Xiaoxiao He

Dye-doped nanoparticles for bioanalysis

Understanding biological processes at the molecular level with accurate quantitation needs advanced bioanalysis. In this review, we describe dye-doped silica nanoparticles (NPs), their synthesis, bioconjugation, and applications in different bioanalysis formats. Silica-based nanomaterials have been developed with optical-encoding capabilities for the selective tagging of a wide range of biomedically important targets, such as bacteria, cancer cells, and individual biomolecules. We also briefly review other closely related nanomaterials, such as quantum dots, Au NPs, and magnetic NPs. We envisage that further development of these NPs will provide a variety of advanced tools for molecular biology, genomics, proteomics, drug discovery, and diagnosis and therapy of infectious disease and cancer.

Activatable aptamer probe for contrast-enhanced in vivo cancer imaging based on cell membrane protein-triggered conformation alteration

Aptamers have emerged as promising molecular probes for in vivo cancer imaging, but the reported “always-on” aptamer probes remain problematic because of high background and limited contrast. To address this problem, we designed an activatable aptamer probe (AAP) targeting membrane proteins of living cancer cells and achieved contrast-enhanced cancer visualization inside mice. The AAP displayed a quenched fluorescence in its free state and underwent a conformational alteration upon binding to target cancer cells with an activated fluorescence. As proof of concept, in vitro analysis and in vivo imaging of CCRF-CEM cancer cells were performed by using the specific aptamer, sgc8, as a demonstration. It was confirmed that the AAP could be specifically activated by target cancer cells with a dramatic fluorescence enhancement and exhibit improved sensitivity for CCRF-CEM cell analysis with the cell number of 118 detected in 200 μl binding buffer. In vivo studies demonstrated that activated fluorescence signals were obviously achieved in the CCRF-CEM tumor sites in mice. Compared to always-on aptamer probes, the AAP could substantially minimize the background signal originating from nontarget tissues, thus resulting in significantly enhanced image contrast and shortened diagnosis time to 15 min. Furthermore, because of the specific affinity of sgc8 to target cancer cells, the AAP also showed desirable specificity in differentiating CCRF-CEM tumors from Ramos tumors and nontumor areas. The design concept can be widely adapted to other cancer cell-specific aptamer probes for in vivo molecular imaging of cancer.

Concatemeric dsDNA-templated copper nanoparticles strategy with improved sensitivity and stability based on rolling circle replication and its application in microRNA detection.

DNA-templated copper nanoparticles (CuNPs) have emerged as promising fluorescent probes for biochemical assays, but the reported monomeric CuNPs remain problematic because of weak fluorescence and poor stability. To solve this problem, a novel concatemeric dsDNA-templated CuNPs (dsDNA-CuNPs) strategy was proposed by introducing the rolling circle replication (RCR) technique into CuNPs synthesis. In this strategy, a short oligonucleotide primer could trigger RCR and be further converted to a long concatemeric dsDNA scaffold through hybridization. After the addition of copper ions and ascorbate, concatemeric dsDNA-CuNPs could effectively form and emit intense fluorescence in the range of 500-650 nm under a 340 nm excitation. In comparison with monomeric dsDNA-CuNPs, the sensitivity of concatemeric dsDNA-CuNPs was greatly improved with ~10,000 folds amplification. And their fluorescence signal was detected to reserve ~60% at 2.5 h after formation, revealing ~2 times enhanced stability. On the basis of these advantages, microRNA let-7d was selected as the model target to testify this strategy as a versatile assay platform. By directly using let-7d as the primer in RCR, the simple, low-cost, and selective microRNA detection was successfully achieved with a good linearity between 10 and 400 pM and a detection limit of 10 pM. The concatemeric dsDNA-CuNPs strategy might be widely adapted to various analytes that can directly or indirectly induce RCR.

An efficient method for recovery of target ssDNA based on amino-modified silica-coated magnetic nanoparticles

Abstract In this paper, an improved recovery method for target ssDNA using amino-modified silica-coated magnetic nanoparticles (ASMNPs) is reported. This method takes advantages of the amino-modified silica-coated magnetic nanoparticles prepared using water-in-oil microemulsion technique, which employs amino-modified silica as the shell and iron oxide as the core of the magnetic nanoparticles. The nanoparticles have a silica surface with amino groups and can be conjugated with any desired bio-molecules through many existing amino group chemistry. In this research, a linear DNA probe was immobilized onto nanoparticles through streptavidin conjugation using covalent bonds. A target ssDNA(I) (5′-TMR-CGCATAGGGCCTCGTGATAC-3′) has been successfully recovered from a crude sample under a magnet field through their special recognition and hybridization. A designed ssDNA fragment of severe acute respiratory syndrome (SARS) virus at a much lower concentration than the target ssDNA(I) was also recovered with high efficiency and good selectivity.

