An electrogenerated chemiluminescence (ECL) based method is described for the determination of hydrogen peroxide (H2O2) released by living cells. It is making use of a fluorine-doped tin oxide (FTO) electrode that was modified with gold nanoparticles (AuNPs). The AuNP-FTO was fabricated by electrodeposition of AuNP on the surface of the FTO electrode and characterized by scanning electron microscopy and electrochemical techniques. The AuNP-FTO exhibits a significant electrocatalytic activity towards luminol-based ECL system compared to a bare FTO. The calibration plot covers the 10 nM to 1 μM H2O2 concentration range, with a 8 nM detection limit (at a signal-to-noise ratio of 3). The method enabled (a) the monitoring of drug-stimulated production of H2O2 released by living cells including RAW 264.7 macrophage cells and HeLa cells; and (b) the determination of inhibitors of H2O2 secretion. The method, due to strong signal amplification by AuNPs, provides a viable tool for determination of H2O2 in biological systems.

•An Ir(III) complex and a Ru complex were employed as potential-resolved ECL labels.•A multiplexed biosensor was fabricated by co-immobilizing Ir(III) and Ru complex labeled peptides on a gold electrode.•The biosensor was facile to determine MMP-2 and MMP-7 in a single run with DL of 5 ng/mL and 10  pg/mL, respectively.•The multiplexed ECL biosensor was successfully used in analyzing two matrix metalloproteinases released from living cells.Highly selective bioassays are extremely important in biological and chemical sensing. Herein, a selective biosensor incorporating sensitive electrogenerated chemiluminescence (ECL) is reported for simultaneous detection of two matrix metalloproteinases (MMPs) that are related to pathological development and progression of cancer. An Ir complex ((dfppy)2Ir(dcbpy)PF6, Ir1) and a Ru complex ((Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2, Ru1) were employed as potential resolved ECL labels. It was found that the Ru1 and Ir1 in the presence of tripropylamine could produce two well-separated strong ECL emissions at +0.9 V and +1.4 V vs Ag/AgCl, respectively, with a large difference of ECL peak potential (∼0.5 V) at the gold electrode, which provided an access for potential-resolved detection of dual targets. A multiplexed biosensor was simply fabricated by co-immobilizing the Ir1 and Ru1 labeled peptides on a gold electrode via self-assembling technique. It was facile to determine MMP-2 and MMP-7 in a single run with detection limit as low as 5 ng/mL and 10 pg/mL, respectively. The advantages of the multiplexed ECL biosensor were further exemplified by analyzing two MMPs released from living HeLa, K562, HCT116 and macrophage cells. The multiplexity of the biosensor will open a new door for facile and rapid detection of multiple biomarkers.Download high-res image (129KB)Download full-size image

•A PLA-ECL bioassay was developed using gold nanoparticles and quenching mechanism.•A detection limit of 0.04 ng/mL was obtained for AFP.•AFP was detected in serum samples with one-step recognition and good accuracy.•The PLA-ECL bioassay shows a proof-of-concept for POCT of protein.A proximity hybridization-regulated electrogenerated chemiluminescence (PLA-ECL) bioassay was developed for the detection of α-fetoprotein (AFP) on basis of the sensitization of gold nanoparticles (AuNPs) and target-induced quenching mechanism. Ru(bpy)32+ was used as ECL signal while ferrocene (Fc) was used as ECL quencher. Ru(bpy)32+ was electrostatically adsorbed into the AuNPs/Nafion film prepared by casting the mixture of Nafion and AuNPs onto the surface of glassy carbon electrode (GCE) to form an ECL platform (Ru(bpy)32+/AuNPs/Nafion/GCE), which displayed strong ECL emissions. A recognition platform was fabricated by self-assembling a capture DNA via thiol-gold bond on the surface of Ru(bpy)32+/AuNPs/Nafion/GCE. After sandwich immunoassay and proximity hybridization assay among capture DNA, AFP, a pair of antibody-oligonucleotide conjugates and a signal probe (DNA-Fc), Fc in DNA-Fc was brought close to the surface of electrode in conjunction with target induced ECL quenching. The ECL intensity decreased with the increasing concentration of the AFP and AFP was monitored with a linear range of 0.05–50 ng/mL along with a detection limit of 0.04 ng/mL. The ECL bioassay is successfully applied to the detection of AFP in serum samples with one-step recognition, short operating time and good accuracy. This method displays great potential for point-of-care testing and commercial application.Download high-res image (175KB)Download full-size image

