Weibing Qiang

Bioinspired polydopamine nanospheres: a superquencher for fluorescence sensing of biomolecules

The strong fluorescence quenching ability towards a wide spectrum of fluorescent dyes of bioinspired polydopamine nanospheres was shown for the first time. Up to 97% quenching efficiency via energy transfer and/or electron transfer was obtained towards four kinds of fluorophores, aminomethylcoumarin acetate (AMCA), 6-carboxyfluorescein (FAM), 6-carboxytetramethylrhodamine (TAMRA) and Cy5. This fluorescence quenching ability compared favorably with that of graphene oxide, the superquencher. The nanospheres (NS) also exhibit different affinities for various ssDNA conformations. Furthermore, FAM-labeled ssDNA was adsorbed onto NS through non-covalent binding to form an ssDNA/NS complex, leading to the quenching of the fluorescence of FAM. This complex was used as a sensing platform for the detection of DNA and proteins based on the fluorescence recovery due to target recognition. The LODs of DNA and thrombin were equal or close to those of GO-based biosensors. The assay is fast, simple and homogeneous, and could be used for fluorescence imaging. The excellent biocompatibility and biodegradability of polydopamine also render it suitable for in vivo applications.

Disposable electrochemical aptasensor array by using in situ DNA hybridization inducing silver nanoparticles aggregate for signal amplification.

Nanomaterials as tracing tags have been widely used in biosensors with high sensitivity and selectivity. In this work, a signal amplification electrochemical aptamer sensing strategy for the detection of protein was designed by combining the hybridization-inducing aggregate of DNA-functionalized silver nanoparticles (AgNPs) and differential pulse stripping voltammetry (DPSV) detection. The multiprobes containing hybridization DNA and aptamers were anchored onto the silver nanoparticles. The protein assay was prepared through the immobilization of capture aptamer that specifically recognizes platelet-derived growth factor (PDGF-BB) on gold nanoparticles modified screen-printed electrode (SPE) array. After a sandwich-type reaction, two kinds of DNA-modified AgNPs were simultaneously added on the electrode surface for specifically recognizing PDGF-BB and forming the AgNPs aggregate caused by in situ hybridization of DNA. Compared to the signal-labeled tag, the tracing aggregate tags showed a strong electroactivity for signal amplification through stripping detection of silver after preoxidation. By using the hybridization-inducing aggregate as electrochemical readouts, the sensor showed wide linear range and low detection limit. The hybridization-inducing AgNPs aggregate were further used as tracing tags in multiplied proteins assays for PDGF-BB and thrombin by using the SPE array chip as sensing platform. The cross-talk between different aptamer-modified electrodes on the same array was avoided because of the advantage of labeled AgNPs. The array detection was also applied in the logic gate operation. The proposed method described here is ideal for multianalytes determination in clinical diagnostics with good analytical performance.

Fluorescence Enhancement of Silver Nanoparticle Hybrid Probes and Ultrasensitive Detection of IgE

An ultrasensitive protein assay method was developed based on silver nanoparticle (AgNP) hybrid probes and metal-enhanced fluorescence. Two aptamer based silver nanoparticles, Aptamer/Oligomer-A/Cy3-modified AgNPs (Tag-A) and Aptamer/Oligomer-B/Cy3-modified AgNPs (Tag-B) were hybridized to form a silver nanoparticle aggregate that produced a red shift and broadening of the Localized Surface Plasmon Resonance (LSPR) peak. The enhanced fluorescence resulted from the increased content of Cy3 molecules and their emission resonance coupled to the broadened localized surface plasmon (LSP) of AgNP aggregate. The separation distance between Cy3 and AgNPs was 8 nm which was the most optimal for metal enhanced fluorescence and the separation distance between adjacent AgNPs was about 16 nm and this was controlled by the lengths of oligomer-A and oligomer-B. The protein array was prepared by covalently immobilizing capture antibodies on aldehyde-coated slide. After addition of protein IgE sample, two kinds of aptamer-modified AgNPs (Tag-A and Tag-B) were employed to specifically recognize IgE and form the AgNP aggregate on the arrays based on their hybridization. The detection property of the aptamer-modified AgNP aggregate was compared to two other modified aptamer-based probes, aptamer-modified Cy3 and Tag-A. The modified AgNP hybrid probe (Tag-A and Tag-B) showed remarkable superiority in both sensitivity and detection limit due to the formed AgNP aggregate. The new hybrid probe also produced a wider linear range from 0.49 to 1000 ng/mL with the detection limit reduced to 40 pg/mL (211 fM). The presented method showed that the newly designed strategy of combining aptamer-based nanomaterials to form aggregates results in a highly sensitive optical detection method based on localized surface plasmon.

