Near-Infrared Light-Powered and DNA Nanocage-Confined Catalytic Hairpin Assembly Nanobiosensor with a Nucleic Acid Restriction Behavior and Reinforced Enzymatic Resistance for Robust Imaging Assay in Live Biosystems.

While DNA amplifier-built nanobiosensors featuring a DNA polymerase-free catalytic hairpin assembly (CHA) reaction have shown promise in fluorescence imaging assays within live biosystems, challenges persist due to unsatisfactory precision stemming from premature activation, insufficient sensitivity arising from low reaction kinetics, and poor biostability caused by endonuclease degradation. In this research, we aim to tackle these issues. One aspect involves inserting an analyte-binding unit with a photoinduced cleavage bond to enable a light-powered notion. By utilizing 808 nm near-infrared (NIR) light-excited upconversion luminescence as the ultraviolet source, we achieve entirely a controllable sensing event during the biodelivery phase. Another aspect refers to confining the CHA reaction within the finite space of a DNA self-assembled nanocage. Besides the accelerated kinetics (up to 10-fold enhancement) resulting from the nucleic acid restriction behavior, the DNA nanocage further provides a 3D rigid skeleton to reinforce enzymatic resistance. After selecting a short noncoding microRNA (miRNA-21) as the modeled low-abundance sensing analyte, we have verified that the innovative NIR light-powered and DNA nanocage-confined CHA nanobiosensor possesses remarkably high sensitivity and specificity. More importantly, our sensing system demonstrates a robust imaging capability for this cancer-related universal biomarker in live cells and tumor-bearing mouse bodies, showcasing its potential applications in disease analysis.

Photoresponsive CHA-Integrated Self-Propelling 3D DNA Walking Amplifier within the Concentration Localization Effect of DNA Molecular Framework Enables Highly Efficient Fluorescence Bioimaging.

While three-dimensional (3D) DNA walking amplifiers hold considerable promise in the construction of advanced DNA-based fluorescent biosensors for bioimaging, they encounter certain difficulties such as inadequate sensitivity, premature activation, the need for exogenous propelling forces, and low reaction rates. In this contribution, a variety of profitable solutions have been explored. First, a catalytic hairpin assembly (CHA)-achieved nonenzymatic isothermal nucleic acid amplification is integrated to enhance sensitivity. Subsequently, one DNA component is simply functionalized with a photocleavage-bond to conduct a photoresponsive manner, whereby the target recognition occurs only when the biosensor is exposed to an external ultraviolet light source, overcoming premature activation during biodelivery. Furthermore, a special self-propelling walking mechanism is implemented by reducing biothiols to MnO2 nanosheets, thereby propelling forces that are self-supplied to a Mn2+-reliant DNAzyme. By carrying the biosensing system with a DNA molecular framework to induce a unique concentration localization effect, the nucleic acid contact reaction rate is notably elevated by 6 times. Following these, an ultrasensitive in vitro detection performance with a limit of detection down to 2.89 fM is verified for a cancer-correlated microRNA biomarker (miRNA-21). Of particular importance, our multiple concepts combined 3D DNA walking amplifier that enables highly efficient fluorescence bioimaging in live cells and even bodies, exhibiting a favorable application prospect in disease analysis.

Multiple miRNA Detection through a Suspended Microbead Array Encoded by Triple-Color Upconversion Luminescent Nanotags via Bi-Beam Splitter Hybrid-Multitrap Optical Tweezers.

