Ultrasensitive electrochemical immunosensor employing glucose oxidase catalyzed deposition of gold nanoparticles for signal amplification

This paper describes a novel enzymatic amplification strategy for ultrasensitive electrochemical immunosensing. This approach utilizes glucose oxidase for the enzymatic deposition of gold nanoparticles onto an indium tin oxide (ITO) electrode surface using a novel gold developer solution consisting of 20 mM of glucose, 20 mM of NaSCN, 0.5 M of p-benzoquinone (PBQ) and 1 mM of AuCl(4)(-) dissolved in 0.1 M of pH 7.5 phosphate buffer solution. The amount of gold deposited was quantified electrochemically by monitoring the reduction of gold oxide in an aqueous solution of 0.5 M of H(2)SO(4), which was correlated to the amount of antigens in the solution. The effectiveness of this strategy was demonstrated experimentally through the construction of an immunosensor for the detection of mouse IgG using a sandwich immunoassay in a linear dynamic range of 5 pg/ml to 50 ng/ml. A good mean apparent recovery in the range of 88-102% was obtained over the entire linear dynamic range of the sensor response in the serum samples. This suggested that the immunosensor would be useful for the testing of proteins in real clinical samples.     

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.     

Electrochemical immunosensor of platelet-derived growth factor with aptamer-primed polymerase amplification

A new method for the determination of platelet-derived growth factor BB (PDGF-BB) was developed using an electrochemical immunosensor with an aptamer-primed, long-strand circular detection probe. Rabbit anti-human PDGF-B polyclonal antibody was immobilized on the electrode to serve as the capture antibody. The detection probe was synthesized via polymerase extension along a single-stranded circular plasmid DNA template with a primer headed by the anti-PDGF-B aptamer. In the presence of the analyte, the aptamer-primed circular probe was captured on the electrode via the formation of an antibody/PDGF-BB/aptamer sandwiched complex. The electroactivity indicator methylene blue was adsorbed on the electrode surface via the analyte-sandwiched complex with long-strand circular DNA, thus yielding a strong electrochemical signal for the quantification of PDGF-BB. This strategy allowed electrochemical detection with enormous signal amplification arising from the long-strand localized circular probe. The oxidation peak current of methylene blue in square wave voltammetric measurements showed a linear dependence on the concentration of PDGF-BB in the range from 50 to 500 ng mL⁻¹, with a detection limit as low as18 pg mL⁻¹.     

A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin

A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH₃)₆]³⁺ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1–18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin.     

Enzymatic signal amplification of molecular beacons for sensitive DNA detection

Molecular beacons represent a new family of fluorescent probes for nucleic acids, and have found broad applications in recent years due to their unique advantages over traditional probes. Detection of nucleic acids using molecular beacons has been based on hybridization between target molecules and molecular beacons in a 1:1 stoichiometric ratio. The stoichiometric hybridization, however, puts an intrinsic limitation on detection sensitivity, because one target molecule converts only one beacon molecule to its fluorescent form. To increase the detection sensitivity, a conventional strategy has been target amplification through polymerase chain reaction. Instead of target amplification, here we introduce a scheme of signal amplification, nicking enzyme signal amplification, to increase the detection sensitivity of molecular beacons. The mechanism of the signal amplification lies in target-dependent cleavage of molecular beacons by a DNA nicking enzyme, through which one target DNA can open many beacon molecules, giving rise to amplification of fluorescent signal. Our results indicate that one target DNA leads to cleavage of hundreds of beacon molecules, increasing detection sensitivity by nearly three orders of magnitude. We designed two versions of signal amplification. The basic version, though simple, requires that nicking enzyme recognition sequence be present in the target DNA. The extended version allows detection of target of any sequence by incorporating rolling circle amplification. Moreover, the extended version provides one additional level of signal amplification, bringing the detection limit down to tens of femtomolar, nearly five orders of magnitude lower than that of conventional hybridization assay.     

