Development of a low density colorimetric protein array for cardiac troponin I detection

This work presented a rapid, inexpensive, reliable, and flexible quantitative immunoassay for cardiac troponin I (cTnI). The assay was based on the concepts of one-step dual monoclonal antibody "sandwich" principle, the low density protein array, the nanogold probe, and the silver enhancement on the gold particles. The capture antibody (IgG1) coated supporting nitrocellulose membrane and the colloidal gold-labeled detection antibody (cAu-IgG2) were prepared before the detection. The detection procedure involved two steps, i.e., immunoreaction and silver amplification. The assay needs only small amounts of serum samples of patients, The whole detection procedure of the assay could be fulfilled within 40 min (much faster than the routine enzyme-linked immunosorbent assay (ELISA) that takes usually at least 3 hours for a turnaround test). The detection results could be easily imaged with a simple flatbed scanner or even observed with the naked eye. The assay showed good specific response to cTnI with very little cross-reactivity to the skeletal isoforms of troponin I (sTnl), cardiac troponin T (cTnT), and myoglobin (Mb). A cut-off value of 0.3 ng/ml was obtained from a reference control group (200 normal serum samples). 588 patients' serum samples were assayed simultaneously by routine ELISA and this colloidal gold method to test the validity of the method. The data were analyzed using the statistical package SPSS version 11.0 (SPSS Inc.) There was no significant difference between these two assays (P = 0.66 > 0.05). The agreement between this method (> or < 0.3 ng/ml) and ELISA was 86%.     

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.     

Alkaline phosphatase-catalyzed silver deposition for electrochemical detection

Alkaline phosphatase (AP) is one of the most used enzymatic labels for the development of ELISAs, immunosensors, DNA hybridization assays, etc. This enzyme catalyzes the dephosphorylation of a substrate into a detectable product usually quantified by optical or electrochemical measurements. This work is based on a substrate (3-indoxyl phosphate) that produces a compound able to reduce silver ions in solution into a metallic deposit, which is localized where the enzymatic label AP is attached. The deposited silver is electrochemically stripped into solution and measured by anodic stripping voltammetry. Its application to an enzymatic genosensor on streptavidin-modified screen-printed carbon electrodes for the detection of virulence nucleic acid determinants of autolysin gene, exclusively present on the genome of the human pathogen Streptococcus pneumoniae, is described. Compared with the direct voltammetric detection of indigo carmine, the anodic stripping voltammetry of silver ions is 14-fold more sensitive.     

Graphene oxide sheet-mediated silver enhancement for application to electrochemical biosensors

Functionalized graphene oxide (GO) sheets coupled with a signal amplification method based on the nanomaterial-promoted reduction of silver ions for the sensitive and selective detection of bacteria. This paper aims to develop an electrochemical route combined with GO sheet-mediated Ag enhancement for biological/chemical analyte detection. A linear relationship between the stripping response and the logarithm of the bacterial concentration was obtained using an electrochemical technique for concentrations ranging from 1.8 × 10(2) to 1.8 × 10(8) cfu mL(-1), with a slope of 15.28 and a correlation coefficient of 0.995. Dot blot assay was used as a conventional immunoassay method for comparison with the electrochemical method, as well as to observe the quality of the anti-sulfate-reducing bacteria (SRB) antibody (Ab) used in the immunosensor. The GO sheet-mediated silver enhancement holds great potential for the rapid analysis of protein, DNA, and pathogens.     

Ultrasensitive multiplexed immunoassay with electrochemical stripping analysis of silver nanoparticles catalytically deposited by gold nanoparticles and enzymatic reaction

A novel ultrasensitive multiplexed immunoassay method was developed by combining alkaline phosphatase (ALP)-labeled antibody functionalized gold nanoparticles (ALP-Ab/Au NPs) and enzyme-Au NP catalyzed deposition of silver nanoparticles at a disposable immunosensor array. The immunosensor array was prepared by covalently immobilizing capture antibodies on chitosan modified screen-printed carbon electrodes. After sandwich-type immunoreactions, the ALP-Ab/Au NPs were captured on an immunosensor surface to catalyze the hydrolysis of 3-indoxyl phosphate, which produced an indoxyl intermediate to reduce Ag(+). The silver deposition process was catalyzed by both ALP and Au NPs, which amplified the detection signal. The deposited silver was then measured by anodic stripping analysis in KCl solution. Using human and mouse IgG as model analytes, this multiplexed immunoassay method showed wide linear ranges over 4 orders of magnitude with the detection limits down to 4.8 and 6.1 pg/mL, respectively. Acceptable assay results for practical samples could be obtained. The newly designed strategy avoided cross talk and the need of deoxygenation for the electrochemical immunoassay and, thus, provided a promising potential in clinical applications.     