In recent years, optical tweezers have become a novel tool for biodetection, and to improve the inefficiency of a single trap, the development of multitraps is required. Herein, we constructed a set of hybrid multitrap optical tweezers with the balance of stability and flexibility by the combination of two different beam splitters, a diffraction optical element (DOE) and galvano mirrors (GMs), to capture polystyrene (PS) microbeads in aqueous solutions to create an 18-trap suspended array. A sandwich hybridization strategy of DNA-miRNA-DNA was adopted to detect three kinds of target miRNAs associated with triple negative breast cancer (TNBC), in which different upconversion nanoparticles (UCNPs) with red, green, and blue emissions were applied as luminescent tags to encode the carrier PS microbeads to further indicate the levels of the targets. With encoded luminescent microbeads imaged by a three-channel microscopic system, the biodetection displayed high sensitivity with low limits of detection (LODs) of 0.27, 0.32, and 0.33 fM and exceptional linear ranges of 0.5 fM to 1 nM, 0.7 fM to 1 nM, and 1 fM to 1 nM for miR-343-3p, miR-155, and miR-199a-5p, respectively. In addition, this bead-based assay method was demonstrated to have the potential for being applied in patients' serum by satisfactory standard addition recovery experiment results.

A Photoresponsive and Metal-Organic Framework Encapsulated DNA Tetrahedral Entropy-Driven Amplifier for High-Performance Imaging Intracellular MicroRNA.

The further development of high-performance fluorescent biosensors to image intracellular microRNAs is beneficial to cancer medicine. By virtue of the need for enzymes and hairpin DNA probes, the entropy-driven reaction-assisted signal amplification strategy has shown an enormous potential to accomplish this task. Nevertheless, this good option still meets with poor biostability, low cell uptake efficiency, and unsatisfactory accuracy. On the basis of these challenges, we put forward here a battery of solving pathways. First, the straight DNA probes are anchored onto the vertexes of dual DNA tetrahedrons, and thus the enzyme resistance of the whole sensing system is observably enhanced. A metal-organic framework (ZIF-8 nanoparticle), which can be effectively dissociated into a weakly acidic environment, then is employed as an additional delivery vehicle to encapsulate such a DNA tetrahedron sustained biosensor and finally bring about a more efficient endocytosis. Last, a kind of photocleavage-linker triggered photoresponsive manner is incorporated to achieve an exceptional precise target identification, by which the biosensor can only be initiated under the irradiation of an externally mild 365 nm ultraviolet light source. In accordance with the above efforts, worthy assay performance toward microRNA-196a has given rise to this newly constructed biosensor, whose sensitivity is down to 2.7 pM and also able to distinguish single-base variation. Beyond that, the amplifier can work as a powerful imaging toolbox to accurately determine the targets in living cells, providing a promising intracellular sensing platform.

Biomimetic Chip Enhanced Time-Gated Luminescent CRISPR-Cas12a Biosensors under Functional DNA Regulation.

Despite that the currently discovered CRISPR-Cas12a system is beneficial for improving the detection accuracy and design flexibility of luminescent biosensors, there are still challenges to extend target species and strengthen adaptability in complicated biological media. To conquer these obstacles, we present here some useful strategies. For the former, the limitation to nucleic acids assay is broken through by introducing a simple functional DNA regulation pathway to activate the unique trans-cleavage effect of this CRISPR system, under which the expected biosensors are capable of effectively transducing a protein (employing dual aptamers) and a metal ion (employing DNAzyme). For the latter, a time-gated luminescence resonance energy transfer imaging manner using a long-persistent nanophosphor as the energy donor is performed to completely eliminate the background interference and a nature-inspired biomimetic periodic chip constructed by photonic crystals is further combined to enhance the persistent luminescence. In line with the above efforts, the improved CRISPR-Cas12a luminescent biosensor not only exhibits a sound analysis performance toward the model targets (carcinoembryonic antigen and Na+) but also owns a strong anti-interference feature to actualize accurate sensing in human plasma samples, offering a new and applicative analytical tool for laboratory medicine.

Photo-gated and self-powered three-dimensional DNA motors with boosted biostability for exceptionally precise and efficient tracing of intracellular survivin mRNA.