Colorimetric detection of platelet-derived growth factors through competitive interactions between proteins and functional gold nanoparticles

We have developed a colorimetric assay-using aptamer modified 13-nm gold nanoparticles (Apt-Au NPs) and fibrinogen adsorbed Au NPs (Fib-Au NPs, 56nm)-for the highly selective and sensitive detection of platelet-derived growth factors (PDGF). Apt-Au NPs and Fib-Au NPs act as recognition and reporting units, respectively. PDGF-binding-aptamer (Apt(PDGF)) and 29-base-long thrombin-binding-aptamer (Apt(thr29)) are conjugated with Au NPs to prepare functional Apt-Au NPs (Apt(PDGF)/Apt(thr29)-Au NPs) for specific interaction with PDGF and thrombin, respectively. Thrombin interacts with Fib-Au NPs in solutions to catalyze the formation of insoluble fibrillar fibrin-Au NPs agglutinates through the polymerization of the unconjugated and conjugated fibrinogen. The activity of thrombin is suppressed once it interacts with the Apt(PDGF)/Apt(thr29)-Au NPs. The suppression decreases due to steric effects through the specific interaction of PDGF with Apt(PDGF), occurring on the surfaces of Apt(PDGF)/Apt(thr29)-Au NPs. Under optimal conditions [Apt(PDGF)/Apt(thr29)-Au NPs (25pM), thrombin (400pM) and Fib-Au NPs (30pM)], the Apt(PDGF)/Apt(thr29)-Au NPs/Fib-Au NPs probe responds linearly to PDGF over the concentration range of 0.5-20nM with a correlation coefficient of 0.96. The limit of detection (LOD, signal-to-noise ratio=3) for each of the three PDGF isoforms is 0.3nM in the presence of bovine serum albumin at 100μM. When using the Apt(PDGF)/Apt(thr29)-Au NPs as selectors for the enrichment of PDGF and for the removal of interferences from cell media, the LOD for PDGF provided by this probe is 35pM. The present probe reveals that the concentration of PDGF in the three cell media is 230 (±20)pM, showing its advantages of simplicity, sensitivity, and specificity.     

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.     

Exonuclease III-based and gold nanoparticle-assisted DNA detection with dual signal amplification

Herein we report a sensitive electrochemical biosensor for DNA detection by making use of exonuclease III and probe DNA functionalized gold nanoparticles. While probe DNA P1 modified on a gold electrode surface can self-hybridize into a stem-loop structure with an exonuclease III-resistant 3' overhang end, in the presence of target DNA, P1 may also hybridize with the target DNA to form a duplex region. Therefore, exonuclease III may selectively digest P1 from its 3'-hydroxyl termini until the duplex is fully consumed. Since a single target DNA can trigger exonuclease III digestion of numerous P1 strands, the first signal amplification is achieved. On the other hand, since the digested P1, exposing its complementary sequence to probe DNA P2, can further hybridize with P2 that has been previously modified on the surface of gold nanoparticles, many nanoparticles loaded with numerous DNA strands are immobilized onto the electrode surface. Consequently, large amount of electroactive molecules [Ru(NH(3))(6)](3+) can bind with the DNA strands to produce an intense electrochemical response as the second signal amplification. Based on the studies with cyclic voltammetry (CV) and chronocoulometry (CC) techniques, the proposed biosensor can sensitively detect specific target DNA at a picomolar level with high specificity.     

Ultrasensitive electrochemical strategy for NT-proBNP detection with gold nanochains and horseradish peroxidase complex amplification

A novel electrochemical immunosensor for sensitive detection of cardiac biomarker N-terminal pro-B-type natriuretic peptide (NT-proBNP) is fabricated based on the nanostructural gold and carbon nanotubes composite as desirable platform for the capture antibodies immobilization and gold nanochains (AuNCs) and horseradish peroxidase (HRP) complex labeled secondary antibodies (AuNCs-HRP-Ab(2)) for signal amplification. The gold nanochains were prepared by the employment of L-ascorbic acid (AA) as a mediator and template. With the surface area enhancement by nanostructural gold functionalized carbon nanotubes composite, the amount of immobilized primary antibodies (Ab(1)) can be enhanced. More importantly, enhanced sensitivity can be achieved by introducing the multibioconjugates of AuNCs-HRP-Ab(2) onto the electrode surface through "sandwich" immunoreactions. The linear range extended from 0.02 to 100 ng/mL with a correlation coefficient of R=0.997 and a limit of detection reaching 6 pg/mL at a signal-to-noise ratio of 3:1, which is well below the commonly accepted concentration threshold (0.1 ng/mL) used in clinical diagnosis. The specificity, regeneration and stability test demonstrated the feasibility of the developed immunoassay, which gives the attractive characteristics to be a candidate for the detection of NT-proBNP and other proteins of interest in both fundamental and applied research.     

