Search

A double covalent coupling method for the fabrication of a highly sensitive and reusable electrogenerated chemiluminescence (ECL) chemical sensor for the detection of tertiary amines and ECL aptamer-based (ECL-AB) biosensor for the detection of cocaine is reported. The ECL sensors were constructed by covalent coupling of amino-containing Ru[(bpy).sub.3.sup.2+] derivatives (Rul, Ru[(bpy).sub.3.sup.2+] = tris(2,2'-bipyridyl)ruthenium(II)) or cocaine aptamerRu1 to the surface of a paraffin-impregnated graphite electrode that had been covalently modified with a monolayer of 4-aminobenzene sulfonic acid via electrochemical oxidations. ECL performance of the newly developed chemical sensors was evaluated using trin-propylamine (TPrA) and metoclopramide (MCP) as model analytes. The sensors exhibited excellent sensitivity, stability, and reproducibility with a detection limit of 30 nM for TPrA and 2.0 nM for MCP, and relative standard deviations (RSDs) of 2.1% over 90 cyclic potential cycles (0 to 1.50 V vs Ag/AgCI) and 2.6% over 45 cycles (0.60 to +1.30 V vs Ag/AgCI) at 400 mV/s for 50 nM TPrA and 200 nM MCP, respectively. For the ECL-AB biosensor, it showed an extremely low detection limit of 10 pM for cocaine, and offered a good selectivity toward cocaine, heroin, and caffeine. This detection limit was about 4-6 orders of magnitude lower than that reported on the basis of alternating current (AC) voliammetry and optical aptamer-based cocaine biosensors. Additionally, the ECL-AB biosensor was highly reusable (RSD = 2.8%, n = 7) and possessed long-term storage stability (96.8% initial ECL recovery over 21 days storage). A binding constant of 4.6 [+ or -] 0.3 X [10.sup.9] [M.sup.-1] between cocaine and its aptamer was estimated using an ECL based Langmuir isotherm approach. Wide ranging applications of the presently reported strategy in fabricating various chemical sensors or biosensors are expected. 10.1021/ac9029289

A highly selective electrogenerated chemiluminescence (ECL) biosensor for the detection of target single-strand DNA (ss-DNA) was developed using hairpin DNA as the recognition element and ruthenium complex as the signal-producing compound. The ECL-based DNA biosensor was fabricated by self-assembling the ECL probe of thiolated hairpin DNA tagged with ruthenium complex on the surface of a gold electrode. In the absence of target ss-DNA, the ECL probe immobilized on the surface of the electrode was in the folded configuration in which its termini were held in close proximity to the electrode, and thus a strong ECL signal could be generated. In the presence of target ss-DNA, a stem-loop of the ECL probe on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the tag moving away from the electrode surface, which in turn decreased the ECL signal. The ECL intensity of the DNA biosensor generated a 'switch off' mode, which decreased with an increase of the concentration of target DNA, and a detection limit of 9 x [10.sup.-11] M complementary target ss-DNA was achieved. Single mismatched target ss-DNA was effectively discriminated from complementary target ss-DNA. The effect of different loop lengths of the hairpin DNA on the selectivity of the ECL DNA biosensor has been investigated. This work demonstrated that the sensitivity and specificity of an ECL DNA biosensor could be greatly improved using a hairpin DNA species which has an appropriate stem and loop length as the recognition element.

