Danke Xu

Ultrasensitive Electrochemical Detection For DNA Arrays Based on Silver Nanoparticle Aggregates

Multiplexed DNA target detection is of great significance in many fields including clinical diagnostics, environmental monitoring, biothreat detection and forensics. Although the emergence of DNA chip technology has accelerated this process, it is still a challenge to perform ultrasensitive DNA assay at low attomol concentrations so that DNA detection can be directly achieved without a PCR protocol. In this work, an oligonucleotide-functionalized silver nanoparticle tag has been successfully developed for multiplexed DNA electrochemical detection with ultrahigh sensitivity. The multiprobes containing oligo(d)A and the reporting probes were anchored onto the silver nanoparticles, followed by hybridizing with the silver nanoparticle conjugate modified with oligo(d)T. The hybridization-induced tag was found to show an aggregated nanostructure 10 times larger than the individual nanoparticle, as revealed by TEM. For sandwich-based assays, the tag was specifically coupled to a gold electrode surface via target DNA. Compared to a single nanoparticle label, this novel tag has shown excellent electroactive property and produces 10(3)-fold amplification in the differential pulse voltammetric (DPV) method. Hepatitis B virus (HBV) sequence was employed as a sample model, and we have achieved a detection limit of 5 aM ( approximately 120 molecules in 40 muL volume), demonstrating ultrasensitive measurement for DNA. The property of the electrochemical process involving silver aggregates was further investigated and the integrative oxidation of the silver tag was observed. We further demonstrated the multiplexed DNA target detection using array chips functionalized with Herpes simplex virus (HSV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) sequences, which shows effective recognition of the relative sequences individually or simultaneously. The method offers a uniquely new approach for DNA detection with ultrahigh sensitivity as well as advantages of rapidity, throughput, and miniaturization.

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.

An impedance array biosensor for detection of multiple antibody–antigen interactions

Electrochemical impedance spectroscopy (EIS) combined with a gold electrode array was developed to detect multiple antibody-antigen interactions. Hepatitis B surface antigen (HBsAg), as a model sample, was employed to evaluate the characteristics of the biosensor. The array was fabricated by immobilizing antibodies on the self-assembled molecules surface of the electrodes. The surface characteristics of the array during the binding process including the antibody-antigen conjugation and the sandwich complex with HRP-labeled antibody, as well as the precipitation layer, were characterized by atomic force microscopy (AFM) and electrochemical impedance spectroscopy, respectively. A linear relationship between electron-transfer resistance and the concentrations of HBsAg ranged from 10 pg ml(-1) to 1 ng ml(-1) and the detection limit of 10 pg ml(-1) was obtained. 100 pg ml(-1) antigen samples, such as rat IgG, HBsAg and HBeAg, as well as the antigen mixture, were incubated with the relative antibody-modified electrodes on the array. No obvious cross-talk reaction was observed. All these results confirm the feasibility of applying electrochemical impedance spectroscopy to the electrode array.

Determination of purine bases by capillary zone electrophoresis with wall-jet amperometric detection

Abstract A new and sensitive method for the simultaneous determination of purine bases using capillary zone electrophoresis with vertical wall-jet amperometric detection was developed. The separation of adenine, guanine, hypoxanthine, xanthine and uric acid was performed in an uncoated silica capillary (50cm × 25 μ m i.d.). A bundle of carbon fibers was used to fabricate the working electrode and the assay was optimized with respect to detection potential, pH and buffer concentration. The dependence of peak heights for these purine bases on their concentrations from 1.0 fmol to 0.2 pmol was linear. Under optimized conditions, the proposed method has been applied to analyze salmon testes DNA and human plasma with satisfactory results.

Identification and quantitative determination of uric acid in human urine and plasma by capillary electrophoresis with amperometric detection

Application of capillary zone electrophoresis with electrochemical detection to the identification and quantitative determination of uric acid in human urine as well as plasma is described. This work was carried out in a 30 cm x 25 microm I.D. fused-silica capillary with tricine buffer and a carbon fiber bundle was employed as a working electrode, the working voltage in amperometric detection was set at +0.80 V (vs. SCE). The sample constituent is identified by stopped flow-linear sweep voltammetry. Under optimal conditions, a lower detection limit of 0.48 fmol was obtained for uric acid.

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.