for the optical detection of DNA and for the multiplexed analysis of genes. Two different sensing modules including Ag NCs as luminescence labels are described. One sensing module involves the assembly of a three-component sensing module composed of a nucleic acid-stabilized Ag NC and a quencher-modified nucleic acid hybridized with a nucleic acid scaffold that is complementary to the target DNA. The luminescence of the Ag NCs is quenched in the sensing module nanostructure. The strand displacement of the scaffold by the target DNA separates the nucleic acid-functionalized Ag NCs, leading to the turned-on luminescence of the NCs and to the optical readout of the sensing process. By implementing two different-sized Ag NC-modified sensing modules, the parallel multiplexed analysis of two genes (the Werner Syndrome gene and the HIV, human immunodeficiency, gene), using 615 and 560 nm luminescent Ag NCs, is demonstrated. The second sensing module includes the nucleic acid functionalized Ag NCs and the quencher-modified nucleic acid hybridized with a hairpin DNA scaffold. The luminescence of the Ag NCs is quenched in the sensing module. Opening of the hairpin by the target DNA triggers the luminescence of the Ag NCs, due to the spatial separation of the Ag NCs/quencher units. The system is applied for the optical detection of the BRAC1 gene. In addition, by implementing two-sized Ag NCs, the multiplexed analysis of two genes by the hairpin sensing module approach is demonstrated.

Ag nanocluster/DNA hybrids: functional modules for the detection of nitroaromatic and RDX explosives.

Luminescent Ag nanoclusters (NCs) stabilized by nucleic acids are implemented as optical labels for the detection of the explosives picric acid, trinitrotoluene (TNT), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The sensing modules consist of two parts, a nucleic acid with the nucleic acid-stabilized Ag NCs and a nucleic acid functionalized with electron-donating units, including L-DOPA, L-tyrosine and 6-hydroxy-L-DOPA, self-assembled on a nucleic acid scaffold. The formation of donor-acceptor complexes between the nitro-substituted explosives, exhibiting electron-acceptor properties, and the electron-donating sites, associated with the sensing modules, concentrates the explosives in close proximity to the Ag NCs. This leads to the electron-transfer quenching of the luminescence of the Ag NCs by the explosive molecule. The quenching of the luminescence of the Ag NCs provides a readout signal for the sensing process. The sensitivities of the analytical platforms are controlled by the electron-donating properties of the donor substituents, and 6-hydroxy-L-DOPA was found to be the most sensitive donor. Picric acid, TNT, and RDX are analyzed with detection limits corresponding to 5.2 × 10(-12) M, 1.0 × 10(-12) M, and 3.0 × 10(-12) M, respectively, using the 6-hydroxy-L-DOPA-modified Ag NCs sensing module.

DNA sensors and aptasensors based on the hemin/G-quadruplex-controlled aggregation of Au NPs in the presence of L-cysteine.

L-cysteine induces the aggregation of Au nanoparticles (NPs), resulting in a color transition from red to blue due to interparticle plasmonic coupling in the aggregated structure. The hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme catalyzes the aerobic oxidation of L-cysteine to cystine, a process that inhibits the aggregation of the NPs. The degree of inhibition of the aggregation process is controlled by the concentration of the DNAzyme in the system. These functions are implemented to develop sensing platforms for the detection of a target DNA, for the analysis of aptamer-substrate complexes, and for the analysis of L-cysteine in human urine samples. A hairpin DNA structure that includes a recognition site for the DNA analyte and a caged G-quadruplex sequence, is opened in the presence of the target DNA. The resulting self-assembled hemin/G-quadruplex acts as catalyst that controls the aggregation of the Au NPs. Also, the thrombin-binding aptamer folds into a G-quadruplex nanostructure upon binding to thrombin. The association of hemin to the resulting G-quadruplex aptamer-thrombin complex leads to a catalytic label that controls the L-cysteine-mediated aggregation of the Au NPs. The hemin/G-qaudruplex-controlled aggregation of Au NPs process is further implemented for visual and spectroscopic detection of L-cysteine concentration in urine samples.

Analysis of telomerase by the telomeric hemin/G-quadruplex-controlled aggregation of au nanoparticles in the presence of cysteine.