Sub-femtomolar electrochemical detection of DNA using surface circular strand-replacement polymerization and gold nanoparticle catalyzed silver deposition for signal amplification

A highly sensitive method was developed for detection of target DNA. This method combined circular strand-displacement polymerization (CSRP) with silver enhancement to achieve dual signal amplification. After molecular beacon (MB) hybridized with target DNA, the reporter gold nanoparticle (Au NPs) was attached to an electrode surface by hybridization between Au NP labeled primer and stem part of the MB to initiate a polymerization of DNA strand, which led to the release of target and another polymerization cycle. Thus the CSRP produced the multiplication of target-related reporter Au NPs on the surface. The Au NPs then catalyzed silver deposition for subsequent stripping analysis of silver. The dual signal amplification offered a dramatic enhancement of the stripping response. This signal could discriminate perfect matched target DNA from 1-base mismatch DNA. The dynamic range of the sequence-specific DNA detection was from 10(-16) to 10(-12)molL(-1) with a detection limit down to sub-femtomolar level. This proposed method exhibited an efficient amplification performance, and would open new opportunities for sensitive detection of other biorecognition events.     

Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme-linked aptamer

Here, we describe the fabrication of an electrochemical immunoglobulin E (IgE) aptasensor using enzyme-linked aptamer in the sandwich assay method and thionine as redox probe. In this protocol, 5′-amine-terminated IgE aptamer and thionine were covalently attached on glassy carbon electrode modified with carbon nanotubes/ionic liquid/chitosan nanocomposite. Furthermore, another IgE aptamer was modified with biotin and enzyme horseradish peroxidase (HRP), which attached to the aptamer via biotin–streptavidin interaction. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry were performed at each stage of the chemical modification process to confirm the resulting surface changes. The presence of IgE induces the formation of a double aptamer sandwich structure on the electrode, and the electrocatalytic reduction current of thionine in the presence of hydrogen peroxide was measured as the sensor response. Under optimized conditions and using differential pulse voltammetry as the measuring technique, the proposed aptasensor showed a low detection limit (6 pM) and high sensitivity (1.88 μA nM⁻¹). This aptasensor also exhibited good stability and high selectivity for IgE detection without an interfering effect of some other proteins such as bovine serum albumin (BSA) and lysozyme. The application of the aptasensor for IgE detection in human serum sample was also investigated. The proposed protocol is quite promising as an alternative sandwich approach for various protein assays.     

Simultaneous electrochemical detection of multiple biomarkers using gold nanoparticles decorated multiwall carbon nanotubes as signal enhancers

In this work, a novel sandwich-type electrochemical immunosensor has been developed for simultaneous detection of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) based on metal ion labels. Gold nanoparticles decorated multiwall carbon nanotubes (AuNPs@MWCNTs) were used as carriers to immobilize secondary antibodies and distinguishable electrochemical tags of Pb²⁺ and Cd²⁺ to amplify the signals. Due to the intrinsic property of high surface-to-volume ratio, the AuNPs@MWCNTs could load numerous secondary antibodies and labels. Therefore, the multiplexed immunoassay exhibited good sensitivity and selectivity. Experimental results revealed that this sandwich-type immunoassay displayed an excellent linear response, with a linear range of 0.01 to 60 ng mL^–1 for both analytes and detection limits of 3.0 pg mL^–1 for CEA and 4.5 pg mL^–1 for AFP (at a signal-to-noise ratio of 3). The method was successfully applied for the determination of AFP and CEA levels in clinical serum samples.     

