Aptamer–Au NPs conjugates-enhanced SPR sensing for the ultrasensitive sandwich immunoassay

The goal of this work is to explore the amplification effect of aptamer–gold nanoparticles (Au NPs) conjugates for ultrasensitive detection of large biomolecules by surface plasmon resonance (SPR). A novel sandwich immunoassay is designed to demonstrate the amplification effect of aptamer–Au NPs conjugates by using human immunoglobulin E (IgE) as model analyte. Human IgE, captured by immobilized goat anti-human IgE on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 1 ng/ml after anti-human IgE aptamer–Au NPs conjugates is used as amplification reagent. Meanwhile, the non-specific adsorption of aptamer–Au NPs conjugates on goat anti-human IgE is confirmed by SPR spectroscopy and then it is minimized by treating aptamer–Au NPs conjugates with 6-mercaptohexan-1-ol (MCH). These results confirm that aptamer–Au NPs conjugates is a powerful sandwich element and an excellent amplification reagent for SPR-based sandwich immunoassay.     

Ultrasensitive electrochemical aptasensor for thrombin based on the amplification of aptamer-AuNPs-HRP conjugates

Successful development of an ultrasensitive and highly specific electrochemical aptasensor for thrombin based on amplification of aptamer-gold nanoparticles-horseradish peroxidase (aptamer-AuNPs-HRP) conjugates was reported. In this electrochemical protocol, aptamer1 (Apt1) was immobilized on core/shell Fe(3)O(4)/Au magnetic nanoparticles (AuMNPs) and served as capture probe. Aptamer2 (Apt2) was dual labeled with AuNPs and HRP and used as detection probe. In the presence of thrombin, the sandwich format of AuMNPs-Apt1/thrombin/Apt2-AuNPs-HRP was fabricated. Remarkable signal amplification was realized by taking the advantage of AuNPs and catalytic reactions of HRP. Other proteins, such as human serum albumin, lysozyme, fibrinogen, and IgG did not show significant interference with the assay for thrombin. Linear response to thrombin concentration in the range of 0.1-60 pM and lower detection limit down to 30 fM (S/N=3) was obtained with the proposed method. This electrochemical aptasensor is simple, rapid (the whole detection period for a thrombin sample is less than 35 min), sensitive and highly specific, it shows promising potential in protein detection and disease diagnosis.     

Enzyme-based ultrasensitive electrochemical biosensors

Signal amplification in conventional enzyme-based biosensors is not high enough to achieve the ultrasensitive detection of biomolecules. In recent years, signal amplification has been improved by combining enzymatic reactions with redox cycling or employing multienzyme labels per detection probe. Electrochemical-chemical redox cycling and electrochemical-chemical-chemical redox cycling allow ultrasensitive detection simply by including one or two more chemicals in a solution without the use of an additional enzyme and/or electrode. Multiple horseradish peroxidase labels on magnetic bead carriers provide high signal enhancement along with a multiplex detection possibility. In both cases, the detection procedures are the same as those in conventional enzyme-based electrochemical sensors.     

Sensitive detection of proteins using assembled cascade fluorescent DNA nanotags based on rolling circle amplification

A novel cascade fluorescence signal amplification strategy based on the rolling circle amplification (RCA)-aided assembly of fluorescent DNA nanotags as fluorescent labels and multiplex binding of the biotin-streptavidin system was proposed for detection of protein target at ultralow concentration. In the strategy, fluorescent DNA nanotags are prepared relying on intercalating dye arrays assembled on nanostructured DNA templates by intercalation between base pairs. The RCA product containing tandem-repeat sequences could serve as an excellent template for periodic assembly of fluorescent DNA nanotags, which were presented per protein recognition event to numerous fluorescent DNA nanotags for assay readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human IgG as a model protein, the designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a three-decade concentration range from 1.0 pM to 1.0 fM with a limit of detection as low as 0.9 fM. By comparison with the assay of multiple labeling antibodies with the dye/DNA conjugate, the limit of detection was improved by 4 orders. The designed signal amplification strategy would hold great promise as a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.     