Benefiting from the outstanding signal amplification effect and the admirable construction flexibility, the currently proposed DNA motors (particularly DNA walkers) based biosensing concepts have provided a forceful fluorescence imaging tool for intracellular detection. Even so, this promising sensing means is not only subject to poor controllability and prone to produce false signals but also requires exogenous powering forces owing to the common employment of DNAzyme. In response to these challenges, we are herein motivated to present some meaningful solving strategies. For one thing, the surfaces of gold nanoparticles are conducted with a photo-gated walking behavior by introducing a photocleave mode, under which the light-switchable DNA walkers are capable of being selectively activated via an external ultraviolet source to faultlessly prevent the sensing frame from being pre-initiated during cellular uptake and intracellular delivery. For another, the intracellular biothiols are consumed by MnO2 nanosheets to effectively avoid the competitions to Au-S bonds to eliminate potential false outputs and also self-supply sufficient cofactors (Mn2+) to actualize a self-powered operation pattern as well as facilitate the endocytosis process. Following these breakthroughs, a favorable analysis performance towards a model tumor biomarker (survivin mRNA) is endowed with the newly raised biosensor, whose sensitivity is low to pM level with a sound specificity for identifying single base mismatching. Moreover, the significantly improved autonomous three-dimensional DNA walkers can be used to determine and dynamically trace the targets in live cancer cells with an exceptional precise and efficient manner, commendably impelling the sensing ability of DNA motors in biological specimens.

Light-Activated and Self-Driven Autonomous DNA Nanomachine Enabling Fluorescence Imaging of MicroRNA in Living Cells with Exceptional Precision and Efficiency.

Owing to their favorable design flexibility and eminent signal amplification ability, DNA nanomachine-supported biosensors have provided an attractive avenue for intracellular fluorescence imaging, especially for DNA walkers. However, this promising option not only suffers from poor controllability but also needs to be supplied with additional driving forces on account of the frequent employment of metal ion-dependent DNAzymes. Aiming at overcoming these obstacles, we introduce some fruitful solutions. On one hand, innovative light-activated walking behavior induced by a photocleavage mode is established on the surfaces of gold nanoparticles, and such a photoselective sensing system can be perfectly prevented from pre-activating during the intracellular delivery process and made to achieve target identification only under irradiation using a moderate ultraviolet light source. On the other hand, this light-switchable sensing frame is encapsulated within a dissociable metal-organic framework (ZIF-8) to facilitate endocytosis and ensure sufficient internal cofactors (Zn2+) to realize a self-driven pattern in the acidic environment of the cell lysosome. Based on the abovementioned efforts, the newly constructed autonomous three-dimensional DNA walkers present satisfactory sensitivity (a limit of detection of down to 19.4 pM) and specificity (even distinguishing single-base changes) toward a model biomarker (microRNA-21). More importantly, the sensing method allows determination of the variations in targets in living cancer cells with exceptional precision and efficiency, offering a powerful assay platform for intracellular imaging.

Spectrally Combined Encoding for Profiling Heterogeneous Circulating Tumor Cells Using a Multifunctional Nanosphere-Mediated Microfluidic Platform.

Comprehensive phenotypic profiling of heterogeneous circulating tumor cells (CTCs) at single-cell resolution has great importance for cancer management. Herein, a novel spectrally combined encoding (SCE) strategy was proposed for multiplex biomarker profiling of single CTCs using a multifunctional nanosphere-mediated microfluidic platform. Different cellular biomarkers uniquely labeled by multifunctional nanosphere barcodes, possessing identical magnetic tags and distinct optical signatures, enabled isolation of heterogeneous CTCs with over 91.6 % efficiency and in situ SCE of phenotypes. By further trapping individual CTCs in ordered microstructures on chip, composite single-cell spectral signatures were conveniently and efficiently obtained, allowing reliable spectral-readout for multiplex biomarker profiling. This SCE strategy exhibited great potential in multiplex profiling of heterogeneous CTC phenotypes, offering new avenues for cancer study and precise medicine.