Formaldehyde (FA), the smallest molecular aldehyde with strong reducing properties, could regulate body homeostasis endogenously during physiological and pathological processes. The effective near-infrared (NIR) fluorescent probe is needed as a visualizer of FA in biologic organisms. In this work, a novel NIR fluorescent Probe - NHNH 2 was designed on the basis of Probe - NH 2 via introducing a strong nucleophilic hydrazine group, which can be used as a quenching and recognizing moiety for the detection of FA. With the treatment of FA, the hydrazine group of Probe - NHNH 2 undergoes condensation and achieves a turn-on NIR fluorescence signal at a wavelength of 706 nm. The spectroscopic performance of Probe-NHNH 2 for FA was evaluated, and it exhibited high sensitivity and selectivity for the detection of FA in solution. Moreover, compared to the amine moiety-based Probe - NH 2, which our group reported, we found that hydrazine moiety-based Probe - NHNH 2 , exhibited a better reaction time of within 10 min and a lower detection limit of 0.68 μM, reflecting that the reaction of FA with hydrazine moiety is faster and more sensitive than that of FA with the amino group. More importantly, Probe - NHNH 2 was successfully applied to real-time imaging of endogenous FA by reacting with effective stimulant tetrahydrofolate and scavenger sodium bisulfite in zebrafish and mice. It is expected that we can provide a new rapid, sensitive NIR fluorescence theoretical basis for FA detection and in vivo imaging applications.

In this work, a simple electrochemiluminescence (ECL) imaging method based on the cell shield of the ECL emission was developed for the morphological and quantitative analysis of living cells under external stimulation. ECL images of MCF-7 cells cultured on or captured at the glassy carbon electrode (GCE) surface in a solution of tris(2,2'-bipyridyl)ruthenium(II)-tri- n -propylamine were recorded. Important morphological characteristics of living cells, including cell shape, cell area, average cell boundary, and junction distance between two adjacent cells, were directly obtained using the developed negative ECL imaging method. The ECL images revealed gradual morphological changes in cells on the GCE surface. During the course of H 2 O 2 stimulation of cells on the GCE surface, cells shrunk, rounded up, disengaged from surrounding cells, and finally detached from the electrode surface. During the course of electrical stimulation (0.8 V), the cells on the GCE surface exhibited aggregation as demonstrated by increases in the average cell boundary and decreases in the junction distance between two adjacent cells. Additionally, a quantitative method for the sensitive determination of MCF-7 cells with a limit of detection of 29 cells/mL was developed using the negative ECL imaging strategy. This work demonstrates that the proposed negative ECL imaging strategy is a promising approach to assess important morphological characteristics of living cells during the course of external stimulation and to obtain quantitative information on cell concentrations in solution.

A separation-free electrogenerated chemiluminescence (ECL) immunoassay for biomarkers has been developed incorporating target assistant proximity hybridization and dynamically competitive hybridization of a DNA ECL signal probe for the first time. In this work, the biomarkers of acute myocardial infarction including cardiac troponin I (cTnI), cardiac troponin T (cTnT), and myoglobin (Myo) were chosen as the model proteins while the corresponding antibody was utilized as a recognition probe and the DNA5 tagged with ruthenium complex was chosen as an ECL signal probe (DNA5-Ru1). The biosensors were fabricated by covalently coupling the capture probe DNA1 onto the surface of a glassy carbon electrode, and then, a competitor ss-DNA2 was hybridized with DNA1. When the biosensor was incubated in the solution containing a target protein, the recognition probes (DNA3-Ab1 and DNA4-Ab2), DNA5-Ru1, and the coreactant tri- n -propylamine, the target protein was bounded with two antibodies of the recognition probes and thus induced the sufficient proximity hybridization of DNA3 with DNA1, DNA4 with DNA5-Ru1, and DNA5-Ru1 with DNA1 and the unwinding of the competitor DNA2 with DNA1, and ECL measurement was performed in separation-free format. It was found that the hybridization base number and length of DNA1 and a competing hybridization of DNA5-Ru1 with DNA2 for DNA1 have important effects. The developed ECL method showed a quite low detection limit of 0.4 pg/mL for cTnI, 0.5 pg/mL for cTnT, and 0.5 ng/mL for Myo. The fabricated biosensor exhibited stability and reusability. This work demonstrated that the developed ECL immunoassay is a promising separation-free and flexible strategy for quantitation of multiple proteins using one biosensor.

This Feature simply introduces the history and mechanism of classical electrogenerated chemiluminescence (ECL) systems for the detection of biomolecules, highlights new advances and emerging fields of the ECL biosensing with recent illustrative examples, and presents the challenges and perspectives of ECL biosensing.