We synthesized and studied a new class of A–(π-D)3 type donor–acceptor molecular graphene, triphenothiazinyl triazacoronenes, [2,3,6,7,10,11-hexamethoxy-4,8,12-tri-(10-alkyl-phenothiazine)-1,5,9-triazacoronene] (TPTZ-TAC derivatives). These molecules have been synthesized by employing three electron-rich triphenothiazine (PTZ) groups as electron donors, which were linked to an electron acceptor of an electron-deficient triazacoronene core (2,3,6,7,10,11-hexamethoxy-1,5,9-triazacoronene, TAC). These donor–acceptor molecular graphenes exhibited unique multiple fluorescence and electro-generated chemiluminescence (ECL) emissions that are dependent on the concentration of these molecules, attributed to strong π-stacking interactions. The electrochemical behaviour showed two closely spaced consecutive reversible one-electron oxidations occurring on the PTZ groups and a reversible one-electron reduction localizing on the TAC core. The absorption and fluorescence emission spectra reveal that the electronic properties are affected by the intramolecular charge transfer (ICT) interaction from the PTZ donors to TAC acceptors in the excited state. The effect of π-stacking interaction was noticed for the excimer emission at the lower energy region. The ICT properties of the TPTZ-TACs have been analyzed by concentration-dependence and solvatochromism of fluorescence spectral studies. Remarkably, multiple ECL emissions were produced from the TPTZ-TAC derivatives via a radical ion annihilation and coreactant process through the formation of a charge-transfer excimer state. This work demonstrates that the attachment of electron-rich PTZ groups as electron donors to an electron-deficient TAC core as an electron acceptor, is a promising route to improve the optoelectronic properties of the molecular graphene TAC core. The D–A molecule excimers will represent a new approach to red luminescence and a means to enhance the fluorescence efficiency.

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.

DNA methylation is used to dynamically reprogram cells in the course of early embryonic development in mammals. 5-Hydroxymethylcytosine in DNA (5-hmC-DNA) plays essential roles in the demethylation processes. 5-Methylcytosine in DNA (5-mC-DNA) is oxidized to 5-hmC-DNA by 10–11 translocation proteins, which are relatively high abundance in embryonic stem cells and neurons. A new method was developed herein to quantify 5-hmC-DNA based on selective electrogenerated chemiluminescence (ECL) labeling with the specific oxidation of 5-hmC to 5-fC by KRuO4. A thiolated capture probe (ssDNA, 35-mer) for the target DNA containing 5-hmC was self-assembled on a gold surface. The 5-hmC in the target DNA was selectively transformed to 5-fC via oxidation by KRuO4 and then subsequently labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI). The ABEI-labeled target DNA was hybridized with the capture probe on the electrode, resulting in a strong ECL emission. An extremely low detection limit of 1.4 × 10–13 M was achieved for the detection of 5-hmC-DNA. In addition, this ECL method was useful for the quantification of 5-hmC in serum samples. This work demonstrates that selective 5-hmC oxidation in combination with an inherently sensitive ECL method is a promising tactic for 5-hmC biosensing.