Real-Time Monitoring of Temperature Variations around a Gold Nanobipyramid Targeted Cancer Cell under Photothermal Heating by Actively Manipulating an Optically Trapped Luminescent Upconversion Microparticle.

We demonstrate an effective approach to realize active and real-time temperature monitoring around the gold nanobipyramids (AuNBPs)-labeled cancer cell under 808 nm laser irradiation by combining optical tweezers and temperature-sensitive upconversion microparticles (UCMPs). On the one hand, the aptamer-modified AuNBPs that absorb laser at 808 nm not only act as an excellent photothermal reagent but also accurately and specifically bind the target cancer cells. On the other hand, the single optically trapped NaYF4:Yb3+, Er3+ UCMPs with a 980 nm laser exhibit temperature-dependent luminescence properties, where the intensity ratio of emission 525 and 547 nm varies with the ambient temperature. Therefore, real-time temperature variation monitoring is performed by 3D manipulation of the trapped single UCMP to control its distance from the AuNBPs-labeled cancer cell while being photothermally killed. The results show distance-related thermal propagation because the temperature increase reaches as high as 10 °C at a distance of 5 µm from the cell, whereas the temperature difference drops rapidly to 5 °C when this distance increases to 15 µm. This approach shows that the photothermal conversion from AuNBPs is sufficient to kill the cancer cells, and the temperature increase can be controlled within the micrometer level at a certain period of time. Overall, we present a micrometer-size thermometer platform and provide an innovative strategy to measure temperature at the micrometer level during photothermal killing of cancer cells.

Breaking Through Bead-Supported Assay: Integration of Optical Tweezers Assisted Fluorescence Imaging and Luminescence Confined Upconversion Nanoparticles Triggered Luminescent Resonance Energy Transfer (LRET).

Herein, a conceptual approach for significantly enhancing a bead-supported assay is proposed. For the fluorescence imaging technology, optical tweezers are introduced to overcome the fluid viscosity interference and immobilize a single tested bead at the laser focus to guarantee a fairly precise imaging condition. For the selection of fluorescent materials and the signal acquisition means, a type of innovative luminescence confined upconversion nanoparticle with a unique sandwich structure is specially designed to act as an efficient energy donor to trigger the luminescent resonance energy transfer (LRET) process. By further combining the double breakthrough with a molecular beacon model, the newly developed detection strategy allows for achieving a pretty high LRET ratio (≈ 88%) to FAM molecules and offering sound assay performance toward miRNA analysis with a detection limit as low as the sub-fM level, and is capable of well identifying single-base mismatching. Besides, this approach not only is able to accurately qualify the low-abundance targets from as few as 30 cancer cells but also can be employed as a valid cancer early warning tool for performing liquid biopsy.

Combining Holographic Optical Tweezers with Upconversion Luminescence Encoding: Imaging-Based Stable Suspension Array for Sensitive Responding of Dual Cancer Biomarkers.

Establishment of a stable analytical methodology with high-quality results is an urgent need for screening cancer biomarkers in early diagnosis of cancer. In this study, we incorporate holographic optical tweezers with upconversion luminescence encoding to design an imageable suspension array and apply it to conduct the detection of two liver cancer related biomarkers, carcinoembryonic antigen and alpha fetal protein. This bead-based assay is actualized by forming a bead array with holographic optical tweezers and synchronously exciting the upconversion luminescence of corresponding trapped complex beads fabricated with a simple one-step sandwich immunological recognition. Owing to the fact that these flowing beads are stably trapped in the focal plane of the objective lens which tightly converges the array of the laser beams by splitting a 980 nm beam using a diffraction optical element, a fairly stable excitation condition is achieved to provide reliable assay results. By further taking advantage of the eminent encoding capability of upconversion nanoparticles and the extremely low background signals of anti-Stokes luminescence, the two targets are well-identified and simultaneously detected with quite sound sensitivity and specificity. Moreover, the potential on-demand clinical application is presented by employing this approach to respond the targets toward complex matrices such as serum and tissue samples, offering a new alternative for cancer diagnosis technology.

