Magnetic bead-based chemiluminescent metal immunoassay with a colloidal gold label

A novel, sensitive chemiluminescent (CL) immunoassay has been developed by taking advantage of a magnetic separation/mixing process and the amplification feature of colloidal gold label. First, the sandwich-type complex is formed in this protocol by the primary antibody immobilized on the surface of magnetic beads, the antigen in the sample, and the second antibody labeled with colloidal gold. Second, a large number of Au3+ ions from each gold particle anchored on the surface of magnetic beads are released after oxidative gold metal dissolution and then quantitatively determined by a simple and sensitive Au3+-catalyzed luminol CL reaction. Third, this protocol is evaluated for a noncompetitive immunoassay of a human immunoglobulin G, and a concentration as low as 3.1 x 10(-12) M is determined, which is competitive with colloidal gold-based anodic stripping voltammetry (ASV), colorimetric ELISA, or immunoassays based on fluorescent europium chelate labels. The high performance of this protocol is related to the sensitive CL determination of Au3+ ion (detection limit of 2 x 10(-10) M), which is 25 times higher than that by ASV at a single-use carbon-based screen-printed electrode. From the analytical chemistry point of view, this protocol will be quite promising for numerous applications in immunoassay and DNA hybridization.     

A renewable electrochemical magnetic immunosensor based on gold nanoparticle labels

A particle-based renewable electrochemical magnetic immunosensor was developed by using magnetic beads and gold nanoparticle labels. Anti-IgG antibody-modified magnetic beads were attached to a renewable carbon paste transducer surface by magnet that was fixed inside the sensor. Gold nanoparticle labels were capsulated to the surface of magnetic beads by sandwich immunoassay. Highly sensitive electrochemical stripping analysis offers a simple and fast method to quantify the capatured gold nanoparticle tracers and avoid the use of an enzyme label and substrate. The stripping signal of gold nanoparticles is related to the concentration of target IgG in the sample solution. A transmission electron microscopy image shows that the gold nanoparticles were successfully capsulated to the surface of magnetic beads through sandwich immunoreaction events. The parameters of immunoassay, including the loading of magnetic beads, the amount of gold nanoparticle conjugate, and the immunoreaction time, were optimized. The detection limit of 0.02 microg ml(-1) of IgG was obtained under optimum experimental conditions. Such particle-based electrochemical magnetic immunosensors could be readily used for simultaneous parallel detection of multiple proteins by using multiple inorganic metal nanoparticle tracers and are expected to open new opportunities for disease diagnostics and biosecurity.     

DNA-based hybridization chain reaction for amplified bioelectronic signal and ultrasensitive detection of proteins

This work reports a novel electrochemical immunoassay protocol with signal amplification for determination of proteins (human IgG here used as a model target analyte) at an ultralow concentration using DNA-based hybridization chain reaction (HCR). The immuno-HCR assay consists of magnetic immunosensing probes, nanogold-labeled signal probes conjugated with the DNA initiator strands, and two different hairpin DNA molecules. The signal is amplified by the labeled ferrocene on the hairpin probes. In the presence of target IgG, the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles. The carried DNA initiator strands open the hairpin DNA structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Numerous ferrocene molecules are formed on the neighboring probe, each of which produces an electrochemical signal within the applied potentials. Under optimal conditions, the immuno-HCR assay presents good electrochemical responses for determination of target IgG at a concentration as low as 0.1 fg mL(-1). Importantly, the methodology can be further extended to the detection of other proteins or biomarkers.     

Ultrasensitive electrochemical detection of nucleic acids by template enhanced hybridization followed with rolling circle amplification

An ultrasensitive protocol for electrochemical detection of DNA is designed with quantum dots (QDs) as a signal tag by combining the template enhanced hybridization process (TEHP) and rolling circle amplification (RCA). Upon the recognition of the molecular beacon (MB) to target DNA, the MB hybridizes with assistants and target DNA to form a ternary 'Y-junction'. The target DNA can be dissociated from the structure under the reaction of nicking endonuclease to initiate the next hybridization process. The template enhanced MB fragments further act as the primers of the RCA reaction to produce thousands of repeated oligonucleotide sequences, which can bind with oligonucleotide functionalized QDs. The attached signal tags can be easily read out by square-wave voltammetry after dissolving with acid. Because of the cascade signal amplification and the specific TEHP and RCA reaction, this newly designed protocol provides an ultrasensitive electrochemical detection of DNA down to the attomolar level (11 aM) with a linear range of 6 orders of magnitude (from 1 × 10(-17) to 1× 10(-11) M) and can discriminate mismatched DNA from perfect matched target DNA with high selectivity. The high sensitivity and specificity make this method a great potential for early diagnosis in gene-related diseases.     

