Aptamer-Engineered Cu2O Nanocubes as a Surface-Modulated Catalytic Optical Sensor for Lung Cancer Cell Detection.

Herein, fine and homogeneous Cu2O nanocubes are synthesized and sensitized with a hairpin-structured AS1411 aptamer for the establishment of a biosensor for lung cancer cell detection. The Apt-Cu2O nanocubes feature a recognition function in identifying a cancer-associated surface nucleolin protein. The intrinsic reduction catalytic ability is also confirmed by the use of two benchmark substrates, methylene blue (MB) and 4-nitrophenol (4-NP). The aptamer grafting on Apt-Cu2O nanocubes is able to greatly prevent nonspecific-protein binding and to show specificity toward the nucleolin protein. The specific binding resulting from nucleolin protein leads to less exposure of the active area of the Apt-Cu2O nanocubes, so the catalytic ability of Apt-Cu2O nanocubes is thus diminished. The modulated catalytic ability led to less generation of the reduced 4-AP product, and the change in absorption of 4-AP allows the quantification of the nucleolin protein with a detection limit of 0.47 nM. The as-developed biosensor is applied to the detection of nucleolin-overexpressed A549 lung cancer cells, presenting a sensitive detection limit down to 20 cells. This may be ascribed to the clustering of surface nucleolin protein in a lipid raft membrane of cancer cells, as evidenced by a notable binding of Apt-Cu2O nanocubes on the cancer cell surface. Real human serum samples spiked with cancer cells were also investigated, and a recovery rate of 87 ± 2.4% for 20 extracted cells validates the surface-modulated Apt-Cu2O nanocubes-based catalytic optical biosensor as a promising tool for the detection of circulating tumor cells. The establishment of the Apt-Cu2O nanocubes may allow for further studies on their use as a potential theranostics tool for cancer therapy.

Imaging of Cancer Cells and Dictated Cytotoxicity Using Aptamer-Guided Hybridization Chain Reaction (HCR)-Generated G-Quadruplex Chains.

DNA nanotechnology provides powerful tools for developing cancer theranostics. Here we introduce the autonomous surface-nucleolin-guided HCR that leads to the polymerization of G-quadruplex polymer chains, in which the ZnII -protoporphyrin IX is intercalated. We demonstrate that MDA-MB-231 (Triple Negative Breast Cancer cells, TNBC) with overexpressed surface nucleolin were able to induce HCR leading to the formation of the ZnII PPIX-loaded G-quadruplex polymer chains, while the M10 epithelial breast cells served as control. The ZnII PPIX-loaded nanowires allow the selective imaging of TNBC, and their permeation into the TNBC leads to selective cytotoxicity and guided photodynamic therapy toward the cancer cells due to structural perturbation of the membranes. The aptamer-guided HCR-generated G-quadruplex polymer chains may serve as a versatile tool to target TNBC featuring poor prognosis and high pathological risk of recurrence, thus offering a promising theranostic platform.

Exploiting the Catalytic Ability of Polydopamine-Remodeling Gold Nanoparticles toward the Naked-Eye Detection of Cancer Cells at a Single-Cell Level.

In this study, a catalytic polydopamine-remodeling gold nanoparticle sensitized with an antinucleolin AS1411 probe (pAu nanoprobe) is synthesized, where the surface of the gold nanoparticles (AuNPs) is modified with a spontaneous self-polymerization of a polydopamine coating that imparts the probe functionalization ability and antispecific protein binding while the intrinsic catalytic property of the AuNPs is preserved. The functionalized AS1411 probe exerts specific recognition with nucleolin protein that is found to be overexpressed on the surface of breast cancer cells (MDA-MB-231). Scanning electron microscopy (SEM) confirms that the specific binding of the pAu nanoprobe occurs at the cancer cell surface. Taking advantage of the catalytic ability of the pAu nanoprobe in reducing blue-colored methylene blue (MB) to colorless leuco-MB, a colorimetric biosensing platform is established based on the accessible catalytic active sites on the pAu nanoprobe toward MB. The specific binding inhibits the pAu nanoprobe from efficiently catalyzing the reduction of MB, resulting in a "turn-off" catalytic biosensing platform. The catalytic conversion of MB is inversely proportional to the concentration of the nucleolin protein and the cancer cells, yielding a detection limit of 15 pM of the nucleolin protein and two cancer cells. The presence of five orders of magnitude higher concentration of bovine serum albumin hardly affects the catalytic ability of the pAu nanoprobe, that is, 88% catalytic ability is still preserved, which validates the specificity of the proposed pAu nanoprobe. In particular, a distinct color contrast creates a significant signal-to-noise ratio so as to enable single-cell level detection of two cancer cells by naked-eye judgment. Moreover, the undiluted, real human serum samples spiked with the cancer cells were examined with an impressive recovery of 94 ± 0.3%, which holds great promise in cancer cell screening.