A new amplification strategy for ultrasensitive electrochemical aptasensor with network-like thiocyanuric acid/gold nanoparticles

An ultrasensitive and highly specific electrochemical aptasensor for detection of thrombin based on gold nanoparticles and thiocyanuric acid is presented. For this proposed aptasensor, aptamerI was immobilized on the magnetic nanoparticles, aptamerII was labeled with gold nanoparticles. The magnetic nanoparticle was used for separation and collection, and gold nanoparticle offered excellent electrochemical signal transduction. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle/thrombin/gold nanoparticle was fabricated, and the signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles. A significant sensitivity enhancement had been obtained, and the detection limit was down to 7.82 aM. The presence of other proteins such as BSA and lysozyme did not affect the detection of thrombin, which indicates a high specificity of thrombin detection could be achieved. This electrochemical aptasensor is expected to have wide applications in protein monitoring and disease diagnosis.     

In situ enzymatic silver enhancement based on functionalized graphene oxide and layer-by-layer assembled gold nanoparticles for ultrasensitive detection of thrombin

A highly specific in situ amplification strategy was designed for ultrasensitive detection of thrombin by combining the layer-by-layer (LBL) assembled amplification with alkaline phosphatase (ALP) and gold nanoparticles (Au) mediated silver deposition. High-density carboxyl functionalized graphene oxide (FGO) was introduced as a nanocarrier for LBL assembling of alkaline phosphatase decorated gold nanoparticles (ALP-Au), which was further adopted to label thrombin aptamer II. After sandwich-type reaction, numerous ALP were captured onto the aptasensor surface and catalyzed the hydrolysis of ascorbic acid 2-phosphate (AAP), which in situ generated ascorbic acid (AA), reducing Ag(+) to Ag nanoparticles (AgNPs) for electrochemical readout. Inspiringly, the in situ amplification strategy with ethanolamine as an effective blocking agent showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal, which was favorable to enhance the sensitivity of aptasensor. Our novel dramatic signal amplification strategy, with a detection limit of 2.7fM, showed about 2-3 orders of magnitude improvement in the sensitivity for thrombin detection compared to other universal enzyme-based electrochemical assay.     

Ultrasensitive chemiluminescent immunoassay of Salmonella with silver enhancement of nanogold labels

In this paper, silver enhancement of nanogold labels coupled with chemiluminescence detection was developed for ultrasensitive immunoassay of Salmonella based upon antigen–antibody immunoreaction. Polyclonal rabbit anti‐Salmonella sp. antibodies (pAb) were employed to establish the analytical protocol. The pAb coated onto ELISA microwell plates and Au nanoparticles (Au NPs) conjugated pAb capture target Salmonella to form a sandwich‐type complex. Silver then was in situ deposited around the Au NPs core and resulted in the signal amplification. In consequence, silver was dissolved to form Ag⁺ and a sensitive chemiluminescence based on the Ag⁺–K₂S₂O₈–Mn²⁺–luminol system was coupled for further signal amplification. Under the optimized conditions, the chemiluminescent intensity is proportional to target Salmonella over the range of 5–1038 cfu mL^?1 with a detection limit of 5 cfu mL^?1. The relative standard deviation for 11 measurements of about 50–100 cfu/mL target Salmonella is 4.7%. The proposed method was successfully applied to measure Salmonella in food samples and the results are identical to those of the offical standard method of China. These offer us a more powerful tool for ultrasensitive assay of foodborne pathogens. Copyright © 2010 John Wiley & Son, Ltd.     

Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences.     

DNAzyme-functionalized Pt nanoparticles/carbon nanotubes for amplified sandwich electrochemical DNA analysis

A novel DNAzyme-functionalized Pt nanoparticles/carbon nanotubes (DNAzyme/Pt NPs/CNTs) bioconjugate was fabricated as trace tag for ultrasensitive sandwich DNA detection. The Pt NPs/CNTs were prepared via layer-by-layer (LBL) assembly of the Pt NPs and polyelectrolyte on the carboxylated CNTs, followed by the functionalization with the DNAzyme and reporter probe DNA through the platinum-sulfur bonding. The subsequent sandwich-type DNA specific reaction would confine numerous DNAzyme/Pt NPs/CNTs bioconjugate onto the gold electrode surface for amplifying the signal. In the presence of 3,3',5,5' tetramethylbenzidine (TMB) which could be oxidized by the DNAzyme, electrochemical signals could be generated by chronoamperometry via the interrogation of reduction electrochemical signal of oxidized TMB. The constructed DNA sensor exhibited a wide linear response to target DNA ranging from 1.0fM to 10pM with the detection limit down to 0.6fM and exhibited excellent selectivity against even a single base mismatch. In addition, this novel DNA sensor showed fairly good reproducibility, stability, and reusability.     