A novel kind of quantum dots, sulfur quantum dots (S dots), is synthesized by simply treating sublimated sulfur powders with alkali using polyethylene glycol-400 as passivation agents. The synthesized S dots exhibit excellent aqueous dispersibility, eminent photostability and temperature dependent photoluminescence (PL). An "assemble-fission" mechanism is proposed for the S dots formation in which "assembling" and "fission" are involved and contest each other. The ultimate morphologies of the S dots are dependent on the balance of the two forces. Guided by the assemble-fission mechanism, weakening the assembling effect is beneficial for obtaining monodisperse S dots, which can be achieved by pretreating of sulfur powder with nitric acid. PL wavelength of the S dots has been successfully tuned between green and blue light (from 550 to 440 nm) by simply controlling reaction time. A satisfactory quantum yield of 3.8% is obtained. Significant electrochemiluminescence of the S dots is observed in an annihilation reaction. Chemiluminescence from the S dots has been observed by direct oxidation. Taking advantage of unique and inherent antimicrobial activity of the sulfur particles, it is believed that this new emerging luminescent nanomaterial is highly promising in the development of new types of optoelectronic devices and tracer for live cells, in vivo imaging and diagnostics.

A simple electrochemiluminescence (ECL) immunoassay based on a proximity hybridization-regulated strategy was developed for highly sensitive and specific detection of cell surface protein and protein-overexpressing cancer cells. A biosensor was fabricated by self-assembling a thiolated capture ss-DNA3 (partially hybridize with ss-DNA1 and ss-DNA2) and blocking with 6-mercapto-1-hexanol on a gold electrode surface. Target protein was simultaneously bound by two ss-DNA-tagged antibody probes (DNA1-Ab1 and DNA2-Ab2), while DNA1 and DNA2 were brought in sufficient proximity and hybridized with capture DNA3 on the surface of the biosensor. After ECL signal reagent Ru(phen) 3 2+ was intercalated into the hybridized ds-DNAs, ECL measurement was performed in the coreactant solution. A "signal on" proximity hybridization-regulated ECL immunoassay for alpha-fetoprotein (AFP) was developed. The ECL intensity increased with the increase of AFP concentration in the range of 0.05-20.0 ng/mL with a detection limit of 6.2 pg/mL. Moreover, the developed ECL method was successfully used to detect AFP-overexpressing cancer cells (MCF-7 cancer cells as model) with a detection limit of 620 cells/mL (∼60 MCF-7 cells in 100 μL of cell suspension) and discriminate AFP expression on different types of the living cell surface. This work for the first time reports a proximity hybridization-regulated ECL immunoassay for the detection of the cell surface protein on a living cell surface with good specificity and sensitivity. This simple, specific, and sensitive strategy is greatly promising for the detection of proteins and specific cells.

Organic nanoparticles (NPs) from donor-acceptor based coumarin derivatives, 6-[4-(N,N-diphenylamino)phenyl]-3-ethoxycarbonyl coumarin (DPA-CM), with an average size of 5.82 nm, were synthesized by a facile reprecipitation method using water as a poor solvent and tetrahydrofuran as a good solvent. Red-shifted absorption, blue-shifted photoluminescence emission, and aggregation-induced enhanced electrochemiluminescence (ECL) emission were observed for the DPA-CM NPs in aqueous solution compared with the original DPA-CM in organic solution. The aggregation-induced enhanced ECL emission is ascribed to the combined effects of the small size of the DPA-CM NPs, the restricted conformational relaxation in the NPs, and the good stability of the cationic radical of DPA-CM. A strong and stable ECL emission is obtained at the DPA-CM NPs modified glassy carbon electrode in the presence of tri-n-propylamine, and the ECL intensity of the DPA-CM NPs modified electrode is quenched linearly in the range of 0.05-50 μM with detection limit of 0.04, 0.2, and 0.4 μM for ascorbic acid, uric acid, and dopamine, respectively. This work shows an example of donor-acceptor based organic NPs as ECL emitters and their analytical applications to monitor biomolecules.