Enhanced electrochemical detection of DNA hybridization with carbon nanotube modified paste electrode

A novel electrochemical genesensor using twice hybridization enhancement of gold nanoparticles based on carbon paste modified electrode is described. The carbon nanotube modified carbon paste electrode (CNTPE) and mesoporous molecular sieve SBA-15 modified carbon paste electrode (MSCPE) were investigated. The assay relies on the immobilization of streptavidin-biotin labeled target oligonucleotides onto the electrode surface and its hybridization to the gold nanoparticle-labeled DNA probe. After twice hybridization enhanced connection of gold nanoparticles to the hybridized system, the differential pulse voltammetry (DPV) signal of total gold nanoparticles was monitored. It was found that the adsorption of oligonucleotide and hybridized DPV signal on CNTPE were both enhanced in comparison with that of pure carbon paste electrode (CPE). But this trend was reverse on MSCPE. The DPV detection of twice hybridized gold nanoparticles indicated that the sensitivity of the genesensor enhanced about one order of magnitude compared with one-layer hybridization. One-base mismatched DNA and complementary DNA could be distinguished clearly. However, no distinct advantage of MSCPE over CPE was found.     

Triple signal amplification of graphene film, polybead carried gold nanoparticles as tracing tag and silver deposition for ultrasensitive electrochemical immunosensing

A triple signal amplification strategy was designed for ultrasensitive immunosensing of cancer biomarker. This strategy was achieved using graphene to modify immunosensor surface for accelerating electron transfer, poly(styrene-co-acrylic acid) microbead (PSA) carried gold nanoparticles (AuNPs) as tracing tag to label signal antibody (Ab(2)) and AuNPs induced silver deposition for anodic stripping analysis. The immunosensor was constructed by covalently immobilizing capture antibody on chitosan/electrochemically reduced graphene oxide film modified glass carbon electrode. The in situ synthesis of AuNPs led to the loading of numerous AuNPs on PSA surface and convenient labeling of the tag to Ab(2). With a sandwich-type immunoreaction, the AuNPs/PSA labeled Ab(2) was captured on the surface of an immunosensor to further induce a silver deposition process. The electrochemical stripping signal of the deposited silver nanoparticles in KCl was used to monitor the immunoreaction. The triple signal amplification greatly enhanced the sensitivity for biomarker detection. The proposed method could detect carcinoembryonic antigen with a linear range of 0.5 pg mL(-1) to 0.5 ng mL(-1) and a detection limit down to 0.12 pg mL(-1). The immunosensor exhibited good stability and acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics.     

Electrochemical DNA biosensor based on conducting polyaniline nanotube array

Most of the recent developments in ultrasensitive detection of nucleic acid are based on the gold nanoparticles and carbon nanotubes as a medium of signal amplification. Here, we present an ultrasensitive electrochemical nucleic acid biosensor using the conducting polyaniline (PANI) nanotube array as the signal enhancement element. The PANI nanotube array of a highly organized structure was fabricated under a well-controlled nanoscale dimension on the graphite electrode using a thin nanoporous layer as a template, and 21-mer oligonucleotide probes were immobilized on these PANI nanotubes. In comparison with gold nanoparticle- or carbon nanotube-based DNA biosensors, our PANI nanotube array-based DNA biosensor could achieve similar sensitivity without catalytic enhancement, purification, or end-opening processing. The electrochemical results showed that the conducting PANI nanotube array had a signal enhancement capability, allowing the DNA biosensor to readily detect the target oligonucleotide at a concentration as low as 1.0 fM (approximately 300 zmol of target molecules). In addition, this biosensor demonstrated good capability of differentiating the perfect matched target oligonucleotide from one-nucleotide mismatched oligonucleotides even at a concentration of 37.59 fM. This detection specificity indicates that this biosensor could be applied to single-nucleotide polymorphism analysis and single-mutation detection.     