Development of lateral flow immunoassay system based on superparamagnetic nanobeads as labels for rapid quantitative detection of cardiac troponin I

In this study, superparamagnetic nanobeads (SPMNBs) were used as labels to establish a lateral flow immunoassay system for rapid, quantitative detection of cardiac troponin I (cTnI). First, the immunobeads were prepared by coupling monoclone antibody specific to cTnI onto SPMNBs. Then some factors which may influence the detection sensitivity of this lateral flow immunoassay system were studied, such as the amount of antibody immobilized in the Test line, sample volume and the amount of antibody per SPMNB. Finally, lateral flow test strips for detection of cTnI were established and applied to testing standard samples of different cTnI concentrations, which were also measured by Enzyme-linked Immunosorbent Assay (ELISA) in parallel. Furthermore, testing time and stability of magnetic signal were also investigated in this study. Results showed that this lateral flow test for cTnI had a high detection sensitivity of 0.01 ng/ml with a wide detection range of 5 orders of magnitude. The testing time was less than 15 min, and no significant decline of magnetic signal was observed in 140 days. Therefore, this lateral flow immunoassay system using SPMNB as labels will be a new point of care test (POCT) for rapid, quantitative detection of cardiac troponin I.     

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.     

Electrochemical sensor for organophosphate pesticides and nerve agents using zirconia nanoparticles as selective sorbents

An electrochemical sensor for detection of organophosphate (OP) pesticides and nerve agents using zirconia (ZrO2) nanoparticles as selective sorbents is presented. Zirconia nanoparticles were electrodynamically deposited onto the polycrystalline gold electrode by cyclic voltammetry. Because of the strong affinity of zirconia for the phosphoric group, nitroaromatic OPs strongly bind to the ZrO2 nanoparticle surface. The electrochemical characterization and anodic stripping voltammetric performance of bound OPs were evaluated using cyclic voltammetric and square-wave voltammetric (SWV) analysis. SWV was used to monitor the amount of bound OPs and provide simple, fast, and facile quantitative methods for nitroaromatic OP compounds. The sensor surface can be regenerated by successively running SWV scanning. Operational parameters, including the amount of nanoparticles, adsorption time, and pH of the reaction medium have been optimized. The stripping voltammetric response is highly linear over the 5-100 ng/mL (ppb) methyl parathion range examined (2-min adsorption), with a detection limit of 3 ng/mL and good precision (RSD = 5.3%, n = 10). The detection limit was improved to 1 ng/mL by using 10-min adsorption time. The promising stripping voltammetric performances open new opportunities for fast, simple, and sensitive analysis of OPs in environmental and biological samples. These findings can lead to a widespread use of electrochemical sensors to detect OP contaminates.     

Anodic stripping voltammetry coupled on-line with inductively coupled plasma mass spectrometry: optimization of a thin-layer flow cell system for analyte signal enhancement

Parameters affecting analyte signal enhancement in anodic stripping voltammetry-inductively coupled plasma mass spectrometry (ASV-ICP-MS), using a thin-layer ASV cell and microconcentric nebulization (MCN), have been examined. Silver was used as a test analyte and was deposited at a glassy carbon working electrode. The MCN allowed use of solution flow rates that were beneficial to optimum electrolytic performance of the thin-layer cell. High analyte deposition efficiencies obtained with the thin-layer cell, combined with minimal sample consumption of the MCN, allowed substantial signal enhancement (>400 times higher than continuous nebulization level) to be obtained with 2-3 mL of sample and deposition times of less than 30 min. Signal enhancement was strongly influenced by the opposing effect of flow rate on the electrolytic deposition efficiency (deposition efficiency decreases with increasing flow rate) and on the quantity of analyte delivered to the cell (analyte mass throughput increases with increasing flow rate). Excellent linearity for stripping peak heights was demonstrated for a wide range of analyte deposition times and for peak heights and peak areas (r > 0.999) over a wide concentration range (25 ng/L-20 μg/L). Precision was good (RSD typically <3% for n = 3-6) except for a high Ag blank contributed to by corrosion of the counter electrode and by Ag diffusion from the reference electrode into the cell. Details of the flow manifold and ASV cells are discussed, along with relevant performance characteristics of the MCN.     