A sensitive electrogenerated chemiluminescence (ECL) bioassay was developed for the detection of two protein kinases incorporating the peptide phosphorylation and a versatile ECL probe. Cyclic adenosine monophosphate-dependent protein kinase (PKA) and casein kinase II (CK2) were used as proof-of-concept targets while a PKA-specific peptide (CLRRASLG) and a CK2-specific peptide (CRRRADDSDDDDD) were used as the recognition substrates. Taking advantage of the ability of protein A binding with the Fc region of a variety of antibodies with high affinity, a ruthenium derivative-labeled protein A was utilized as a versatile ECL probe for bioassay of multiple protein kinases. A specific peptide substrate toward target protein kinase was first self-assembled on the surface of gold electrode and then serine in the specific peptide on the electrode was phosphorylated by target protein kinase in the presence of adenosine-5'-triphosphate. After recognition of the phosphorylated peptide by monoclonal antiphosphoserine antibody, the versatile ECL probe was specifically bound to the antiphosphoserine antibody on the electrode surface. The ECL bioassay was developed successfully in the individual detection of PKA and CK2 with detection limit of 0.005 U/mL and 0.004 U/mL, respectively. In addition, the ECL bioassay was applied to quantitative analysis of the kinase inhibitors and monitoring drug-triggered kinase activation in cell lysates. Moreover, an ECL imaging bioassay using electron-multiplying charged coupled device as detector on the gold electrode array was developed for the simultaneous detection of PKA and CK2 activity from 0.01 U/mL to 0.4 U/mL, respectively, at one time. This work demonstrates that the ingenious design and use of a versatile ECL probe are promising to simultaneous detection of multiple protein kinases and screening of kinase inhibitor.

The electrochemistry and electrogenerated chemiluminescence (ECL) of three 1,3,5-tri(anthracen-10-yl)-benzene-centered starburst oligofluorenes (T1-T3) are reported in this paper. The compounds T1-T3 contain 1,3,5-tri(anthracen-10-yl)-benzene as a core with fluorene as an arm from monofluorene to trifluorene groups (n = 1-3), generating a rigid three-dimensional structure. The electrochemical behaviors of these compounds are likely to be tuned by the fluorene arms. In cyclic voltammograms, both the oxidation and reduction of T1 and T2 are characterized by three reversible one-electron transfers from the core while the oxidation and reduction of T3 are characterized by six reversible one-electron transfers from the core and the arms in acetonitrile:benzene (v:v = 1:1) solvent. The second oxidation and reduction waves of T1 show three reversible one-electron transfers from three fluorene arms, while the second and third oxidation and reduction waves of T2 and T3 exhibit three reversible one-electron transfers from the six fluorene arms. The multiple electron transfers in one molecule are confirmed by chronoamperometry at an ultramicroelectrode, simulations and DFT calculations. The T1-T3 compounds display strong absorption in UV-vis and blue fluorescence emission. Strong blue ECL emissions can be generated from T1, T2 and T3 under ion annihilation condition, which is assigned as S-route.

A novel electrogenerated chemiluminescence (ECL) bioanalytic system based on biocleavage of a ECL probe and homogeneous detection was designed and utilized for the first time for highly sensitive quantification of proteases to overcome drawbacks from probes directly immobilized on electrodes and commercial ECL biosystems, based on bioaffinity reactions. Prostate-specific antigen (PSA) was taken as a model analyte and ruthenium complex-tagged specific peptide (CHSSKLQK) was designed as an ECL probe (peptide-Ru1). ECL bioconjugated magnetic beads were synthesized through a simple solid-phase synthesis. When analyte PSA was introduced into the suspension of ECL bioconjugated magnetic beads, a biocleavage of the peptide occurred and the cleaved Ru1 part was released from the magnetic beads. ECL measurement was carried out in the presence of co-reactant tripropylamine, using two models. One is homogeneous ECL detection on a bare graphite pencil electrode (PGE), and the other is enriching ECL detection after the cleaved Ru1 part of the peptide was concentrated into the surface film of Nafion/gold nanoparticles modified PGE (AuNPs/Nafion/PGE). The extremely low detection limit of 80 fg/mL and high reproducibility (relative standard deviation (RSD) of 5.4% for six measurements of 0.5 pg/mL) for the detection of PSA were achieved at AuNPs/Nafion/PGE. This work demonstrates that the bioanalytic system designed can not only quantify proteases with high sensitivity and selectivity, but also diminish the complicated electrode process and improve the reproducibility by conducting the biocleavage and transduction steps at different surfaces. It can be easily extended for ECL analysis of other proteases in this system and other detection techniques, including optics and electrochemistry.