Electrochemical detection of DNA hybridization using biometallization

We demonstrate the amplified detection of a target DNA based on the enzymatic deposition of silver. In this method, the target DNA and a biotinylated detection DNA probe hybridize to a capture DNA probe tethered onto a gold electrode. Neutravidin-conjugated alkaline phosphatase binds to the biotin of the detection probe on the electrode surface and converts the nonelectroactive substrate of the enzyme, p-aminophenyl phosphate, into the reducing agent, p-aminophenol. The latter, in turn, reduces metal ions in solutions leading to deposition of the metal onto the electrode surface and DNA backbone. This process, which we term biometallization, leads to a great enhancement in signal due to the accumulation of metallic silver by a catalytically generated enzyme product and, thus, the electrochemical amplification of a biochemically amplified signal. The anodic stripping current of enzymatically deposited silver provides a measure of the extent of hybridization of the target oligomers. This biometallization process is highly sensitive, detecting as little as 100 aM (10 zmol) of DNA. We also successfully applied this method to the sequence-selective discrimination between perfectly matched and mismatched target oligonucleotides including a single-base mismatched target.     

Gold nanoparticle-based quantitative electrochemical detection of amplified human cytomegalovirus DNA using disposable microband electrodes

An electrochemical DNA detection method has been developed for the sensitive quantification of an amplified 406-base pair human cytomegalovirus DNA sequence (HCMV DNA). The assay relies on (i) the hybridization of the single-stranded target HCMV DNA with an oligonucleotide-modified Au nanoparticle probe, (ii) followed by the release of the gold metal atoms anchored on the hybrids by oxidative metal dissolution, and (iii) the indirect determination of the solubilized AuIII ions by anodic stripping voltammetry at a sandwich-type screen-printed microband electrode (SPMBE). Due to the enhancement of the AuIII mass transfer by nonlinear diffusion during the electrodeposition time, the SPMBE allows the sensitive determination of AuIII in a small volume of quiescent solution. The combination of the sensitive AuIII determination at a SPMBE with the large number of AuIII released from each gold nanoparticle probe allows detection of as low as 5 pM amplified HCMV DNA fragment.     

Signal amplification strategy using gold/N ‐trimethyl chitosan/iron oxide magnetic composite nanoparticles as a tracer tag for high‐sensitive electrochemical detection

This study presents a novel signal amplification method for high‐sensitive electrochemical immunosensing. Gold (Au)/N ‐trimethyl chitosan (TMC)/iron oxide (Fe₃ O₄) (shell/shell/core) nanocomposite was used as a tracing tag to label antibody. The tag was shown to be capable of amplifying the recognition signal by high‐density assembly of Au nanoparticles (NPs) on TMC/Fe₃ O₄ particles. The remarkable conductivity of AuNPs provides a feasible pathway for electron transfer. The method was found to be simple, reliable and capable of high‐sensitive detection of human serum albumin as a model, down to 0.2 pg/ml in the range of 0.25–1000 pg/ml. Findings of the present study would create new opportunities for sensitive and rapid detection of various analytes.Inspec keywords: gold, filled polymers, conducting polymers, iron compounds, magnetic particles, nanoparticles, nanocomposites, nanosensors, electrochemical sensors, proteins, molecular biophysics, biomagnetism, biosensorsOther keywords: signal amplification strategy, gold‐N‐trimethyl chitosan‐iron oxide magnetic composite nanoparticles, tracer tag, high‐sensitive electrochemical detection, high‐sensitive electrochemical immunosensing, antibody, high‐density assembly, AuNP conductivity, electron transfer, human serum albumin, FeO‐Au     