Anodic stripping voltammetry enhancement by redox magnetohydrodynamics

The effect of an external magnetic field on linear scan anodic stripping voltammetry (ASV) in solutions of 10(-6)-10(-7) M concentrations of lead, cadmium, and copper at mercury films on glassy carbon electrodes has been investigated. A high concentration of Hg(2+) was added to the analyte solution to induce a large cathodic current during the deposition step. Therefore, a large Lorentz force from the net flux of charge through the magnetic field resulted in convection due to magnetohydrodynamics. The faster delivery of analytes to the mercury film electrode during deposition caused an increase in the anodic stripping peaks. The effect of varying Hg(2+) concentrations (0-60 mM) and magnetic field strengths (0-1.77 T) on the enhancement of the stripping peaks was investigated. Enhancements as large as 129% for peak currents and 167% for peak areas were observed. An enhancement of approximately 100% was observed when 60 mM Fe(3+) replaced high concentrations of Hg(2+). This method of convection exhibits promise for small-volume ASV analysis with possible improved limits of detection and decreased preconcentration times.     

Response to cardiac markers in human serum analyzed by guided-mode resonance biosensor

Cardiac markers in human serum with concentrations less than 0.1 ng/mL were analyzed by use of a guided-mode resonance (GMR) biosensor. Cardiac troponin I (cTnI), creatine kinase MB (CK-MB), and myoglobin (MYO) were monitored in the serum of both patients and healthy controls. Dose-response curves ranging from 0.05 to 10 ng/mL for cTnI, from 0.1 to 10 ng/mL for CK-MB, and from 0.03 to 1.7 μg/mL for MYO were obtained. The limits of detection (LOD) for cTnI, CK-MB, and MYO were less than 0.05, 0.1, and 35 ng/mL, respectively. Analysis time was 30 min, which is short enough to meet clinical requirements. Antibody immobilization and the hydrophilic properties of the guided-mode resonance filter (GMRF) surface were investigated by X-ray photoelectron spectroscopy (XPS) and by monitoring the peak wavelength shift and water contact angle (CA). Both assays used to evaluate the surface density of the immobilized antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) and a sandwich immunogold assay, showed that the antibodies were successfully immobilized and sufficiently aligned to detect the low concentration of biomarkers. Our results show that the GMR biosensor will be very useful in developing low-cost portable biosensors that can screen for cardiac diseases.     

Cathodic Stripping Voltammetry of Selenium(IV) at a Silver Disk Electrode

A new, simple, and reproducible method is described for the determination of selenium(IV) based on differential pulse cathodic stripping voltammetry. The optimized experimental conditions are as follows: selenium(IV) ions in an acidic medium (0.06 M HCl-0.07 M HNO(3)) are electrodeposited on a rotating silver disk electrode as silver selenide at -0.4 V vs SCE for 30 min; the deposit is then cathodically stripped in another solution (2 M NaOH) at a scan rate of 50 mV s(-1) to -1.2 V vs SCE. The cathodic stripping results in only a single well-defined peak at about -0.95 V vs SCE. The calibration (peak height vs selenium concentration) graph is linear up to at least 40 ng mL(-1) of selenium(IV) and passes through the origin, with a relative standard deviation of 2.7% for 20 ng mL(-1) (n = 5). The detection limit (3σ) is 0.20 ng mL(-1). The possible interferences have been evaluated. Dissolved oxygen does not affect the peak height of selenium. The electrode can be used repeatedly at least 20 times with excellent reproducibility without further polishing. The proposed method is an improvement over the existing cathodic stripping techniques.     

Amplified oligonucleotide sensing in microliter volumes containing copper ions by solution streaming

We present a simple, cost-effective design for amplifying oligodeoxynucleotide (ODN) sensing, in microliter ODN volumes containing copper ions, by solution streaming (bubbling). The inert gas streaming (bubbling) at a constant pressure of 0.04 bar drives the motion of a 30-microL ODN droplet containing a three-electrode circuit (inverted drop microcell), and in the presence of copper ions offers an approximately 50-times improvement in the detection of ODN samples. The detection of ODNs at the carbon paste electrode is based on the enhancement of the oxidation peaks of purine bases (adenine and guanine) by the anodic stripping of the electrochemically accumulated complex of Cu(I) with purine base residues of acid hydrolyzed ODN samples (Cu(I)-ahODN complex). We used the proposed method for (i) the determination of the percentage content of adenine and guanine units within analyzed ODN samples at subnanomolar concentrations (related to monomer content) and (ii) the detection of the (TTC)n triplet expansion using magnetic DNA hybridization with reporter probes containing guanine units (the TTC trinucleotide repeat expansion is associated with serious hereditary diseases, including Friedreich ataxia).     