Highly sensitive electrochemical detection of immunospecies based on combination of Fc label and PPD film/gold nanoparticle amplification

A highly sensitive electrochemical immunoassay strategy based on the combination of ferrocene (Fc) label and poly(o-phenylenediamine) (PPD) film/gold nanoparticle (GNP) amplification for the detection of immunospecies is proposed using human IgG as the model analyte. A gold electrode is firstly modified with an electropolymerized film of poly(o-phenylenediamine), which provides a stable matrix with abundant amino-groups for the fabrication of sensing interface. Using glutaraldehyde as a cross-linker, cystamine is coupled onto the modified electrode. Subsequently, gold nanoparticle monolayer is assembled onto the resulting surface. Making use of the unique properties of gold nanoparticles, antibodies can be self-assembled onto the surface-confined gold nanoparticles via amine-Au affinity with a high loading amount and reserve high immunological activity. After the introduction of model analyte, the ferrocene (Fc)-labeled antibody is immobilized on the sensing interface by antibody-antigen specific reaction, resulting in a redox current signal. The peak current is proportional to the amount of the analyte. Under the optimized experimental conditions, the proposed sensing strategy provides a wide linear dynamic range from 25 to 1000pg/mL with a low detection limit of 10pg/mL. In addition, good reproducibility, high selectivity and stability are achieved. In particular, the extremely high stability of both poly(o-phenylenediamine) and gold nanoparticle monolayer allows the designed biosensing interface to withstand harsh regeneration treatment, making it reusable.     

Enzyme-free signal amplification for electrochemical detection of Mycobacterium lipoarabinomannan antibody on a disposable chip

Vesicular exocytosis plays an important role in many physiological processes. The dense-core vesicles release of chromaf?n cells is a suitable model for the presynaptic process in neurosecretory cells. In this study, light-addressable potentiometric sensor (LAPS) was introduced as a label-free recording method for vesicle release by the local extracellular acidification. The chromaf?n cells are directly cultured on the sensor surface. After cells and LAPS hybrid system is established, the events of vesicular exocytosis are recorded. Protons stored in the vesicles and co-released with transmitters, induced a brief acidic shifts in the cell-sensor cleft. Under the stimulation of the KCl and acetylcholine (Ach), the signals presented the different amplitude and exocytosis rate, and reflected the specific features of the exocytosis. The result indicates that neurosecretory cell-based biosensor will provide a useful platform for neurosecretion mechanism research by monitoring the exocytotic activities with extracellular acidification sensing.     

A DNA intercalation-based electrochemical method for detection of Chlamydia trachomatis utilizing peroxidase-catalyzed signal amplification

A sensitive electrochemical DNA detection method for the diagnosis of sexually transmitted disease (STD) caused by Chlamydia trachomatis was developed. The method utilizes a DNA-intercalating agent and a peroxidase promoted enzymatic precipitation reaction and involves the following steps. After hybridization of the target C. trachomatis gene with an immobilized DNA capture probe on a gold electrode surface, the biotin-tagged DNA intercalator (anthraquinone) was inserted into the resulting DNA duplex. Subsequently, the polymeric streptavidin/peroxidase complex was applied to the biotin-decorated electrode. Peroxidase catalyzed 4-chloronaphthol to produce insoluble product, which is precipitated on the electrode surface in the presence of hydrogen peroxide. Cyclic voltammograms with the gold electrode exhibited a peak current of ferrocenemethanol in electrolyte, which decreased in a proportional way to increasing concentration of target DNA owing to insulation of electrode surface by the growing insoluble precipitate. Using this strategy, we were able to detect picomolar concentrations of C. trachomatis gene in a sample taken from a real patient.     

