Simultaneous electrochemical immunoassay of three liver cancer biomarkers using distinguishable redox probes as signal tags and gold nanoparticles coated carbon nanotubes as signal enhancers

A novel strategy for simultaneous determination of three liver cancer biomarkers based on three redox labels with distinct voltammetric peaks was described. Gold nanoparticles coated carbon nanotubes were used as carriers to immobilize redox probes labeled antibodies and to amplify the signals.     

Polymerization-assisted signal amplification for electrochemical detection of biomarkers

We present a novel immunosensor by using polymerization-assisted signal amplification strategy coupled with electrochemical detection. A sandwich immunoassay process was used to immobilize a polymerization reaction center, the initiator-conjugated polyclonal prostate specific antigen (PSA) or polyclonal carcinoembryonic antigen (CEA) antibodies on the surface of the electrode. Activator generated electron transfer for atom transfer radical polymerization (AGET ATRP) subsequently triggered the local accumulation of glycidyl methacrylate (GMA) monomers. Growth of long chain polymers provided excess epoxy groups for electrochemical tags aminoferrocene (FcNH(2)) coupling, which in turn significantly increased the loading of the signal molecules and enhanced the electrochemical readouts. The detection limit was ～0.14 pg mL(-1) for PSA and ～0.10 pg mL(-1) for CEA in PBS buffers. The proposed immunosensor was highly sensitive, selective and has a good match to the clinical electrochemiluminescent method. This suggested that the polymerization-assisted immunosensing strategy could be used as an effective method to significantly enhance signal output of the sandwich immunoassays and acted as a promising platform for the clinical screening of cancer biomarkers.     

Hierarchical dendritic gold microstructure-based aptasensor for ultrasensitive electrochemical detection of thrombin using functionalized mesoporous silica nanospheres as signal tags

A sensitive electrochemical approach for the detection of thrombin was designed by using densely packed hierarchical dendritic gold microstructures (HDGMs) with secondary and tertiary branches as matrices, and thionine-functionalized mesoporous silica nanospheres as signal tags. To prepare the signal tags, the positively charged thionine (as an indicator) was initially adsorbed onto the mesoporous silica nanoparticles (MSNs). Then [AuCl₄]⁻ ions were in situ reduced on the thionine-modified MSNs by ascorbic acid to construct nanogold-decorated MSNs (GMSNs). The formed GMSNs were employed as label of the aminated aptamers. The assay was carried out in PBS, pH 7.4 with a sandwich-type assay mode by using the assembled thionine in the GMSNs as indicators. Compared with the pure silica nanoparticles, mesoporous silica could provide a larger surface for the immobilization of biomolecules and improve the sensitivity of the aptasensor. Under optimal conditions, the electrochemical aptasensors exhibited a wide linear range from 0.001 to 600 ng mL⁻¹ (i.e. 0.03 pM to 0.018 μM thrombin) with a low detection limit (LOD) of 0.5 pg mL⁻¹ (≈15 fM) thrombin at 3σ. No obvious non-specific adsorption was observed during a series of analyses to detect target analyte. The precision, selectivity and stability of the aptasensors were acceptable. Importantly, the methodology was evaluated with thrombin spiked samples in blank fetal calf serum, and the recoveries were 94.2–112%, indicating an exciting potential for thrombin detection.     

Copper-enhanced gold nanoparticle tags for electrochemical stripping detection of human IgG

We demonstrate herein a novel electrochemical protocol for quantification of human IgG based on the precipitation of copper on gold nanoparticle tags and a subsequent electrochemical stripping detection of the dissolved copper. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction. Goat anti-human IgG was immobilized on the wells of microtiter plates. The human IgG analyte was first captured by the primary antibody and then sandwiched by secondary antibody labeled with gold nanoparticles. The copper enhancer solution was then added to deposite copper on the gold nanoparticle tags. After dissolved with HNO₃, the released copper ions were then quantified by ASV. The detection limit is 0.5 ng/mL by 3σ-rule. In order to investigate the feasibility of the newly developed technique to be applied for clinical analysis, several standard human IgG serum specimens were also examined by the method. To our knowledge, the copper enhancing procedure is the first time to be developed for immunoassay. The new strategy of using copper-enhanced gold nanoparticle tags for electrochemical stripping detection holds great promise for immunoassay and DNA detection.     