Nanoparticle-Based Biocompatible and Long-Life Marker for Lysosome Labeling and Tracking

In this paper, a novel biocompatible and long-life lysosome labeling and tracking method based on dye entrapped silica nanoparticles (DSiNPs) has been put forward. Through colocalization studies using LysoTracker Green as the standard lysosome marker, it has been demonstrated that DSiNPs selectively accumulated in lysosomes of Hela cells and the photostability of DSiNPs associated with lysosomes was detectable, at least, 30 times as long as that of LysoTracker Green involved in lysosomes. By comparison with LysoTracker Green and Alexa 488-dextran, the fluorescence of DSiNPs could be detected over a 5-day postrecultivation period and the staining pattern in lysosomes could be well retained after cell fixation and permeabilization. In addition, results from MTT assays showed that DSiNPs did not affect the viability of Hela cells at the concentration for lysosome labeling. Primary applications of DSiNPs were then further performed in lysosome tracking in chloroquine-treated Hela cells, and lysosome labeling of differnet cell lines, including MCF-7 cells, MEAR cells, and MSC cells. These results indicated that DSiNPs, therefore, can be used as a biocompatible, long-life, and highly photostable lysosome marker for lysosome-related studies.

Iodide-Responsive Cu–Au Nanoparticle-Based Colorimetric Platform for Ultrasensitive Detection of Target Cancer Cells

Colorimetric analysis is promising in developing facile, fast, and point-of-care cancer diagnosis techniques, but the existing colorimetric cancer cell assays remain problematic because of dissatisfactory sensitivity as well as complex probe design or synthesis. To solve the problem, we here present a novel colorimetric analytical strategy based on iodide-responsive Cu-Au nanoparticles (Cu-Au NPs) combined with the iodide-catalyzed H2O2-TMB (3,3,5,5-tetramethylbenzidine) reaction system. In this strategy, bimetallic Cu-Au NPs prepared with an irregular shape and a diameter of ∼15 nm could chemically absorb iodide, thus indirectly inducing colorimetric signal variation of the H2O2-TMB system. By further utilizing its property of easy biomolecule modification, a versatile colorimetric platform was constructed for detection of any target that could cause the change of Cu-Au NPs concentration via molecular recognition. As proof of concept, an analysis of human leukemia CCRF-CEM cells was performed using aptamer Sgc8c-modified Cu-Au NPs as the colorimetric probe. Results showed that Sgc8c-modified Cu-Au NPs successfully achieved a simple, label-free, cost-effective, visualized, selective, and ultrasensitive detection of cancer cells with a linear range from 50 to 500 cells/mL and a detection limit of 5 cells in 100 μL of binding buffer. Moreover, feasibility was demonstrated for cancer cell analysis in diluted serum samples. The iodide-responsive Cu-Au NP-based colorimetric strategy might not only afford a new design pattern for developing cancer cell assays but also greatly extend the application of the iodide-catalyzed colorimetric system.

A novel gene carrier based on amino-modified silica nanoparticles

Uniform-sized amino-modified silica nanoparticles have been prepared by the controlled synchronous hydrolysis of tetraethoxysilane and N-(β-amimoethyl)-γ-aminopropyltriethoxysilane in water nanodroplet of the wa-ter-in-oil microemulsion. These nanoparticles display positive charge potential at definited pH. This is due to the presence of amino groups on the surface of the nanoparticles. Nanoparticles-plasmid DNA complexes can easily form through electrostatical binding between the positive charges of the amino-modified silica nanoparticles and the negative charges of the plasmid DNA. The complexes can be also dissociated under alkaline pH or high ionic strength conditions. And enzymatic digestion of the plasmid DNA is almost inhibited by these nanoparticles complexes. A novel non-viral gene carrier based on the amino-modified silica nanoparticles is proposed under the combination of nanotechnology, biotechnology and gene engineering technology. The plasmid DNA can successfully cross various systemic barriers to COS-7 cells as well as mediate high expression of Green Fluorescence Protein (GFP) gene in cells by use of this novel gene carrier.

A facile graphene oxide-based DNA polymerase assay.