Highly sensitive gold nanoparticles biosensor chips modified with a self-assembled bilayer for detection of Con A

In this paper, an improved method for detection of Concanavalin A (Con A) with label-free optical biosensors is reported. 1-Dodecanethiol (DDT) was self-assembled onto gold nanoparticles which were deposited on glass slides, and then glycolipid molecules were inserted into dodecanethiol by physical interactions only. The recognition between Con A and carbohydrate was observed by UV-vis spectrophotometry. The absorption spectrum shifted when Con A was bound to the sugar residues of glycolipids immobilized onto nanogold slides, while almost no spectrum change was observed when another nonspecific protein molecule met the nanogold slides. The self-assembled bilayer on nanogold substrates had very high sensitivity for Con A, the minimum detection concentration of Con A can be down to 0.1 nM. In addition to the ultra sensitivity for investigating carbohydrate-lectin interaction, the self-assembled bilayer structure, is expected to replace many receptors which require time-consuming organic syntheses for the fixation to the transducer. The simplicity and sensitivity of this biosensor architecture once again show the prospect of nanogold application in biosensor.     

Silica nanoparticles based label-free aptamer hybridization for ATP detection using hoechst33258 as the signal reporter

In this work, we have developed a simple and sensitive method for ATP detection using silica nanoparticles (NPs) as the platform and hoechst33258 as the signal reporter. The ATP-binding aptamers hybridize with the probe DNA (DNA(p)) immobilized NPs to form the aptamer/DNA(p) duplex on the NPs surface. The conformational change of the aptamer leads to the decrease of the aptamer/DNA(p) duplex on the NPs due to the ATP-binding aptamer switches its structure from the aptamer/DNA(p) duplex to the aptamer/target complex in the presence of ATP. ATP detection can be easily realized by separating the silica nanoparticles and adding the hoechst33258 of intercalating to aptamer/DNA(p) (dsDNA). Good selectivity between ATP and CTP, GTP or UTP has been demonstrated, which is due to the specific recognition between ATP aptamer and ATP. The K(d) was estimated to be ～1mM from 0 to 4mM and a liner response was observed from 0 to 0.2mM with a detection limit of ～20μM. Compared with other methods, the carboxyl-modified silica nanoparticles (～60nm) prepared by the reverse microemulsion method can serve as a stable and sensitive sensor platform because of their smaller size and facile conjugation with amine-containing molecules. In addition, the high sensitivity and selectivity of hoechst33258 was employed for the ssDNA and dsDNA determination, which takes advantage of the label-free aptamer and lower cost.     

Combined tyramide signal amplification and quantum dots for sensitive and photostable immunofluorescence detection.

Conventional immunofluorescence detection of biologically relevant proteins and antigens in tissue sections is often limited by relatively weak signals that fade rapidly on illumination. We have developed an immunohistochemical protocol that combines the sensitivity of tyramide signal amplification with the photostability of quantum dots to overcome these limitations. This simple method provides a sensitive and stable fluorescence immunohistochemical alternative to standard chromogen detection.     

Colorimetric detection of bisphenol A based on unmodified aptamer and cationic polymer aggregated gold nanoparticles

In this study, a colorimetric method was exploited to detect bisphenol A (BPA) based on BPA-specific aptamer and cationic polymer-induced aggregation of gold nanoparticles (AuNPs). The principle of this assay is very classical. The aggregation of AuNPs was induced by the concentration of cationic polymer, which is controlled by specific recognition of aptamer with BPA and the reaction of aptamer and cationic polymer forming “duplex” structure. This method enables colorimetric detection of BPA with selectivity and a detection limit of 1.50 nM. In addition, this colorimetric method was successfully used to determine spiked BPA in tap water and river water samples.     

纳米金探针在基因检测中的应用

传统的核酸分析中常采用放射性元素、荧光色素以及酶标记等基因探针,这些探针都存在着一些不足之处。近年来,纳米金探针作为一种新型的基因探针,己引起了广泛的关注。该探针具有优良的光谱特征和光化学稳定性,对核酸的非特异吸附性小,与核酸等生物大分子结合后不改变生物分子的活性。将纳米金探针用于基因检测,具有操作简便、快速、安全、实验成本低等优点。本文就纳米金探针的发展过程、纳米金探针的制备、检测原理及其在基因分析中的应用等几个方面作了系统而全面地概述,同时介绍了纳米金探针的最新研究进展,并对其发展前景作了简要评述。