A novel electrogenerated chemiluminescence peptide-based biosensor (ECL-PB) for the determination of prostate-specific antigen (PSA) was developed on the basis of target-induced cleavage of a specific peptide within Nafion film incorporated with gold nanoparticles (AuNPs) and ECL emitting species. A specific peptide (CHSSKLQK) was used as a molecular recognition element; tris(2,2'-ripyridine) dichlororuthenium(II) (Ru(bpy)3(2+)) was used as ECL emitting species, and ferrocene carboxylic acid (Fc) was employed as ECL quencher. The ECL-PB biosensor was fabricated by casting the mixture of Nafion and AuNPs onto the surface of glassy carbon electrode to form AuNPs/Nafion film, and then, Ru(bpy)3(2+) was electrostatically adsorbed into the AuNPs/Nafion film; finally, the peptide-tagged with ferrocene carboxylic acid (Fc-peptide) was self-assembled onto the surface of the AuNPs. When PSA was present, it specifically cleaved the Fc-peptide, leading the quencher to leave the electrode and resulting in the increase of the ECL intensity obtained from the resulted electrode in 0.1 M phosphate buffer saline (pH 7.4) containing tri-n-propylamine. The results showed that the increased ECL intensity was directly linear to the logarithm of the concentration of PSA in the range from 5.0 × 10(-12) to 5.0 × 10(-9) g/mL. An extremely low detection limit of 8 × 10(-13) g/mL was achieved because of the signal amplification through AuNPs and the ECL background suppression through Fc as ECL quencher. This work demonstrates that the combination of the direct transduction of peptide cleavage events with the highly sensitive ECL method is a promising strategy for the design of enzymatic cleavage-based ECL biosensors with high sensitivity and selectivity.

An ultrasensitive electrogenerated chemiluminescence peptide-based (ECL-PB) method for the determination of cardiac troponin I (TnI) incorporating amplification of signal reagent-encapsulated liposome was reported for the first time. A synthesized short linear specific binding peptide (FYSHSFHENWPSK) was employed as a molecular recognition element for TnI, which was a reliable biomarker for detecting cardiac injury. Liposomes assembled using a standard sonication procedure were designed as the carrier of ECL signal reagents [bis(2,2'-bipyridine)-4,4'-dicarboxybipyridine ruthenium-di(N-succinimidyl ester) bis(hexafluorophosphate)] for signal amplification. The magnetic capture peptides for the enrichment of the target protein and magnetic separation were synthesized by covalently attaching the peptides to the surface of magnetic beads via an acylation reaction, and the liposome peptides were synthesized by covalently attaching the peptides to the signal reagent-encapsulated liposomes. In the presence of TnI, sandwich-type conjugates were generated in incubation of the magnetic capture peptides and the liposome peptides. After a magnetic separation, the sandwich-type conjugates were treated with ethanol and, thus, a great number of the ECL reagents were released and measured by the ECL method at a bare glassy carbon electrode with a potential pulse of +1.15 V versus Ag/AgCl in the presence of tri-n-propylamine. The increased ECL intensity has good linearity with the logarithm of the TnI concentration in the range from 10 pg/mL to 5.0 ng/mL, with an extremely low detection limit of 4.5 pg/mL. The proposed ECL-PB method was successfully applied to the detection of TnI in human serum samples. This work demonstrated that the employment of the magnetic capture peptides for the enrichment of the target proteins and magnetic separation and the liposome peptides for the signal amplification and polyvalent binding motifs may open a new door to ultrasensitive detection of proteins in clinical analyses.