Electrochemical coding for multiplexed immunoassays of proteins

An electrochemical immunoassay protocol for the simultaneous measurements of proteins, based on the use of different inorganic nanocrystal tracers is described. The multiprotein electrical detection capability is coupled to the amplification feature of electrochemical stripping transduction (to yield fmol detection limits) and with an efficient magnetic separation (to minimize nonspecific adsorption effects). The multianalyte electrical sandwich immunoassay involves a dual binding event, based on antibodies linked to the nanocrystal tags and magnetic beads. Carbamate linkage is used for conjugating the hydroxyl-terminated nanocrystals with the secondary antibodies. Each biorecognition event yields a distinct voltammetric peak, whose position and size reflects the identity and level, respectively, of the corresponding antigen. The concept is demonstrated for a simultaneous immunoassay of beta(2)-microglobulin, IgG, bovine serum albumin, and C-reactive protein in connection with ZnS, CdS, PbS, and CuS colloidal crystals, respectively. These nanocrystal labels exhibit similar sensitivity. Such electrochemical coding could be readily multiplexed and scaled up in multiwell microtiter plates to allow simultaneous parallel detection of numerous proteins or samples and is expected to open new opportunities for protein diagnostics and biosecurity.     

Instantaneous derivatization technology for simultaneous and homogeneous determination of multiple DNA targets

There are potential advantages, in terms of simplicity and speed, for detecting DNA hybridization steps directly without using any external labels, especially for the multiplexed assays. In the current paper, we describe the use of a carrier-resolved label-free multiplexed assay for the simultaneous detection of multiple DNA targets. Herein we demonstrate that this protocol, using three homogeneous carriers thermosensitive poly(N-isopropylacrylamide), polystyrene beads, and magnetic beads, respectively, for simultaneous determination of three short DNA fragments specific to hepatitis B virus. Briefly, one hybridization occurs between a mixture of three different capture probe DNAs immobilized onto three carriers and three targets in a single vessel, and then chemiluminescence (CL) detection proceeds via an instantaneous derivatization reaction between the specific CL reagent 3,4,5-trimethoxylphenylglyoxal (TMPG) and the guanine nucleotide-rich regions within the target DNA. An excellent linearity is found within the range between 0.1 and 6.0 pmol with the lowest detection limit of 100 fmol. In contrast to current encoding strategies, every hybridization signal for the corresponding DNA target in our protocol is uniquely immobilized onto one carrier vehicle with a unique and intrinsic physical-chemical signature. Moreover, an instantaneous derivatization reaction is employed for the label-free determination of three targets in a single vessel. In addition, a simple CL setup is employed to read the carrier code instead of an expensive and complicated flow cytometer or imaging system commonly used for multiplexed assays. Further signal amplification is achieved by employing three amplified DNAs for second hybridization, which include a guanine nucleobase-rich sequence domain for the generation of light and an additional tethered nucleic acid domain complementary with one of the target DNA as an amplification platform. Such simple amplified CL transduction allows detection of DNA targets down to the 15-fmol level. This new protocol also provided a good capability in discriminating perfectly complementary DNA from single-base mismatches and noncomplementary sequences. Overall, the protocol described here may have value in a variety of clinical, environmental, and biodefense applications for which the accurate quantitative analysis of multiple DNA targets is desired.     

Open bridge-structured gold nanoparticle array for label-free DNA detection

We focused on changes in the electrical property of the open bridge-structured gold nanoparticles array consisting of 46-nm parent and 12-nm son gold nanoparticles by hybridization and applied it for a simple electrical DNA detection. Since a target DNA of a 24-mer oligonucleotide was added to the probe DNA modified 12-nm Au nanoparticles, which was arranged on the gap between the 46-nm Au particles, the response was read by an electrical readout system. Even in a simple measuring method, we obtained a rapid response to the cDNA with a high S/N ratio of 30 over a wide concentration range and a detection limit of 5.0 fmol. Moreover, the array discriminated 1-base mismatches, regardless of their location in the DNA sequence, which enabled us to detect single-nucleotide polymorphism, which is one of the important diagnoses, without any polymerase chain reaction amplification, sophisticated instrumentation, or fluorescent labeling through an easy-to-handle electrical readout system.     