Breath biosensing: using electrochemical enzymatic sensors for detection of biomarkers in human breath

Detection of biomarkers for disease by noninvasive methods is critical for the early diagnosis and screening of disease, enabling prompt treatment. Breath biosensors are a viable option as the exhaled breath contains several biomarkers linked to lung cancer, oxidative stress, diabetes, and other diseases. Breath analysis has been achieved by advanced analytical techniques such as gas chromatography and infrared spectroscopy. However, electrochemical enzymatic breath biosensors offer a cost-effective, sensitive platform for biomarker detection without complex analysis and interpretation by trained laboratory personnel. This review aims to summarize recent advances in the field of electrochemical enzymatic breath biosensors and offer future opportunities from other applications of nonelectrochemical enzymatic breath biosensors.     

Poly(o-phenylenediamine)-carried nanogold particles as signal tags for sensitive electrochemical immunoassay of prolactin

A novel class of redox-active molecular tags, poly(o-phenylenediamine)-carried nanogold particles (GPPDs), was first synthesized and functionalized with horseradish peroxidase-anti-prolactin conjugates (HRP-anti-PRL). Thereafter, a specific sandwich-type electrochemical immunoassay was designed for determination of prolactin (PRL) by using GPPD-labeled HRP-anti-PRL conjugates as molecular tags on anti-PRL antibody-modified glassy carbon electrode. Compared with pure gold nanoparticles and poly(o-phenylenediamine) microspheres, the as-prepared GPPDs increased the surface coverage of the nanostructures, and enhanced the immobilization amount of biomolecules. Several labeling protocols compromising GPPD-labeled HRP-anti-PRL, nanogold particles-labeled HRP-anti-PRL and poly(o-phenylenediamine) microspheres-labeled HRP-anti-PRL, were investigated for detection of PRL, and improved analytical features were obtained with the GPPD-based strategy. With the GPPD labeling method, dependence of the electrochemical signals on the incubation time and pH of the assay solution were also studied. The strong attachment of HRP-anti-PRL to the GPPDs resulted in a good repeatability and intermediate reproducibility down to 9.8%. The dynamic concentration range spanned from 0.5 to 180 ng mL⁻¹ PRL with a detection limit of 0.1 ng mL⁻¹ at the 3S_blank level. No significant differences at the 95% confidence level were encountered in the analysis of 10 spiked blank cattle serum samples between the developed immunoassay and enzyme-linked immunosorbent assay method for determination of PRL.     

Silver nanoparticles and magnetic beads with electrochemical measurement as a platform for immunosensing devices

The simplicity and analytical utility of silver nanoparticles used as immunolabels with screen-printed measurement electrodes is illustrated by demonstrating an appropriate analytical signal for myoglobin (a protein marker for muscle damage) across a range of concentrations of physiological interest for distinguishing potential myocardial infarctions from normal background levels in serum. Silver nanoparticles were used as labels on one of a pair of anti-myoglobin clones while the other clone was covalently attached to magnetic beads. The two clones were selected so as to bind to different sites on the target protein and allow the formation of complexes containing both magnetic beads and silver nanoparticles. The magnetic beads enabled protein captured from test samples to be separated from other components, while the silver nanoparticles enabled the protein to be quantified. An oxidising potential, applied to screen-printed carbon electrodes, was used to dissolve silver without the need for an external oxidising agent. Silver ions released in the process were subsequently accumulated at the measurement electrodes by applying a suitable reducing potential and, finally, analytical signals were obtained by integrating the charges passed when accumulated silver was stripped from the electrodes by applying a potential ramp. The magnitudes of the measured charges were indicative of the concentrations of myoglobin in each of the test solutions.     

Nanosilver and DNA-functionalized immunosensing probes for electrochemical immunoassay of alpha-fetoprotein

A simple and sensitive electrochemical immunosensor for a one-step immunoassay for alpha-fetoprotein (AFP) was designed using silver nanoparticles and double-stranded DNA as matrices. The detection was based on the change in the electron transfer resistance before and after the antigen-antibody reaction by using electrochemical impedance spectroscopy. Under optimal conditions, the resistance shift of the immunosensor is proportional to the AFP concentration in the range 3.5 –360 ng·mL^?1 with a detection limit of 1.5 ng·mL^?1 (at 3σ). The immunosensor exhibits high sensitivity, good reproducibility and stability. Results obtained for clinical serum samples by the immunosensor are in accordance with those determined by spectrophotometric enzyme-linked immunosorbent assays.     