Telomeres are guanosine-rich nucleic-acid chains that fold, in the presence of K(+) ions and hemin, into the telomeric hemin/G-quadruplex structure, exhibiting horseradish peroxidase mimicking functions. The telomeric hemin/G-quadruplex structures catalyze the oxidation of thiols (e.g., l-cysteine) into disulfides (e.g., cystine). As l-cysteine stimulates the aggregation of Au nanoparticles (NPs), accompanied by absorbance changes from red (individual Au NPs) to blue (aggregated Au NPs), the process is implemented to quantitatively analyze the activity (content) of telomerase, a versatile biomarker for cancer cells. Telomerase extracted from 293T cancer cells catalyzes, in the presence of a dNTPs mixture and an appropriate primer probe, the telomerization process, leading to the generation of catalytic telomeric hemin/G-quadruplex chains that control the l-cysteine-mediated aggregation of Au NPs. The extent of aggregation is thus controlled by the concentration of telomerase. The method enabled the detection of telomerase with a detection limit of 27 cells/µL. The spectral changes accompanying the aggregation of Au NPs are further supported by transmission electron microscopy imaging.

Fluorescence detection of DNA, adenosine-5'-triphosphate (ATP), and telomerase activity by zinc(II)-protoporphyrin IX/G-quadruplex labels.

The zinc(II)-protoporphyrin IX (ZnPPIX) fluorophore binds to G-quadruplexes, and this results in the enhanced fluorescence of the fluorophore. This property enabled the development of DNA sensors, aptasensors, and a sensor following telomerase activity. The DNA sensor is based on the design of a hairpin structure that includes a "caged" inactive G-quadruplex sequence. Upon opening the hairpin by the analyte DNA, the resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event (detection limit 5 nM). Addition of Exonuclease III to the system allows the recycling of the analyte and its amplified analysis (detection limit, 200 pM). The association of the ZnPPIX to G-quadruplex aptamer-substrate complexes allowed the detection of adenosine-5'-triphosphate (ATP, detection limit 10 µM). Finally, the association of ZnPPIX to the G-quadruplex repeat units of telomers allowed the detection of telomerase activity originating from 380 ± 20 cancer 293T cell extract.

Electron-transfer quenching of nucleic acid-functionalized CdSe/ZnS quantum dots by doxorubicin: a versatile system for the optical detection of DNA, aptamer-substrate complexes and telomerase activity.

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.

Optical, electrical and surface plasmon resonance methods for detecting telomerase activity.

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.

CdSe/ZnS quantum dots-G-quadruplex/hemin hybrids as optical DNA sensors and aptasensors.

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.

Control of biocatalytic transformations by programmed DNA assemblies.

This study demonstrates the self-assembly of inhibitor/enzyme-tethered nucleic acid fragments or enzyme I-, enzyme II-modified nucleic acids into functional nanostructures that lead to the controlled inhibition of the enzyme or the activation of an enzyme cascade. In one system, the anti-cocaine aptamer subunits are modified with monocarboxy methylene blue (MB(+)) as the inhibitor and with choline oxidase (ChOx). The cocaine-induced self-assembly of the aptamer subunits complex results in the inhibition of ChOx by MB(+). In a further configuration, two nucleic acids of limited complementarity are functionalized at their 3' and 5' ends with glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively, or with MB(+) and ChOx. In the presence of a target DNA sequence, synergistic complementary base-pairing occurs, thus leading to stable supramolecular Y-shaped nanostructures of the nucleic acid units. A GOx/HRP bienzyme cascade or the programmed inhibition of ChOx by MB(+) is demonstrated in the resulting nucleic acid nanostructures. A quantitative theoretical model that describes the nucleic acid assemblies and that results in the inhibition of ChOx by MB(+) or in the activation of the GOx/HRP cascade, respectively, is provided.

Supramolecular cocaine-aptamer complexes activate biocatalytic cascades.

The anti-cocaine aptamer was fragmented into two nucleic acids, (1) and (2). The nucleic acid (1) was tethered at its 5'-end to aminoethyl nicotinamide adenine dinucleotide, amino-NAD(+), or to horseradish peroxidase, HRP. The nucleic acid (2) was functionalized at its 3'-end with alcohol dehydrogenase, AlcDH, or with glucose oxidase, GOx. In the presence of cocaine, the supramolecular NAD(+)/AlcDH/cocaine-aptamer complex is formed, and the biocatalytic oxidation of ethanol is activated. Similarly, in the presence of cocaine, the GOx/HRP/cocaine-aptamer complex is formed, and this activates the biocatalytic cascade where glucose is oxidized by GOx to yield gluconic acid and H(2)O(2), and the resulting hydrogen peroxide activates the HRP-biocatalyzed oxidation of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate), ABTS(2-). The systems may be considered as biomimetic prototypes for systems biology.

Self-assembly of supramolecular aptamer structures for optical or electrochemical sensing.

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.