CdS nanoparticles‐ enhanced chemiluminescence and determination of baicalin in pharmaceutical preparations

CdS nanoparticles (CdS NPs) of different sizes were synthesized by the citrate reduction method. It was found that CdS NPs could enhance the chemiluminescence (CL) of the luminol‐potassium ferricyanide system and baicalin could inhibit CdS NPs‐enhanced luminol‐potassium ferricyanide CL signals in alkaline solution. Based on this inhibition, a flow‐injection CL method was established for determination of baicalin in pharmaceutical preparations and human urine samples. Under optimized conditions, the linear range for determination of baicalin was 5.0 x 10^?6 to 1.0 x 10^?3 g/L. The detection limit at a signal‐to‐noise ratio of 3 was 1.7 x 10 ^?6 g/L. CL spectra, UV‐visible spectra and transmission electron microscopy (TEM) were used to investigate the CL mechanism. The method described is simple, selective and obviates the need of extensive sample pretreatment. Copyright © 2012 John Wiley & Sons, Ltd.     

Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing

A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H?O?. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL.     

Highly sensitive electrochemical detection of cocaine on graphene/AuNP modified electrode via catalytic redox-recycling amplification

We demonstrated a new strategy for highly sensitive electrochemical detection of cocaine by using two engineered aptamers in connection to redox-recycling signal amplification. The graphene/AuNP nanocomposites were electrochemically deposited on a screen printed carbon electrode to enhance the electron transfers. The cocaine primary binding aptamers were self-assembled on the electrode surface through sulfur-Au interactions. The presence of the target cocaine and the biotin-modified secondary binding aptamers leads to the formation of sandwich complexes on the electrode surface. The streptavidin-conjugated alkaline phosphatases (ALPs) were used as labels to generate quantitative signals. The addition of the ALP substrate and the co-reactant NADH results in the formation of a redox cycle between the enzymatic product and the electrochemically oxidized species and the signal is thus significantly amplified. Because of the effective modification of the sensing surface and signal amplification, low nanomolar (1 nM) detection limit for cocaine is achieved. The proposed aptamer-based sandwich sensing approach for amplified detection of cocaine thus opens new opportunities for highly sensitive determination of other small molecules.     

The strategy of signal amplification for ultrasensitive detection of hIgE based on aptamer-modified poly(di-acetylene) supramolecules

Herein, we demonstrate three strategies of signal amplification for ultrasensitive detection of human immunoglobulin E (hIgE) based on poly(di-acetylene) supramolecules. To fabricate the ultrasensitive PDA biosensor, ethylenediamine as an interlinker and aptamer as a receptor were introduced into the chip fabrication process. Using the prepared PDA liposome biosensor, the hIgE could be detected up to below 1.0 ng/ml by a primary response. In order to accomplish more ultrasensitive detection of protein on a PDA biosensor, polyclonal hIgE antibody was employed as an external mechanical force for the inducement of a secondary response. As a result, a PDA liposome biosensor sensitivity as high as 0.01 ng/ml for the target hIgE was obtained, with a sensitivity which is one hundred times of that of the method without signal amplification. These results indicate that the proposed strategies were capable of ultrasensitive quantitative and qualitative analyses of biomolecules without non-specific binding of non-target proteins.     

Bi-enzyme functionlized hollow PtCo nanochains as labels for an electrochemical aptasensor

In this work, a new signal amplification strategy based on hollow PtCo nanochains (HPtCoNCs) functionalized by bi-enzyme-horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) and glucose oxidase (GOD), as well as ferrocene-labeled secondary thrombin aptamer (Fc-TBA 2), is developed to construct a highly sensitive electrochemical aptasensor. The HRP-DNAzyme contains a special G-quadruplex structure with an intercalated hemin. With the surface area enlarged by HPtCoNCs, the amount of immobilized Fc-TBA 2, hemin and GOD can be enhanced. Under the enzyme catalysis of GOD, d-glucose is rapidly oxidized into gluconic acid accompanying with the generation of H?O?, which is further electrocatalyzed by Pt nanoparticles and HPR-DNAzyme to improve the electrochemical signal of Fc. With several amplification factors mentioned above, a wide linear ranged from 0.001 to 30 nM is acquired with a relatively low detection limit of 0.39 pM for thrombin. The present work demonstrates that using HPtCoNCs as labels is a promising way to amplify the analysis signal and improve the sensitivity of aptasensors.     