•A sensitive and versatile ECL biosensing platform is developed for monitoring protein kinase activity and inhibition.•Ru(bpy)32+ functionalized gold nanoparticles are used as thiol-versatile signal probe.•The strategy exhibits unique advantages of high sensitivity, good selectivity and versatility.•The strategy is promising for multiple protein kinase assay and kinase inhibitor profiling.A novel, sensitive and versatile electrogenerated chemiluminescence biosensing platform is developed for monitoring activity and inhibition of protein kinase based on Ru(bpy)32+ functionalized gold nanoparticles (Ru(bpy)32+-AuNPs) mediated signal transduction. Ru(bpy)32+-AuNPs were formed by functionalizing AuNPs with Ru(bpy)32+ through electrostatic interactions and were used as thiol-versatile signal probe. Casein kinase II (CK2) and cAMP-dependent protein kinase (PKA), two classical protein kinase implicated in disease, were chosen as model protein kinases while a CK2-specific peptide (CRRRADDSDDDDD) and a PKA-specific peptide (CLRRASLG) were employed as molecular substrate for CK2 and PKA, respectively. The specific peptide was self-assembled onto the gold electrode via Au–S bond to form ECL biosensor. Upon thiophosphorylation of the peptide on the electrode in the presence of protein kinase and co-substrate adenosine-5’-(γ-thio)-triphosphate, Ru(bpy)32+-AuNPs was assembled onto the thiophosphorylated peptides via Au–S bond. The Ru(bpy)32+-AuNPs attached on electrode surface produce detectable ECL signal in the presence of coreactant tripropylamine. This strategy is promising for multiple protein kinase assay and kinase inhibitor profiling with high sensitivity, good selectivity and versatility. The ECL intensity is proportional to the activity of CK2 in the range of 0.01–0.5 unit/mL with a low detection limit of 0.008 unit/mL and to the activity of PKA in the range of 0.01–0.4 unit/mL with a detection limit of 0.005 unit/mL. Additionally, this assay was applied to the detection of CK2 in serum samples and the inhibition of CK2 and PKA. This work demonstrates that the developed ECL method can provide a sensitive and versatile platform for the detection of kinase activity and drug-screening.

•Two cyclometalated iridium(III) complex, (pq)2Ir(sa) and (pq)2Ir(psa), were synthesized and characterized.•(pq)2Ir(sa) and (pq)2Ir(psa) show good solubility in water, lower oxidation potential and higher ECL efficiency.•Small amount of water can greatly increase the ECL intensity of two complexes in acetonitrile solvent.•The water-enhanced ECL pathway in the presence of TPA is proposed as a double coreactant ECL pathway.Two cyclometalated iridium(III) complexes, (pq)2Ir(sa) and (pq)2Ir(psa), with 2-phenylquinoline (pq) as the C^N main ligand and succinylacetone (sa) or 4,6-dioxo-6-phenylhexanoic acid (psa) as the O^O ancillary ligand, were synthesized at first time. The solubility was reached down to 0.1 mM for (pq)2Ir(sa) and 10 μM for (pq)2Ir(psa) in acetonitrile-water (v/v = 5:95) solution. The photophysical, electrochemical and electrogenerated chemiluminescence (ECL) properties of (pq)2Ir(sa) and (pq)2Ir(psa) in acetonitrile were mainly investigated. The oxidation peak potential of (pq)2Ir(sa) and (pq)2Ir(psa) appears at + 0.79 V and + 0.8 V vs SCE. (pq)2Ir(sa) and (pq)2Ir(psa) display orange ECL emission with a maximum wavelength at 621 nm and 610 nm, respectively. ECL efficiencies of (pq)2Ir(sa) and (pq)2Ir(psa) in acetonitrile containing tripropylamine were calculated to be 21.3-folds and 35.4-folds higher than that of Ru(bpy)32 +. It was surprised to find that small amount of water can greatly increase the ECL intensity of these two complexes in acetonitrile containing tripropylamine. The ECL methods for the determination of trace water in organic solvent were developed in the range from 0.02 to 4.0% (v/v) and 0.05 to 4.0% (v/v) with detection limit of 0.01% and 0.03% for (pq)2Ir(sa) and (pq)2Ir(psa), respectively. It was found that the O^O ancillary ligand played a key function in the water-enhanced ECL emission. A double coreactant ECL pathway is proposed for the water-enhanced ECL emission. This work demonstrates that (pq)2Ir(sa) and (pq)2Ir(psa) have lower potential and high ECL coefficient, which are promising for the effective use as ECL emitter in analytical application.