Rapid diagnostic barcode system for codetection of multiple protein markers

An ultrasensitive immunodiagnostic readout method based on an electrochemical analysis is presented. Different inorganic quantum dot (QD) nanocrystals (ZnS, CdS, and PbS) are tagged to antibodies for the on-site voltammetric stripping measurements of multiple antigen targets. The multiprotein electrical sensing capability is coupled to the amplification feature of anodic stripping voltammetric transduction and with an efficient magnetic removal (to minimize nonspecific adsorption and cross-reactivity effects). Sandwich-immunoassay formats were performed using model proteins (/spl beta//sub 2/-microglobulin, myoglobin, and human serum albumin). These encoding QD tracers with distinct redox potential yield highly sensitive and selective stripping peaks at -1.11 V (Zn), -0.67 V (Cd), and -0.52 V (Pb) at the mercury-film screen printed carbon electrode (versus Ag/AgCl reference). The position and size of these peaks reflect the identity and risk level of the corresponding antigen marker. The favorable signal-to-noise characteristics of the response for the initial 25-ng/mL mixture indicate a detection limit of ca. 10 ng/mL far below the early warning range and allow a reliable determination of very low protein concentrations. Such analog peaks of the QDs were converted to simple and rapid barcode signals. The digital readout system can code 215 electrically tuned barcodes to mark different protein analytes and to be useful for a wireless communication system.     

Antimony film electrode for electrochemical stripping analysis

In this work, an antimony film electrode (SbFE) is reported for the first time as a possible alternative for electrochemical stripping analysis of trace heavy metals. The SbFE was prepared in situ on a glassy carbon substrate electrode and employed in combination with either anodic stripping voltammetry or stripping chronopotentiometry in nondeaerated solutions of 0.01 M hydrochloric acid (pH 2). Several key operational parameters influencing the electroanalytical response of SbFE were examined and optimized, such as deposition potential, deposition time, and composition of the measurement solution. The SbFE exhibited well-defined and separated stripping signals for both model metal ions, Cd(II) and Pb(II), surrounded with low background contribution and a relatively large negative potential range. The electrode revealed good linear behavior in the examined concentration range from 20 to 140 microg L-1 for both test metal ions, with a limit of detection (3sigma) of 0.7 microg L-1 for Cd(II) and 0.9 microg L-1 for Pb(II) obtained after a 120 s deposition step, and good reproducibility, with a relative standard deviation (RSD) of +/-3.6% for Cd(II) and +/-6.2% for Pb(II) (60 microg L-1, n = 12). When comparing the SbFE with the commonly used mercury film electrode and recently introduced bismuth film electrode, the newly proposed electrode offers a remarkable performance in more acidic solutions (pH < or = 2), which can be advantageous in electrochemical analysis of trace heavy metals, hence contributing to the wider applicability of electrochemical stripping techniques in connection with "mercury-free" electrodes.     

Nanoscale control of Ag nanostructures for plasmonic fluorescence enhancement of near-infrared dyes

Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based detection techniques. Metal induced fluorescence enhancement offers the possibility of increasing the sensitivity of protein detection in clinical applications. We report the use of tunable plasmonic silver nanostructures for the fluorescence enhancement of a near-infrared (NIR) dye (Alexa Fluor 790). Extensive fluorescence enhancement of ～2 orders of magnitude is obtained by the nanoscale control of the Ag nanostructure dimensions and interparticle distance. These Ag nanostructures also enhanced fluorescence from a dye with very high quantum yield (7.8 fold for Alexa Fluor 488, quantum efficiency (Qy) = 0.92). A combination of greatly enhanced excitation and an increased radiative decay rate, leading to an associated enhancement of the quantum efficiency leads to the large enhancement. These results show the potential of Ag nanostructures as metal induced fluorescence enhancement (MIFE) substrates for dyes in the NIR “biological window” as well as the visible region. Ag nanostructured arrays fabricated by colloidal lithography thus show great potential for NIR dye-based biosensing applications.      