Cadmium sulfide-modified GCE for direct signal-amplified sensing of DNA hybridization

A novel DNA hybridization sensor based on nanoparticle CdS modified glass carbon electrode (GCE) was constructed and characterized coupled with Cyclic Voltammogram (CV) and Differential Pulse Voltammogram (DPV) techniques. The mercapto group-linked probe DNA was covalently immobilized onto the CdS layer and exposed to oligonucleotide (ODN) target for hybridization. The structure of DNA sensor was characterized by X-ray diffraction (XRD), field-emission microscopy (FESEM) and X-ray photoelectron spectra (XPS). Sensitive electrical readout achieved by CV and DPV techniques shown that when the target DNA hybridized with probe CdS-ODN conjugates and the double helix formed on the modified electrode, a significant increased response was observed comparing with the bare electrodes. The selectivity of the sensor was tested using a series of matched and certain-point mismatched sequences with concentration grads ranging from 10(-6) microM to 10(1) microM. The signal was in good linear with the minus logarithm of target oligonucleotide concentration with detection limit <1 pM and the optimized target DNA concentration was 10(-6) microM for the signal amplification. Due to great surface properties, the additional negative charges and space resistance of as-prepared CdS nanoparticles, the sensor was able to robustly discriminate the DNA hybridization responses with good sensitivity and stability.     

In-situ produced ascorbic acid as coreactant for an ultrasensitive solid-state tris(2,2'-bipyridyl) ruthenium(II) electrochemiluminescence aptasensor

Herein, an ultrasensitive solid-state tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)(3)(2+)) electrochemiluminescence (ECL) aptasensor using in-situ produced ascorbic acid as coreactant was successfully constructed for detection of thrombin. Firstly, the composite of Ru(bpy)(3)(2+) and platinum nanoparticles (Ru-PtNPs) were immobilized onto Nafion coated glass carbon electrode, followed by successive adsorption of streptavidin-alkaine phosphatase conjugate (SA-ALP) and biotinylated anti-thrombin aptamer to successfully construct an ECL aptasensor for thrombin determination. In our design, Pt nanoparticles in Ru(bpy)(3)(2+)-Nafion film successfully inhibited the migration of Ru(bpy)(3)(2+) into the electrochemically hydrophobic region of Nafion and facilitated the electron transfer between Ru(bpy)(3)(2+) and electrode surface. Furthermore, ALP on the electrode surface could catalyze hydrolysis of ascorbic acid 2-phosphate to in-situ produce ascorbic acid, which co-reacted with Ru(bpy)(3)(2+) to obtain quite fast, stable and greatly amplified ECL signal. The experimental results indicated that the aptasensor exhibited good response for thrombin with excellent sensitivity, selectivity and stability. A linear range of 1 × 10(-15)-1 × 10(-8) M with an ultralow detection limit of 0.33 fM (S/N=3) was obtained. Thus, this procedure has great promise for detection of thrombin present at ultra-trace levels during early stage of diseases.     

Triplex signal amplification for electrochemical DNA biosensing by coupling probe-gold nanoparticles-graphene modified electrode with enzyme functionalized carbon sphere as tracer

An ultrasensitive electrochemical DNA biosensor was constructed by assembling probe labeled gold nanoparticles (ssDNA-AuNP) on electrochemically reduced graphene oxide (ERGO) modified electrode with thiol group tagged (GT) DNA strand (d(GT)(29)SH) and coupling with horseradish peroxidase (HRP) functionalized carbon sphere (CNS) as tracer. The heteronanostructure formed on the biosensor surface appeared relatively good conductor for accelerating the electron transfer, while the HRP tagged CNS provided dual signal amplification for electrochemical biosensing. The triplex signal amplification strategy produced an ultrasensitive electrochemical detection of DNA down to attomolar level (5 aM) with a linear range of 5 orders of magnitude (from 1 × 10(-17)M to 1 × 10(-13)M), and appeared high selectivity to differentiate single-base mismatched and three-base mismatched sequences of DNA. The proposed approach provided a simple and reliable method for DNA detection with high sensitivity and specificity, indicating promising application in bioanalysis and biomedicine.     