Polydopamine-gold composite-based electrochemical biosensor using dual-amplification strategy for detecting pancreatic cancer-associated microRNA.

Herein, we developed a dual-amplification-boosted, translational electrochemical biosensor for the detection of pancreatic cancer-associated microRNA, miR-196b. A biosensing event occurred when a hairpin target probe recognized miR-196b as a result of the opening of an electrode hairpin probe on a polydopmine-gold (PDA-Au) composite-modified screen-printed carbon electrode. The PDA-Au composite was synthesized in a alkaline condition where dopamine polymerized into polydopamine while gold(III) ion was in-situ reduced to gold nanoparticle, characterized by FT-IR, SEM, cyclic voltammetry and gel electrophoresis. The electrode modifier, PDA-Au, provided a functionalizable interface for the sensitization of electrode probes; besides, a suitable hydrophilic interface for an aqueous biosensing reaction, confirmed by water contact angle measurement and cyclic voltammetry. Validated by UV-VIS analysis and gel electrophoresis, the single polymerase created a dual amplification by target-recycling and generation of an catalytic DNAzyme product on the electrode probe-functionalized PDA-Au@SPCE. The isothermal, dual amplification was thereby translated into a TMB/H2O2-resulted electrochemical signal, acquired by scan wave voltammetry for quantifying target miR-196b. The novel electrochemical biosensor offered a detection limit of 0.26 pM, with a nice analytical reproducibility by CV (coefficient of variance) value of 8.5 ± 3.1%, and a recovery rate of 105 ± 4.1% obtained from a spiked, real human serum sample. The proposed biosensor showed great promise as a complementary tool for real sample detection in the clinic.

Diagnosing the RGS11 Lung Cancer Biomarker: The Integration of Competitive Immunoassay and Isothermal Nucleic Acid Exponential Amplification Reaction.

Lung cancer is the primary cause of cancer-associated mortality worldwide, which makes the identification of reliable lung cancer biomarkers a pressing need for early diagnosis and prognosis. RGS11, which is a regulator of G-protein signaling and also a lung cancer biomarker, plays an important role in cancer-related metastasis. However, trace levels of RGS11 (in the range of pg/mL) in serum samples make it difficult to quantify using currently available enzyme-linked immunosorbent assay (ELISA) kits and, therefore, this hinders progress in the discovery of new approaches for treating lung cancer. The aim of this study is to develop a rapid, sensitive, and reliable platform for the detection of RGS11 lung cancer biomarker based on a suspension immunoassay coupled with an isothermal exponential amplification strategy. Our study was initiated by the functionalization of magnetic beads with anti-RGS11 antibodies (Ab-MB) by EDC (1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide)/NHS ( N-hydroxysulfosuccinimide) activation. Ab-MB served as a sensing probe for the competitive immunorecognitions between known concentrations of His-tag RGS11 and unknown concentrations of target RGS11 in serum. The reporter anti-His antibodies, which were modified with primers that induced an isothermal exponential amplification reaction, were subsequently introduced to the reaction mixture that resulted in the formation of immunosandwich complexes. The exponentially amplified DNA duplex that was intercalated with SYBR Green was designated as a signal reporter for the assessment of RGS11 in an inversely proportional relationship. The sensing platform was excellent for the determination of RGS11 with an exceptional detection limit of 148 fg/mL and a linear dynamic range of 0.1-10 pg/mL using a minimal sample volume (20 µL) and with a reaction time of 1.5 h. In addition, we challenged the sensing platform with RGS11-spiked samples (in 2× diluted serum), and an acceptable recovery rate (>90%) was observed. Finally, 24 clinical samples acquired from patients with advanced lung cancer (C), inflammation (I), and heart failure (H) were analyzed by this newly developed sensing platform and a commercial ELISA kit for validation. This sensing platform has potential in biomedical applications for clinically diagnosing liquid biopsy samples for patients with lung cancer. Moreover, the universal design of our proposed system is easily adapted to detect any other protein if a His-tag recombinant protein is available.