Target-triggered signal turn-on detection of prostate specific antigen based on metal-enhanced fluorescence of Ag@SiO2@SiO2-RuBpy composite nanoparticles.

A three-layer core-shell nanostructure consisting of a silver core, a silica spacer, and a fluorescent dye RuBpy-doped outer silica layer was fabricated, and the optimal metal-enhanced fluorescence (MEF) distance was explored through adjusting the thickness of the silica spacer. The results show that the optimal distance is ∼10.4 nm with the maximum fluorescence enhancement factor 2.12. Then a new target-triggered MEF 'turn-on' strategy based on the optimized composite nanoparticles was successfully constructed for quantitative detection of prostate specific antigen (PSA), by using RuBpy as the energy donor and BHQ-2 as the acceptor. The hybridization of the complementary DNA of PSA-aptamer immobilized on the surface of the MEF nanoparticles with PSA-aptamer modified with BHQ-2, brought BHQ-2 in close proximity to RuBpy-doped silica shell and resulted in the decrease of fluorescence. In the presence of target PSA molecules, the BHQ-PSA aptamer is dissociated from the surface of the nanoparticles with the fluorescence switched on. Therefore, the assay of PSA was achieved by measuring the varying fluorescence intensity. The results show that PSA can be detected in the range of 1-100 ng ml-1 with a detection limit of 0.20 ng ml-1 (6.1 pM), which is 6.7-fold increase of that using hollow RuBpy-doped silica nanoparticles. Moreover, satisfactory results were obtained when PSA was detected in 1% serum.

Metal-enhanced fluorescent dye-doped silica nanoparticles and magnetic separation: A sensitive platform for one-step fluorescence detection of prostate specific antigen.

The world health organization figures show prostate cancer in developed countries has been the second primary cause of cancer mortality following lung cancer for the men. So, early and sensitive diagnosis of cancer is very important before it spreads out to the other organs of the body. It is well-known that prostate-specific antigen (PSA) is the most specific and efficient tumor marker for the diagnosis of prostate cancer. Herein, we successfully fabricated core-shell composite fluorescent nanoparticle Ag@SiO2@SiO2-RuBpy which provide a photoluminescence enhancement of up to ~3-fold when the separation distance between the surface of silver core and the center of the third RuBpy doped silica shell is about 10nm. These core-shell MEF-capable nanoparticles have obvious advantages. The interaction between the doped RuBpy molecules in the outer silica layer and the silver core, greatly improves the excitation efficiency and enhances the fluorescence intensity. Importantly, the presence of silica can reduce the self-quenching of RuBpy, which makes larger amounts of RuBpy incorporated into the silica shell. In addition, the shell protects the RuBpy against collisional quenching and irreversible photodegradation and provides abundant hydroxyl for easy conjugation. After that a highly sensitive, specific and reliable strategy based on metal-enhanced fluorescence and magnetic separation was applied for the detection of PSA in both buffer and serum. The process could be rapidly accomplished, in which the immunomagnetic nanospheres (IMNs) and immunofluorescent nanoparticles (IFNs) were used to capture and identify the target molecules simultaneously. A good linear relationship between the fluorescence intensity and the concentration of PSA (0.1-100ng/mL) with a detection limit 27pg/mL was obtained.

One-step separation-free detection of carcinoembryonic antigen in whole serum: Combination of two-photon excitation fluorescence and optical trapping.