T4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay

The use of xanthine oxidase in immunoanalysis has never been reported. We describe here a procedure in which the xanthine oxidase dependent luminescence of luminol is enhanced in the presence of Fe–EDTA complex, providing an highly sensitive assay (3 amol of enzyme) and a long-term signal. This specific amplification has been applied to T₄ and ultrasensitive TSH solid phase immunoassays, with T₄–XO and anti-TSH monoclonal antibody-XO conjugates as tracers. The performances of these assays are at least equivalent to those obtained with iodinated tracers, using the same solid phases and the same calibrators. The major advantages of these immunoassays are: (1) the long-term signal which can be repeatedly recorded over several days, (2) the high detection sensitivity, (3) the long-term stability of the luminescence reagent and (4) the stability of the conjugates.     

Multifunctional Fe₃O₄ core/Ni-Al layered double hydroxides shell nanospheres as labels for ultrasensitive electrochemical immunoassay of subgroup J of avian leukosis virus

A novel electrochemical immunosensor for ultrasensitive detection of subgroup J of avian leukosis virus (ALVs-J) was designed by using graphene sheets (GS)-layered double hydroxides (LDHs) composites modified electrode with multifunctional Fe(3)O(4) core/Ni-Al LDHs shell (LDHs@Fe(3)O(4)) nanospheres as labels. At first, the GS-LDHs were used for the immunosensor platform for improving the electronic transmission rate as well as increasing the surface area to capture a large amount of primary antibodies (Ab(1)). After that, ferrocene (Fc), secondary antibodies (Ab(2)) and horseradish peroxidase (HRP) multifunctional LDHs@Fe(3)O(4) nanospheres were used as labels with high load amount and good biological activity. Subsequently, in presence of H(2)O(2), amplified signals were obtained by an electrochemical sandwich immunoassay protocol. To embody the signal amplification property of the protocol, the analytical properties of various immunosensor platform and labels were compared in detail. Under optimal conditions, the reduction peak currents of the electrochemical immunosensor were proportional to the ALVs-J concentration over the range from 10(2.32) to 10(5.50) TCID(50)/mL with a low detection limit (180 TCID(50)/mL, S/N=3). The resulting immunosensor also displayed a good selectivity, reproducibility and stability.     

4-(dimethylamino)butyric acid@PtNPs as enhancer for solid-state electrochemiluminescence aptasensor based on target-induced strand displacement

A solid-state electrochemiluminescence (ECL) aptasensor based on target-induced aptamer displacement for highly sensitive detection of thrombin was developed successfully using 4-(dimethylamino)butyric acid (DMBA)@PtNPs labeling as enhancer. Such a special aptasensor included three main parts: ECL substrate, ECL intensity amplification and target-induced aptamer displacement. The ECL substrate was made by modifying the complex of Pt nanoparticles (PtNPs) and tris(2,2-bipyridyl) ruthenium (II) (Ru(bpy)(3)(2+)) (Ru-PtNPs) onto nafion@multi-walled carbon nanotubes (nafion@MWCNTs) modified electrode surface. A complementary thrombin aptamer labeled by DMBA@PtNPs (Aptamer II) acted as the ECL intensity amplification. The thrombin aptamer (TBA) was applied to hybridize with the labeled complementary thrombin aptamer, yielding a duplex complex of TBA-Aptamer II on the electrode surface. The introduction of thrombin triggered the displacement of Aptamer II from the self-assembled duplex into the solution and the association of inert protein thrombin on the electrode surface, decreasing the amount of DMBA@PtNPs and increasing the electron transfer resistance of the aptasensor and thus resulting large decrease in ECL signal. With the synergistic amplification of DMBA and PtNPs to Ru(bpy)(3)(2+) ECL, the aptasensor showed an enlarged ECL intensity change before and after the detection of thrombin. As a result, the change of ECL intensity has a direct relationship with the logarithm of thrombin concentration in the range of 0.001-30 nM. The detection limit of the proposed aptasensor is 0.4 pM. Thus, the approach is expected to open new opportunities for protein diagnostics in clinical as well as bioanalysis in general.