The DNA polymerase assay is fundamental for related molecular biology investigations and drug screenings, however, the commonly used radioactive method is laborious and restricted. Herein, we report a novel, simple and cost-effective fluorometric DNA polymerase detection method by utilizing graphene oxide (GO) as a signal switch. In this strategy, in the absence of DNA polymerase, the fluorophore-labeled template ssDNA could be strongly adsorbed and almost entirely quenched by GO. However, as DNA polymerase exists, the polymerized dsDNA product might lead to a much lower quenching efficiency after addition of GO due to the much weaker interaction of dsDNA with GO than ssDNA, thus resulting in a much higher fluorescence signal detected. As proof of concept, the quantitative DNA polymerase activity assay was performed using the Klenow fragment exo(-) (KF(-)) as a model. It was confirmed that, after optimization of detection conditions, KF(-) activity could be sensitively detected through facile fluorescence measurements, with a detection limit of 0.05 U mL(-1) and a good linear correlation between 0.05-2.5 U mL(-1) (R(2) = 0.9928). In addition, this GO-based method was further inspected to evaluate the inhibitive behaviors of several drugs toward KF(-) activity, the result of which firmly demonstrated its potential application in polymerization-targeted drug screening.

The adenine DNA self-assembly of pH- and near-infrared-responsive gold nanorod vehicles for the chemothermal treatment of cancer cells

Despite the remarkable progress in the construction of nanostructures for drug delivery in cancer therapy, multifunctional nanoplatforms with synergistic advantages over any single-model nanostructures are still encouraging. Herein, a dual pH- and near-infrared (NIR)-responsive nanotherapeutic system for the chemothermal treatment of cancer cells was achieved by using adenine DNA coated gold nanorod vehicles (poly(A)/AuNR). The poly(A)/AuNRs were prepared by self-assembling thiolated poly(A) on the surface of the AuNRs via Au-S bonding. In this system, the poly(A) provided the ability to load coralyne, a model chemotherapeutic drug, through adenine-coralyne-adenine specific binding. Under low-pH or high-temperature conditions, adenine-coralyne-adenine would be unstable, leading to the stimuli-responsive release of coralyne for cancer chemotherapy. The AuNRs showed a high efficiency for the conversion of NIR light into heat, providing the fundamental basis of hyperthermal cancer therapy, and also promoting the triggered release of coralyne from poly(A) upon NIR irradiation. The characteristics of the poly(A)/AuNRs have been investigated by using TEM and UV-vis spectroscopy. It was shown that about 160 copies of poly(A) could be assembled on one AuNR prepared with the dimensions of about 30 nm in length and 10 nm in width, and each poly(A)/AuNR could load about 2500 coralyne molecules. The in vitro studies using human hepatoma SMMC-7721 cells demonstrated that the coralyne loaded poly(A)/AuNRs could be endocytosed and demonstrated an efficient operation in a cellular acidic environment and NIR irradiation, leading to significant cytotoxicity through the excellent chemothermal synergistic effects. We believe that this developed multimode nanostructure will provide potential applications for cancer therapy.

Gold nanorod-seeded synthesis of Au@Ag/Au nanospheres with broad and intense near-infrared absorption for photothermal cancer therapy

As a widely-adopted agent for photothermal therapy (PTT), gold nanorods (Au NRs) remain problematic due to the cytotoxicity derived from cetyltrimethylammonium bromide (CTAB) and the comparatively weak and narrow near-infrared (NIR) absorption band. To address this problem, in this study, we propose a shape-controllable and spectrum-adjustable method of synthesis for Au@Ag/Au nanoparticles (NPs) through first coating a Ag nanolayer on the Au NR seed and a subsequent replacement reaction with HAuCl4 to yield a Ag/Au alloy nanoshell. Results from TEM and UV-vis spectra analysis showed that the thickness of the Ag layers directly determined the shape and size of the NPs, and the formation of Ag/Au nanoshells effectively enhanced the NIR absorbance of the NPs. Remarkably, the optimum Au@Ag/Au nanospheres (NSs) with a diameter of ∼40 nm were revealed to have a broad and intense absorption cross section from 400 to 1100 nm, a ∼4.7 times higher hyperthermic effect than Au NRs, and low dark-cytotoxicity. By using A549 lung cancer as the model, a series of in vitro investigations was performed and demonstrated that Au@Ag/Au NSs could efficaciously kill cancer cells under a 980 nm irradiation. The efficacy of PTT could be further improved by increasing the concentration, incubation time or irradiation time of the NSs. Moreover, a preliminary in vivo study also showed that, after injection into the A549 tumor, Au@Ag/Au NSs could cause an obvious necrosis at the irradiation site. Thus, a novel, promising and highly-effective NIR PTT agent has been developed, which might greatly advance the application of PTT in biomedical research.