Biosensor for multiplex detection of two DNA target sequences using enzyme-functionalized Au nanoparticles as signal amplification

Multiplex electrochemical detection of two DNA target sequences in one sample using enzyme-functionalized Au nanoparticles (AuNPs) as catalytic labels for was proposed. This DNA sensor was fabricated using a “sandwich” detection strategy, involving two kinds of capture probes DNA immobilized on glassy carbon electrode (GCE), and hybridization with target DNA sequences, which further hybridized with the reporter DNA loaded on the AuNPs. The AuNP contained two kinds of DNA sequences, one was complementary to the target DNA, while the other was noncomplementary to the target. The noncomplementary sequences were linked with horseradish peroxidase (HRP) and alkaline phosphatase (ALP), respectively. Enhanced detection sensitivity was obtained where the AuNPs carriers increased the amount of enzyme molecules per hybridization. Electrochemical signals were generated from the enzymatic products produced from the substrates catalyzed by HRP and ALP. Under optimal conditions, a 33-mer sequence could be quantified over the ranges from 1.5 × 10⁻¹³ to 5.0 × 10⁻¹² M with a detection limit of 1.0 × 10⁻¹³ M using HRP-AuNP as labels, and a 33-mer sequence could be quantified over the ranges from 4.5 × 10⁻¹¹ M to 1.0 × 10⁻⁹ M with a detection limit of 1.2 × 10⁻¹¹ M using ALP-AuNP as labels.     

Sensitive ferrocene reagents for derivatization of amines for high-performance liquid chromatography with electrochemical detection

Six reagents possessing ferrocene as an electrophore were prepared and evaluated for pre-column derivatization of amino compounds for their determination by high-performance liquid chromatography with electrochemical detection. The utility of these reagents was investigated employing phenethylamine as a model compound. Among these six, N-succinimidyl 3-ferrocenylpropionate was the best with respect to reactivity, stability and electrochemical properties. The developed method was applied to the determination of putrescine formed from ornithine by ornithine decarboxylase.     

An innovative strategy for direct electrochemical detection of microRNA biomarkers

We report an electrochemical method for direct, reagentless, and label-free detection of microRNA, based on a conjugated copolymer, poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-2-carboxyethyl-1,4-naphthoquinone), acting as hybridization transducer. Hybridization between the oligonucleotide capture probe and a microRNA target of 22 base pairs generates an increase in the redox current (“signal-on”), which is evidenced by square wave voltammetry. Selectivity is good, with little hybridization for non-complementary targets, and the limit of detection reaches 650 fM. It is also evidenced that this sensitivity benefits from the high affinity of DNA for RNA.

Hemin/G-quadruplex-based DNAzyme concatamers as electrocatalysts and biolabels for amplified electrochemical immunosensing of IgG1

Hemin/G-quadruplex-based DNAzyme concatamers were utilized as electrocatalysts and biolabels to construct a sandwich-type electrochemical immunosensor for sensitive detection of IgG1 (as a model analyte).     

Os(VI)bipy-based electrochemical assay for detection of specific microRNAs as potential cancer biomarkers

Recently, altered expression levels of microRNAs (miRNAs) – short noncoding RNA molecules which bind to mRNAs and thus regulate gene expression – were observed in many cancer cells. miRNA expression profiling is therefore of great interest, but current standard methods are still considered relatively laborious and expensive. Electrochemistry has a potential to become quick and inexpensive alternative. Here, we describe modification of miRNA with an electroactive complex composed of six-valent osmium and 2,2′-bipyridine, Os(VI)bipy, specifically binding to the 3′-end of the ribose, which is detectable at hanging mercury drop electrode at femtomole level due to an electrocatalytic nature of a resulting signal. By combining miRNA labeling step with magnetic beads-based hybridization assay, detection of specific miRNA sequence from a mixture of other noncomplementary miRNAs was possible.     

Amplified inhibition of the electrochemical signal of ferrocene by enzyme-functionalized graphene oxide nanoprobe for ultrasensitive immunoassay

A nanoprobe-induced signal inhibition mechanism was designed for ultrasensitive electrochemical immunoassay at a chitosan-ferrocene (CS-Fc) based immunosensor. The nanoprobe was prepared by covalently loading signal antibody and high-content horseradish peroxidase (HRP) on the graphene oxide (GO) nanocarrier. The immunosensor was prepared through the stepwise assembly of gold nanoparticles (Au NPs) and capture antibody at a CS-Fc modified electrode. After sandwich immunoreaction, the GO-HRP nanoprobes were quantitatively captured onto the immunosensor surface and thus induced the production of a layer of insoluble film through the enzymatically catalytic reaction of the HRP labels. Both the dielectric immunocomplex formed on the immunosensor surface and the enzymatic precipitate with low electroconductivity led to the electrochemical signal decease of the Fc indicator, which was greatly amplified by the multi-enzyme signal amplification of the nanoprobe. Based on this amplified signal inhibition mechanism, a new ultrasensitive electrochemical immunoassay method was developed. Using carcinoembryonic antigen as a model analyte, this method showed a wide linear range over 5 orders of magnitude with a detection limit down to 0.54 pg/mL. Besides, the immunosensor showed good specificity, acceptable reproducibility and stability as well as satisfactory reliability for the serum sample analysis.     