Direct analysis of biomolecules in complex biological samples remains a major challenge for fluorescence-based approaches due to the interference of background signals. Herein, we report an analytical methodology by exploiting a single low-cost near-infrared sub-nanosecond pulse laser to synchronously actualize optical trapping and two-photon excitation fluorescence for senstive detection of carcinoembryonic antigen (CEA) in buffer solution and human whole serum with no separation steps. The assay is performed by simultaneously trapping and exciting the same immune-conjugated microsphere fabricated with a sandwich immunization strategy. Since the signal is strictly limited in the region of a three-dimensional focal volume where the microsphere is trapped, no obvious background signal is found to contribute the detected signals and thus high signal-to-background data are obtained. As a proof-of-concept study, the constructed platform exhibits good specificity for CEA and the detection limit reaches as low as 8pg/mL (45 fM) with a wide linear range from 0.01 to 60ng/mL in the both cases. To investigate the potential application of this platform in clinical diagnosis, 15 cases of serum samples were analyzed with satisfactory results, which further confirm the applicability of this method.

DNA-stabilized silver nanoclusters and carbon nanoparticles oxide: A sensitive platform for label-free fluorescence turn-on detection of HIV-DNA sequences.

Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1-50nM with a detection limit of 0.40nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity.

A fluorescent aptasensor using double-stranded DNA/graphene oxide as the indicator probe.

We developed a fluorescent aptasensor based on the making use of double-stranded DNA (dsDNA)/graphene oxide (GO) as the signal probe and the activities of exonuclease I (Exo I). This method takes advantage of the stronger affinity of the aptamer to its target rather than to its complementary sequence (competitor), and the different interaction intensity of dsDNA, mononucleotides with GO. Specifically, in the absence of target, the competitor hybridizes with the aptamer, preventing the digestion of the competitor by Exo I, and thus the formed dsDNA is adsorbed on GO surface, allowing fluorescence quenching. When the target is introduced, the aptamer preferentially binds with its target. Thereby, the corresponding nuclease reaction takes place, and slight fluorescence change is obtained after the introduction of GO due to the weak affinity of the generated mononucleotides to GO. Adenosine (AD) was chosen as a model system and tested in detail. Under the optimized conditions, smaller dissociation constant (Kd, 311.0 µM) and lower detection limit (LOD, 3.1 µM) were obtained in contrast with traditional dye-labeled aptamer/GO based platform (Kd=688.8 µM, LOD=21.2 µM). Satisfying results were still obtained in the evaluation of the specificity and the detection of AD in human serum, making it a promising tool for the diagnosis of AD-relevant diseases. Moreover, we demonstrated the effect of the competitor on the LOD, and the results reveal that the sensitivity could be enhanced by using the rational competitor. The present design not only constructs a label-free aptamer based platform but also extends the application of dsDNA/GO complex in biochemical and biomedical studies.

Analysis of cancer marker in tissues with Hadamard transform fluorescence spectral microscopic imaging.

Quantum dots (QDs) probes were used to tag and trace cancer biomarkers in cancer tissues based on the system of home-made Hadamard transform (HT) spectral microscopic imaging, which can be applied to provide high-resolution fluorescence spectrum and image of single cells and tissues. In situ fluorescence imaging for cancer marker proteins, such as estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), proliferating cell nuclear antigen (PCNA) and cytokeratin 20 (CK20) in tumor tissues, were realized by using the HT system to capture quantitative information for these proteins when tumor tissues were immunostained with QDs probes. A method to evaluate tumor malignancy of the specimens based on in situ analysis of distribution of marker proteins was proposed based on the comparative study of positive samples and negative controls. The investigation of ER contents of the cores in breast cancer tissue microarrays (TMAs) shows that the technique of QDs-immunohistochemistry (IHC)/HT spectral imaging is more sensitive than conventional IHC method. The results also demonstrate that the QDs-IHC/HT spectral imaging technique can be applied to visualize and quantitatively measure the subcellular molecules inside tumor tissues, and the coupling of HT spectral imaging to the probing of subcellular molecules with QDs has great potential in biology and medical diagnosis.

A gold nanoparticle-based label free colorimetric aptasensor for adenosine deaminase detection and inhibition assay.