Indium microrod tags for electrochemical detection of DNA hybridization

The preparation and advantages of indium microrod tracers for solid-state electrochemical detection of DNA hybridization are described. The cylindrical metal particles were prepared by a template-directed electrochemical synthetic route involving plating of indium into the pores of a host membrane. The linear relationship between the charge passed during the preparation and the resulting particle size allows tailoring of the sensitivity of the electrical DNA assay. The resulting micrometer-long rods thus offer a greatly lower detection limit (250 zmol), as compared to common bioassays' spherical nanoparticle tags. Indium offers a very attractive electrochemical stripping behavior and is not normally present in biological samples or reagents. Solid-state derivative-chronopotentiometric measurements of the indium tracer have been realized through a "magnetic" collection of the DNA-linked particle assembly onto a thick-film electrode transducer. Factors affecting the performance, including the preparation of the microrods and pretreatment of the transducer surface, were evaluated and optimized. The resulting protocol offers great promise for other affinity bioassays, as well as for electrical coding and identification (through the plating of different metal markers and of multimetal redox-encoded tags).     

Proteolytic Fluorescent Signal Amplification on Gold Nanoparticles for a Highly Sensitive and Rapid Protease Assay

A new strategy for highly sensitive and rapid protease assay is developed by mediating proteolytic formation of oligonucleotide duplexes and using the duplexes for signal amplification. In the presence of matrix metalloprotease‐2 (MMP‐2), fragmentation of the intact DNA–peptide on gold nanoparticles (GNP) by hydrolytic cleavage of a peptide bond within the substrate allows diffusion of DNA away from the GNP and the formation of a DNA/RNA heteroduplex, leading to digestion of RNA by RNase H. Because of the high quenching efficacy of GNP to the fluorophore in RNA and multiple digestions of the RNA, the fluorescence signal recovery is amplified. This method permits the assessment of the activity of MMP‐2 at concentrations as low as 10 pM within 4 h. Compared with the reported protease nanosensors using quantum dots, GNP, and magnetic nanoparticles with the same peptide sequence, the assay time of this method is sixfold faster and the limit of detection is 100‐fold more sensitive. The formulations for proteolytic formations of oligonucleotides duplexes for signal amplification on GNP could lead to the development of more sensitive and rapid protease assay techniques, thus extending the role of proteases as therapeutic targets and disease indicators.     

Nano‐biosensor based on reduced graphene oxide and gold nanoparticles,for detection of phenylketonuria‐associated DNA mutation

Phenylketonuria (PKU)‐associated DNA mutation in newborn children can be harmful to his health and early detection is the best way to inhibit consequences. A novel electrochemical nano‐biosensor was developed for PKU detection, based on signal amplification using nanomaterials, e.g. gold nanoparticles (AuNPs) decorated on the reduced graphene oxide sheet on the screen‐printed carbon electrode. The fabrication steps were checked by field emission scanning electron microscope imaging as well as cyclic voltammetry analysis. The specific alkanethiol single‐stranded DNA probes were attached by self‐assembly methodology on the AuNPs surface and Oracet blue was used as an intercalating electrochemical label. The results showed the detection limit of 21.3 fM and the dynamic range of 80–1200 fM. Moreover, the selectivity results represented a great specificity of the nano‐biosensor for its specific target DNA oligo versus other non‐specific sequences. The real sample simulation was performed successfully with almost no difference than a synthetic buffer solution environment.Inspec keywords: biosensors, nanosensors, nanoparticles, graphene compounds, gold, nanomedicine, DNA, molecular biophysics, biomedical equipment, electrochemical sensors, electrochemical electrodes, field emission scanning electron microscopy, voltammetry (chemical analysis), self‐assembly, biochemistryOther keywords: reduced graphene oxide, gold nanoparticles, phenylketonuria‐associated DNA mutation, newborn children, electrochemical nanobiosensor, signal amplification, nanomaterials, reduced graphene oxide sheet, screen‐printed carbon electrode, field emission scanning electron microscopy imaging, cyclic voltammetry, alkanethiol single‐stranded DNA probes, self‐assembly methodology, Oracet blue, intercalating electrochemical label, Au‐CO     