Synthesis,crystal structures and electrochemical properties of two new metal-centered ferrocene complexes

Two new metal-centered ferrocene complexes Ni(SCN)2(L)4 (1) and Cu(OAc)2(L)2 (2) (L = 1-[1-ferro- cenylmethyl]imidazole) have been synthesized and characterized by elemental analysis, single crystal X-ray diffraction analysis, spectroscopic and cyclic voltammetric measurements. The geometry of Ni(Ⅱ) in 1 is octahedral, with four ligands in the equatorial plan and two thiocyanate anions at the axial site, while that of Cu(Ⅱ) in 2 is a distorted octahedron formed by two chealted OAc- and two ligands. Single c...     

CE-based simultaneous liquid-phase noncompetitive enzyme immunoassay for three tumor markers in human serum using electrochemical detection

A method for indirectly detecting horseradish peroxidase (HRP) was described by CE with electrochemical detection. Details of selection for optimum conditions were presented. The detection limit of free HRP was 1.09 x 10(-12) M or 0.94 zmol (S/N = 3). A novel CE-based liquid-phase binding noncompetitive enzyme immunoassay (CE-EIA) was developed. In this method, after the noncompetitive immunoreaction in liquid phase, the free enzyme (HRP)-labeled antibody (Ab*) and the bound enzyme-labeled complex (Ag-Ab*) were separated and then the system of HRP catalyzing H(2)O(2)/o-aminophenol (OAP) reaction was adopted. Prostate specific antigen (PSA), carcinoembryonic antigen (CEA), and human chorionic gonadotropin (HCG) in human serum samples were detected without any sample preparation, with the detection limits (S/N = 3) of 0.22, 0.17 and 0.30 ng/mL, respectively. This technique has been successfully applied to detect simultaneously PSA, CEA, and HCG in 12 min, upon adding these three antigens into human serum to simulate patient serum. It proves that the CE-EIA technique proposed could be developed into a sensitive and new method for simultaneous clinical assay of multianalytes.     

Simultaneous detection of two lung cancer biomarkers using dual-color fluorescence quantum dots

Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, a novel protocol for performing a simultaneous dual-protein immunoassay, i.e. two lung cancer biomarkers, carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE), based on dual-color QDs, is described. First, two capture antibodies (both with biotin tags), two antigens and two detection antibodies were mixed together and the sandwich complexes were thus formed in the homogeneous solution, and then streptavidin coated polystyrene beads were directly added into the resultant system. Bead aggregation can be made self-limiting by controlling the shaker speed during the immunoassay. A distinct transition occurs between limited and complete aggregation as a function of the shaker speed during the immunoassay. Second, dual-color QDs with emission maxima at 525 and 655 nm were added after washing and reacted with the corresponding detection antibodies. Third, the bead-QD conjugates were dissociated in the dissociation buffer and then free QDs were directly used for the fluorescence detection of CEA and NSE. The results show that CEA and NSE could be sensitively determined with a common 96-well fluorescence plate reader and with equal detection limits down to the 1.0 ng mL(-1) level. Within the calibrated amount, the protocol had excellent precision within 0.53% for each target and was comparable in performance to commercial single-analyte ELISAs. Furthermore, the proposed method has been successfully applied to the determination of dual markers in real samples without cross-reaction, and a good correlation was achieved after comparison with the conventional assay for CEA and NSE in 25 human serum samples.     

Site-specific DNA cleavage of EcoRI endounclease probed by electrochemical analysis using ferrocene capped gold nanoparticles as reporter

An electrochemical method was proposed to study DNA cleavage by EcoRI endonuclease using ferrocene as electrochemical indicator. Ferrocene was not covalently crosslinked to the DNA molecule but co-immobilized on the gold nanoparticles with thiolated target DNA via Au–S bond. EcoRI–DNA interaction was monitored based on the alteration of ferrocene signal before and after digestion with EcoRI. Enzymatic cleavage specificity of EcoRI was investigated, and enzymatic cleavage at DNA-modified electrode was also monitored real-time.