Highly Sensitive Detection of Proteins Based on Metal-Enhanced Fluorescence with Novel Silver Nanostructures

We present a highly sensitive metal enhanced fluorescence (MEF) method based on a novel silver nanostructure fabricated with Cy5-functionalized silver nanoparticles (AgNPs) and AgNO(3). The analytical performance has been demonstrated by microarray detection of streptavidin (SA) and human IgE. The fluorescence intensity can be enhanced substantially with the combined use of AgNPs and fluorescence enhanced solution (FES). Aptamers have been used for the preparation of Tag-C, which demonstrate IgE detection from 0.5 ng/mL to 16 ng/mL, and the limit of detection is determined to be 0.25 ng/mL. SEM images show nanogaps exist in the aggregated silver nanoparticles and the nanogaps allow for the trap of fluorophores in the nanostructures that emit brighter light upon excitation. The silver nanostructures formed by Tags and FES proved to be an excellent platform for MEF of fluorophores whose excitation and emission occurred between 436 nm and 1000 nm. Finite-difference time-domain (FDTD) simulation has been carried out to confirm the enhanced electromagnetic field inside silver nanostructures, leading to strong overlap/resonance coupling and eventual fluorescence enhancement.

Silver nanoparticle-enhanced fluorescence resonance energy transfer sensor for human platelet-derived growth factor-BB detection.

A silver nanoparticle (AgNP)-enhanced fluorescence resonance energy transfer (FRET) sensing system is designed for the sensitive detection of human platelet-derived growth factor-BB (PDGF-BB). Fluorophore-functionalized aptamers and quencher-carrying strands hybridized in duplex are coupled with streptavidin (SA)-functionalized nanoparticles to form a AgNP-enhanced FRET sensor. The resulting sensor shows lower background fluorescence intensity in the duplex state due to the FRET effect between fluorophores and quenchers. Upon the addition of PDGF-BB, the quencher-carrying strands (BHQ-2) of the duplex are displaced leading to the disruption of the FRET effect. As a result, the fluorescent intensity of the fluorophore-aptamer within the proximity of the AgNP is increased. When compared to the gold nanoparticle (AuNP)-based FRET and bare FRET sensors, the AgNP-based FRET sensor showed remarkable increase in fluorescence intensity, target specificity, and sensitivity. Results also show versatility of the AgNP in the enhancement of sensitivity and selectivity of the FRET sensor. In addition, a good linear response was obtained when the PDGF-BB concentrations are in the ranges of 100-500 and 6.2-50 ng/mL with the detection limit of 0.8 ng/mL.

A non-aggregation colorimetric assay for thrombin based on catalytic properties of silver nanoparticles

In this paper, we developed a simple and rapid colorimetric assay for protein detection based on the reduction of dye molecules catalyzed by silver nanoparticles (AgNPs). Aptamer-modified magnetic particles and aptamer-functionalized AgNPs were employed as capture and detection probes, respectively. Introduction of thrombin as target protein could form a sandwich-type complex involving catalytically active AgNPs, whose catalytic activity was monitored on the catalytic reduction of rhodamine B (RhB) by sodium borohydride (NaBH4). The amount of immobilized AgNPs on the complex increased along with the increase of the thrombin concentration, thus the detection of thrombin was achieved via recording the decrease in absorbance corresponding to RhB. This method has adopted several advantages from the key factors involved, i.e., the sandwich binding of affinity aptamers contributed to the increased specificity; magnetic particles could result in rapid capture and separation processes; the conjugation of AgNPs would lead to a clear visual detection. It allows for the detection limit of thrombin down to picomolar level by the naked eye, with remarkable selectivity over other proteins. Moreover, it is possible to apply this method to the other targets with two binding sites as well.

Multifunctional Aptamer–Silver Conjugates as Theragnostic Agents for Specific Cancer Cell Therapy and Fluorescence-Enhanced Cell Imaging

We fabricated a multifunctional theragnostic agent Ag-Sgc8-FAM for apoptosis-based cancer therapy and fluorescence-enhanced cell imaging. For cancer therapy, aptamers Sgc8 and TDO5 acted as recognizing molecules to bind CCRF-CEM and Ramos cells specifically. It was found that aptamer-silver conjugates (Ag-Sgc8, Ag-TDO5) could be internalized into cells by receptor-mediated endocytosis, inducing specific apoptosis of CCRF-CEM and Ramos cells. The apoptosis of cells depended on the concentration of aptamer-silver conjugates, as well as the incubation time between cells and aptamer-silver conjugates. The apoptotic effects on CCRF-CEM and Ramos cells were different. Annexin V/PI staining, AO/PI staining, MTT assays and ROS (reactive oxygen species) detection demonstrated the specific apoptosis of CCRF-CEM and Ramos cells. For fluorescence-enhanced cell imaging, Ag-Sgc8-FAM was prepared. Compared to Sgc8-FAM molecules, Ag-Sgc8-FAM was an excellent imaging agent as numerous Sgc8-FAM molecules were enriched on the surface of AgNPs for multiple binding with CCRF-CEM cells and signal amplification. Moreover, AgNPs could increase the fluorescence intensity of FAM by metal-enhanced fluorescence (MEF) effect. Therefore, aptamer-silver conjugates can be potential theragnostic agents for inducing specific apoptosis of cells and achieving cells imaging in real time.