"Multicolor" electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes

Labeling of oligonucleotide reporter probes (RP) with electroactive markers has frequently been utilized in electrochemical detection of DNA hybridization. Osmium tetroxide complexes with tertiary amines (Os,L) bind covalently to pyrimidine (predominantly thymine) bases in DNA, forming stable, electrochemically active adducts. We propose a technique of electrochemical "multicolor" DNA coding based on RP labeling with Os,L markers involving different nitrogenous ligands (such as 2,2' -bipyridine, 1,10-phenanthroline derivatives or N,N,N',N'-tetramethylethylenediamine). At carbon electrodes the Os,L-labeled RPs produce specific signals, with the potentials of these differing depending on the ligand type. When using Os,L markers providing sufficiently large differences in their peak potentials, parallel analysis of multiple target DNA sequences can easily be performed via DNA hybridization at magnetic beads followed by voltammetric detection at carbon electrodes. Os,L labeling of oligonucleotide probes comprising a segment complementary to target DNA and an oligo(T) tail (to be modified with the osmium complex) does not require any organic chemistry facilities and can be achieved in any molecular biological laboratory. We also for the first time show that this technology can be used for labeling of oligonucleotide probes hybridizing with target DNAs that contain both purine and pyrimidine bases.     

An electrochemical metalloimmunoassay based on a colloidal gold label

A novel, sensitive electrochemical immunoassay has been developed using a colloidal gold label that, after oxidative gold metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a single-use carbon-based screen-printed electrode (SPE). The use of disposable electrodes allows for simplified measurement in 35 microL of solution. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) and a concentration as low as 3 x 10(-12) M was determined, which is competitive with colorimetric ELISA or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is related to the sensitive ASV determination of gold(III) at a SPE (detection limit of 5 x 10(-9) M) and to the release of a large number of gold(III) ions from each gold particle anchored on the immunocomplex (e.g., 1.7 x 10(5) gold atoms are theoretically contained in a 18-nm spherical gold particle).     

Magnetic Bead/Gold Nanoparticle Double‐Labeled Primers for Electrochemical Detection of Isothermal Amplified Leishmania DNA

A novel methodology for the isothermal amplification of Leishmania DNA using labeled primers combined with the advantages of magnetic purification/preconcentration and the use of gold nanoparticle (AuNP) tags for the sensitive electrochemical detection of such amplified DNA is developed. Primers labeled with AuNPs and magnetic beads (MBs) are used for the first time for the isothermal amplification reaction, being the amplified product ready for the electrochemical detection. The electrocatalytic activity of the AuNP tags toward the hydrogen evolution reaction allows the rapid quantification of the DNA on screen‐printed carbon electrodes. Amplified products from the blood of dogs with Leishmania (positive samples) are discriminated from those of healthy dogs (blank samples). Quantitative studies demonstrate that the optimized method allows us to detect less than one parasite per microliter of blood (8 × 10^?3 parasites in the isothermal amplification reaction). This pioneering approach is much more sensitive than traditional methods based on real‐time polymerase chain reaction (PCR), and is also more rapid, cheap, and user‐friendly.     

A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles

Using a fluorescence-based method, we have determined the number of thiol-derivatized single-stranded oligonucleotides bound to gold nanoparticles and their extent of hybridization with complementary oligonucleotides in solution. Oligonucleotide surface coverages of hexanethiol 12-mer oligonucleotides on gold nanoparticles (34 +/- 1 pmol/cm2) were significantly higher than on planar gold thin films (18 +/- 3 pmol/cm2), while the percentage of hybridizable strands on the gold nanoparticles (1.3 +/- 0.3 pmol/cm2, 4%) was lower than for gold thin films (6 +/- 2 pmol/cm2, 33%). A gradual increase in electrolyte concentration over the course of oligonucleotide deposition significantly increases surface coverage and consequently particle stability. In addition, oligonucleotide spacer sequences improve the hybridization efficiency of oligonucleotide-modified nanoparticles from approximately 4 to 44%. The surface coverage of recognition strands can be tailored using coadsorbed diluent oligonucleotides. This provides a means of indirectly controlling the average number of hybridized strands per nanoparticle. The work presented here has important implications with regard to understanding interactions between modified oligonucleotides and metal nanoparticles, as well as optimizing the sensitivity of gold nanoparticle-based oligonucleotide detection methods.