Diagnosing the miR-141 prostate cancer biomarker using nucleic acid-functionalized CdSe/ZnS QDs and telomerase.

The microRNA, miR-141, is a promising biomarker for prostate cancer. We implement here a two-step sensing platform for the sensitive detection of miR-141. The first step involves the use of semiconductor CdSe/ZnS quantum dots (QDs) modified by FRET quencher-functionalized nucleic acids, that include the recognition sequence for miR-141 and a telomerase primer sequence for the second step of the analytical platform. Subjecting the probe-modified QDs to miR-141, in the presence of duplex specific nuclease, DSN, leads to the formation of a miR-141/probe duplex and to its DSN-mediated cleavage, while regenerating the miR-141. The DSN-induced cleavage of the quencher units leads to the activation of the fluorescence of the QDs, thus allowing the optical detection of miR-141 with a sensitivity corresponding to 1.0 × 10-12 M. The nucleic acid residues associated with the QDs after cleavage of the probe nucleic acids by DSN act as primers for telomerase. The subsequent telomerase/dNTPs-stimulated elongation of the primer units forms G-quadruplex telomer chains. Incorporation of hemin in the resulting G-quadruplex telomer chains yields horseradish peroxidase-mimicking DNAzyme units, that catalyze the generation of chemiluminescence in the presence of luminol/H2O2. The resulting chemiluminescence intensities provide a readout signal for miR-141, DL = 2.8 × 10-13 M. The first step of the sensing platform is non-selective toward miR-141 and the resulting fluorescence may be considered only as an indicator for the existence of miR-141. The second step in the sensing protocol, involving telomerase, provides a selective chemiluminescence signal for the existence of miR-141. The two-step sensing platform is implemented for the analysis of miR-141 in serum samples from healthy individuals and prostate cancer carriers. Impressive discrimination between healthy individuals and prostate cancer carriers is demonstrated.

Monitoring the subcellular localization of doxorubicin in CHO-K1 using MEKC-LIF: liposomal carrier for enhanced drug delivery.

Doxorubicin (DOX) is an extensively used anthracycline that has proven to be effective against a variety of human malignant tumors, such as ovarian or breast cancer. While DOX was administered into cultured cancer cell targets (such as CHO-K1) in either free drug form or in drug carrier-associated form (i.e., DOX encapsulated in the drug delivery carrier), various action of mechanisms for DOX were initiated, among which, it has been long believed that DOX enters the nucleus, interacts with DNA in numerous ways, and finally halts cell proliferation. Aside from its therapeutic effect, regrettably DOX treatment may be accompanied by the occurrence of cardiac and liver toxicity and drug resistance that are attributed from cellular processes involving the parent drug or its metabolites. Liposomal formulation of DOX, known to be capable of attenuating direct uptake of reticuloendothelial system (RES) and prolonging the circulation time in blood, demonstrated reduced toxic side-effects. We herein report the development of a modified MEKC-LIF (Micellar electrokinetic chromatography-Laser-induced fluorescence) method suitable for analyzing DOX in biological samples. The MEKC migration buffer, consisting of 10 mM borate, 100 mM sodium dodecyl sulfate (SDS) (pH 9.3), was found to provide an efficient and stable electrophoretic separation and analysis for DOX. Responses were linear in the range of 11.3-725 ng/mL; the limit of quantitation (LOQ) was found to be 43.1 ng/mL (S/N=10) (equivalent to 74.3 nM) and limit of detection (LOD) was calculated as 6.36 ng/mL (S/N=3) (equivalent to 11.0 nM). This approach was subsequently employed to compare the intracellular accumulation in three subcellular fractions of DOX-treated CHO-K1 cells. These fractions form a pellet at <1400 g, 1400-14000 g, and >14000 g and are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosole components, respectively, resulting from treatment of CHO-K1 cells with 25 µM (equivalent to 14.5 µg/mL) of two DOX formats (in free drug form or liposomal form synthesized in current study) for different periods of time. Our results indicated that the most abundant DOX was found in the nuclear-enriched fraction of cells treated for 12 h and 6 h with free and liposomal DOX, respectively, providing direct evidence to confirm the enhanced efficiency of liposomal carriers in delivering DOX into the nucleus. The observations presented herein suggest that subcellular fractionation followed by liquid-liquid extraction and MEKC-LIF could be a powerful diagnostic tool for monitoring intracellular DOX distribution, which is highly relevant to cytotoxicity studies of anthracycline-type anticancer drugs.