A novel strategy for the fabrication of a colorimetric aptasensor using label free gold nanoparticles (AuNPs) is proposed in this work, and the strategy has been employed for the assay of adenosine deaminase (ADA) activity. The aptasensor consists of adenosine (AD) aptamer, AD and AuNPs. The design of the biosensor takes advantage of the special optical properties of AuNPs and the interaction between AuNPs and single-strand DNA. In the absence of ADA, the AuNPs are aggregated and are blue in color under appropriate salt concentration because of the grid structure of an AD aptamer when binding to AD, while in the presence of the analyte, AuNPs remain dispersed with red color under the same concentration of salt owing to ADA converting AD into inosine which has no affinity with the AD aptamer, thus allowing quantitative investigation of ADA activity. The present strategy is simple, cost-effective, selective and sensitive for ADA with a detection limit of 1.526 U L(-1), which is about one order of magnitude lower than that previously reported. In addition, a very low concentration of the inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) could generate a distinguishable response. Therefore, the AuNP-based colorimetric biosensor has great potential in the diagnosis of ADA-relevant diseases and drug screening.

Sensitive multiplexed DNA detection using silica nanoparticles as the target capturing platform.

We present a simple and sensitive method for multiplexed DNA detection by simultaneously capturing two different DNA sequences with a same silica nanoparticle (NP) through a sandwich mode. This biobarcode assay method was demonstrated by using oligonucleotide sequences of 64 bases associated with human papillomavirus (HPV) 16 and 18 L1 genes as model systems. The nonfluorescent carboxyl-modified silica NPs were prepared using water-in-oil (W/O) microemulsion methods. Avidin was immobilized on the surface of the NPs by covalent binding to the carboxyl linkers. The binding capacity of the avidin-covered NPs for ligand biotin was quantified and the results show that about 8 avidin molecules are bound to one nanoparticle. The silica nano-platforms were prepared through the biotin-avidin interaction and the amounts of capture DNA strands for HPV-16 and HPV-18 (C-16 and C-18, respectively) conjugated to the surface of the same NPs were measured using fluorescent dye hoechst33258. The calculated result shows that the amounts of conjugated C-16 and C-18 on 1mg of NPs (9.2 pmol) are about 13.5 pmol and 15.5 pmol, respectively. A one-step hybridization reaction was performed by mixing the silica nano-platforms, HPV-16 and HPV-18 target DNA (T-16 and T-18), fluorescein amidite (FAM) or 6-carboxyl-X-rhodamine (Rox) labeled HPV-16 and HPV-18 probes. The hybrid-conjugated NPs were separated by centrifugation, and T-16 and T-18 were detected by measuring fluorescence signals of FAM and Rox respectively. The results show linear dependence of the fluorescence intensity on target DNA concentration in the range from 0.5 to 9 nM, and the detection limit (3σ) of T-16 and T-18 is 0.17 nM and 0.78 nM, respectively.

Exploring sialic acid receptors-related infection behavior of avian influenza virus in human bronchial epithelial cells by single-particle tracking.

Human respiratory tract epithelial cells are the portals of human infection with influenza viruses. However, the infection pathway of individual avian influenza viruses in human respiratory cells remains poorly reported so far. The single-particle tracking technique (SPT) is a powerful tool for studying the transport mechanism of biomolecules in live cells. In this work, we use quantum dots to label avian influenza H9N2 virus and elaborate on the infection mechanism of the virus in human bronchial epithelial (HBE) cells using a three-dimensional SPT technique. We have found that the H9N2 virus can infect HBE cells directly and the virus infection follows an actin filament- and microtubule-dependent process with a three-stage pattern. The transport behaviors show a high degree of consistency between the sialic acid receptors and the influenza virus. Real-time SPT provides dynamic evidence of the sialic acid receptors-related infection behavior of the avian influenza virus in live cells. The study of the influence of sialic acid receptors on virus infection may contribute to a better understanding of the cross-species transmission of the avian influenza virus.