Nanoparticle-catalyzed reductive bleaching for fabricating turn-off and enzyme-free amplified colorimetric bioassays

Nanoparticle-catalyzed reductive bleaching reactions of colored substrates are emerging as a class of novel indicator reactions for fabricating enzyme-free amplified colorimetric biosensing (turn-off mode), which are exactly opposite to the commonly used oxidative coloring processes of colorless substrates in traditional enzyme-catalyzed amplified colorimetric bioassays (turn-on mode). In this work, a simple theoretical analysis shows that the sensitivity of this colorimetric bioassay can be improved by increasing the amplification factor (kcatΔt), or enhancing the binding affinity between analyte and receptor (Kd), or selecting the colored substrates with high extinction coefficients (ε). Based on this novel strategy, we have developed a turn-off and cost-effective amplified colorimetric thrombin aptasensor. This aptasensor made full use of sandwich binding of two affinity aptamers for increased specificity, magnetic particles for easy separation and enrichment, and gold nanoparticle (AuNP)-catalyzed reductive bleaching reaction to generate the amplified colorimetric signal. With 4-nitrophenol (4-NP) as the non-dye colored substrate, colorimetric bioassay of thrombin was achieved by the endpoint method with a detection limit of 91pM. In particular, when using methylene blue (MB) as the substrate, for the first time, a more convenient and efficient kinetic-based colorimetric thrombin bioassay was achieved without the steps of acidification termination and magnetic removal of particles, with a low detection limit of 10pM, which was superior to the majority of the existing colorimetric thrombin aptasensors. The proposed colorimetric protocol is expected to hold great promise in field analysis and point-of-care applications.

Metal-enhanced fluorescent detection for protein microarrays based on a silver plasmonic substrate.

This paper presents an ultrasensitive fluorescent detection method through fabricating a silver microarray substrate. Silver nanoparticles (AgNPs) and Ag@Au core-shell nanoparticles with different sizes were first synthesized by a seed-mediated growth method and the metal-enhanced fluorescence of these nanoparticles on different fluorescent dyes was investigated. The results indicated that AgNPs could act as a versatile and effective metal-enhanced fluorescence material for various fluorophores, whereas the enhanced fluorescence from Ag@Au was limited only to certain fluorophores. When the AgNPs were functionalized with aptamers and fluorescent dyes, a good analytical performance for simultaneous detection of human IgE and platelet-derived growth factor-BB (PDGF-BB) could be obtained. AgNPs were not only used as detection tags but also used to fabricate the plasmonic microarray substrate to further enhance the sensitivity of fluorescent detection. As a result, a linear response to PDGF-BB concentration was obtained in the concentration range of 16 pg mL(-1) to 50 ng mL(-1), and the detection limit was 3.2 pg mL(-1). In addition, the AgNP modified plasmonic microarrays showed remarkable recovery and no significant interference from human serum when applied to 2 ng mL(-1) PDGF-BB concentration. The plasmonic microarray substrate demonstrated both high specificity and sensitivity for protein microarray detection and this novel approach has great potential for ultrasensitive detection of protein biomarkers in the bio-medical field.

Fast functionalization of silver decahedral nanoparticles with aptamers for colorimetric detection of human platelet-derived growth factor-BB.

Aptamer-silver decahedral nanoparticles (Ag10NPs-aptamer) based detection was developed for protein. Ag10NPs were synthesized by photochemical method. The advantage of Ag10NPs was its tolerance of NaCl which facilitates the functionalization of silver nanoparticles with all kinds of ssDNA. Attaching aptamers to Ag10NPs could be achieved within 2 h, much faster than traditional methods. Human platelet-derived growth factor-BB (PDGF-BB) was used as a model protein to test the binding capacity of aptamers attached on Ag10NPs. Our data showed that the aptamer-Ag10NPs conjugates were successful in detecting human PDGF-BB. Furthermore, we developed an aptamer-Ag10NPs conjugates-based colorimetric sensor to detect PDGF-BB. The results showed a linear relationship between PDGF-BB concentrations (5 ng mL(-1)-200 ng mL(-1)) and ΔOD with excellent detection specificity in serum. Therefore, the sensor based on aptamer-Ag10NPs conjugates was highly effective and sensitive and had great promise for further development and applications.