Optical aptasensors for the analysis of the vascular endothelial growth factor (VEGF).

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.

Development of an immunopredictor for the evaluation of the risk of cardiovascular diseases based on the level of soluble P-selectin.

Due to its physiologic role in modulating adhesive interactions between blood cells and the endothelium during inflammatory processes or at injury sites, the adhesion molecule P-selectin is of great interest. The level of soluble P-selectin in plasma or serum can be detected and used as a clinical predictor for adverse cardiovascular events, leading to the presumption that it is secreted, shed or cleaved from the cell membrane during the process of diseases. Increased levels of soluble P-selectin in the plasma have been shown to be associated with a range of cardiovascular disorders, including coronary artery disease, hypertension and atrial fibrillation. Therefore, it is of huge significance to develop simple, rapid and sensitive methods for the detection of such pathological predictors, not only for facilitating the surveillance of cardiovascular mortality/sudden cardiac death, but also for effectively monitoring the drug potency on platelets based on measurement of P-selectin performed on fixed blood samples following platelet stimulation in whole blood in a remote setting. We herein developed a simple, yet novel and sensitive electrochemical sandwich immunosensor for the detection of P-selectin; it operates through covalent linkage of anti-P-selectin antibody on CNT@GNB nanocomposites-modified disposable screen-printed electrode as the detection platform, with the potassium ferrocyanide-encapsulated, anti-P-selectin-tagged liposomal biolabels as the electrochemical signal probes. The immunorecognition of the sample P-selectin by the liposomal biolabels occurred on the surface of the electrodes; the release of potassium ferrocyanide from the bound liposomal biolabels extensively contributed to the increase in electrochemical signal, which was acquired in HCl solution at +0.32V in square wave voltammetry mode. The resulting sigmoidally shaped dose-response curves possessed a linear dynamic working range from 1×10(-13) to 1×10(-5)g/mL. This liposome-based electrochemical immunoassay provides an amplification approach for detecting P-selectin at trace levels, leading to a detection limit as low as 4.3fg (equivalent to 5µL of 0.85pg/mL solution). A commercially available ELISA kit was used as a reference method to validate the newly-developed assay through the analysis of mouse serum samples. A strong correlation was observed between the two data sets as the R-squared value of 0.997 from the linear regression line. This electrochemical immunosensor will be useful for the detection of P-selectin in biological fluids and tissue extracts.

Antioxidant, anti-inflammatory and anti-browning activities of hot water extracts of oriental herbal teas.

Traditionally, antioxidants are used to scavenge reactive oxygen species (ROS), which are harmful by-products of aerobic metabolism. Inulae Flos, Horsetail, Chinese Leucas, Broomweed and Indian Wikstroemia are five herbal teas commonly consumed by Asians. Our aim was to investigate the hot water extracts of these five herbal teas for their total phenolics/flavonoid contents and antioxidant capacities. Furthermore, with inflammation and hyper-pigmentation considered as two biological processes associated with elevated cellular oxidative stress, Inulae Flos water extract was chosen for further evaluation of its inhibitory effects on the production of LPS-induced inflammatory mediators (such as, TNF-α, IL-6, IL-1ß) in RAW 264.7 cells and its anti-tyrosinase activity. Our findings suggest that Inulae Flos might be an alternative source as a potential antioxidant, and a noteworthy inhibitor of production of pro-inflammatory cytokines in a dose-dependent manner. Moreover, it could also serve as a potential natural food additive to prevent browning.