Sonically assisted electroanalytical detection of ultratrace arsenic

A simple portable handheld electrochemical sensor with an integrated sound source for the detection of ultratrace quantities of arsenic using square wave anodic stripping voltammetry is described. The sensor uses low-frequency sound (250 Hz) during the arsenic deposition step to enhance the sensitivity of the arsenic stripping response. It is found that under quiescent (silent) conditions a detection limit of 2.1 x 10(-7) M with a sensitivity of 0.51 M(-1) A is achievable using a 120-s accumulation period, while applying low-frequency sound using a "sonotrode" reduced this detection limit to 3.7 x 10(-9) M with an increased sensitivity of 27.2 M(-1) A. Thus, the low-frequency sonotrode is shown to increase the sensitivity by ca. 50 times while reducing the limit of detection by 2 orders of magnitude. A study of the effect of copper contamination is carried out as well as analysis in real samples; it is found that although as expected copper detrimentally effects the arsenic limit of detection, it does not rise significantly above 10(-8) M levels.     

Development of a method for total inorganic arsenic analysis using anodic stripping voltammetry and a Au-coated, diamond thin-film electrode

We demonstrate that a Au-coated, boron-doped, diamond thin-film electrode provides a sensitive, reproducible, and stable response for total inorganic arsenic (As(III) and As(V)) using differential pulse anodic stripping voltammetry (DPASV). As is preconcentrated with Au on the diamond surface during the deposition step and detected oxidatively during the stripping step. Au deposition was uniform over the electrode surface with a nominal particle size of 23 +/- 5 nm and a particle density of 109 cm-2. The electrode and method were used to measure the As(III) concentration in standard and river water samples. The detection figures of merit were compared with those obtained using conventional Au-coated glassy carbon and Au foil electrodes. The method was also used to determine the As(V) concentration in standard solutions after first being chemically reduced to As(III) with Na2SO3, followed by the normal DPASV determination of As(III). Sharp and symmetric stripping peaks were generally observed for the Au-coated diamond electrode. LODs were 0.005 ppb (S/N = 3) for As(III) and 0.08 ppb (S/N = 3) for As(V) in standard solutions. An As(III) concentration of 0.6 ppb was found in local river water. The relative standard deviation of the As stripping peak current for river water was 1.5% for 10 consecutive measurements and was less than 9.1% over a 10-h period. Excellent electrode response stability was observed even in the presence of up to 5 ppm of added humic acid. In summary, the Au-coated diamond electrode exhibited better performance for total inorganic As analysis than did Au-coated glassy carbon or Au foil electrodes. Clearly, the substrate on which the Au is supported influences the detection figures of merit.     

Measurement of ultrasmall volumes using anodic stripping voltammetry

This paper discusses a new and effective method for small volume determination. The benefits of working with small volumes are numerous and include using less material, generating less waste, increased functionality in less space, portability, and high throughput. As sample size decreases, measuring its volume becomes more challenging. The most prevalent method for small volume determination is visual inspection. The method presented in this paper is precise, more quantitative, and adaptive to many solution geometries, because it is based on a coulometric approach. Demonstration of this method is performed on volumes of approximately 1 nL containing silver and using a self-contained microcavity device that contains multiple electrodes. The silver is "exhaustively" deposited during a cathodic potential step, followed by anodic stripping voltammetry. It is expected that an even faster and more accurate analysis of volumes much smaller than those evaluated here is easily possible using this approach.     

A novel photoelectrochemical sensor for the organophosphorus pesticide dichlofenthion based on nanometer-sized titania coupled with a screen-printed electrode

A novel photoelectrochemical sensor for detection of the organophosphorus pesticide (OP) dichlofenthion using nanometer-sized titania coupled with a screen-printed electrode is presented. Nonelectroactive dichlofenthion can be indirectly determined through the photocatalytical degradation of dichlofenthion with nanometer-sized titania. The electrochemical characterization and anodic stripping voltammetric performance of dichlofenthion were evaluated using cyclic voltammetric (CV) and differential pulse anode stripping voltammetric (DPASV) analysis, respectively. DPASV analysis was used to monitor the amount of dichlofenthion and provide a simple, fast, and facile quantitative method for dichlofenthion. Operational parameters, including the photocatalysis time, pH of buffer solution, deposition potential, and accumulation time have been optimized. The stripping voltammetric response is linear over the 0.02-0.1 and 0.2-1.0 μmol/L ranges with a detection limit of 2.0 nmol/L. The assay result of dichlofenthion in green vegetable with the proposed method was in acceptable agreement with that of the gas chromatograph-mass spectrometer (GC-MS) method. The promising sensor opens a new opportunity for fast, portable, and sensitive analysis of OPs in environmental samples.