•An electrochemical biosensor for the determination of carcinoembryonic antigen was developed.•Lectins including wheat-germ agglutinin, Lens culinaris agglutinin, concanavalin A were employed as molecular recognition elements.•The detection limit for CEA was 0.03 ng/mL, 0.05 ng/mL, and 0.01 ng/mL using Con A, WGA, and LCA as molecular recognition elements, respectively.•A lectin-based biosensor array was used to evaluate the glycan expression of CEA N-glycan.•A biosensor array was used to discriminate CEA between healthy and cancer serum samples.An electrochemical lectin-based biosensor array employing lectin as molecular recognition elements was developed for sensitive detection and discrimination of carcinoembryonic antigen (CEA) based on dual signal amplification of gold nanoparticles and enzymatic catalysis. The electrochemical biosensor was fabricated by self-assembly of cysteamine on the surface of gold nanoparticle-modified screen-printed carbon electrodes and subsequently covalently coupling lectins on the surface of the cysteamine-modified electrode via the amidation reaction. A lectin–target-antibody sandwich-type conjugate was formed when the biosensor was successively reacted with target protein and horseradish peroxidase labeled anti-CEA antibody probe. In the presence of hydroquinone and hydrogen peroxide, the resulted biosensor produced an obvious catalysis current signal. The catalytic current at −0.25 V was linear with the concentration of CEA in the range of 1 ng/mL to 10 ng/mL with detection limit of 0.03 ng/mL and 0.05 ng/mL using concanavalin A and wheat-germ agglutinin as molecular recognition element, respectively, and in the range of 0.5 ng/mL to 7 ng/mL with detection limit of 0.01 ng/mL using Lens culinaris agglutinin as molecular recognition element. Additionally, the lectin-based biosensor array was successfully applied to evaluate the glycan expression of CEA N-glycan and discriminate CEA between healthy and cancer serum samples. The lectin-based biosensor array endows a feasibility tool for clinical diagnosis in complex biological systems and shows great promise for cancer detection and further pathological study of carcinogenesis.

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.

Electrogenerated chemiluminescence (ECL) with different emission colors is important in the development of multichannel analytical techniques. In this report, five new heteroleptic iridium(III) complexes were synthesized, and their photophysical, electrochemical, and ECL properties were studied. Here, 2-(2,4-difluorophenyl)pyridine (dfppy, complex 1), 2-phenylbenzo[d]thiazole (bt, complex 2), and 2-phenylpyridine (ppy, complex 3) were used as the main ligands to tune the emission color, while avobenzone (avo) was used as the ancillary ligand. For comparison, complexes 4 and 5 with 2-phenylpyridine and 2-phenylbenzo[d]thiazole as the main ligand, respectively, and acetyl acetone (acac) as the ancillary ligand were also synthesized. All five iridium(III) complexes had strong intraligand absorption bands (π–π*) in the UV region (below 350 nm) and a featureless MLCT (d−π*) transition in the visible 400–500 nm range. Multicolored emissions were observed for these five iridium(III) complexes, including green, orange, and red for complexes 4, 5, 2, 1, 3, respectively. Density functional theory calculations indicate that the electronic density of the highest occupied molecular orbital is entirely located on the C^N ligands and the iridium atom, while the formation of the lowest unoccupied molecular orbital (LUMO) is complicated. The LUMO is mainly assigned to the ancillary ligand for complexes 1 and 3 but to the C^N ligand for complexes 2, 4, and 5. Cyclic voltammetry studies showed that all these complexes have a reversible oxidation wave, but no reduction waves were found in the electrochemical windows of CH2Cl2. The E1/2ox values of these complexes ranged from 0.642 to 0.978 V for complexes 3, 4, 2, 5, 1, (in increasing order) and are all lower than that of Ru(bpy)32+. Most importantly, when using tripropylamine as a coreactant, complexes 1–5 exhibited intense ECL signals with an emission wavelength centered at 616, 580, 663, 536, and 569 nm, respectively. In addition, complexes 1, 2, and 5 displayed approximately 2, 11, and 214 times higher ECL efficiencies than Ru(bpy)32+ under identical conditions.

•A cyclometalated iridium(III) complex, (dfppy)2Ir(PyBiz), was synthesized and characterized.•Intense green ECL emissions from (dfppy)2Ir(PyBiz) were observed in annihilation and coreactant processes.•Green ECL emission of (dfppy)2Ir(PyBiz) can be distinguished from red emission of Ru(bpy)32 + in a mixed solution.A novel cyclometalated iridium(III) complex, (dfppy)2Ir(PyBiz), was synthesized by using 2-(2,4-difluorophenyl)pyridine (dfppy) as the C^N main ligand and 2-(2-pyridyl)benzimidazolato-N,N′ (PyBiz) as the N^N ancillary ligand. The photophysical, electrochemical and electrogenerated chemiluminescence (ECL) properties of (dfppy)2Ir(PyBiz) were investigated. (dfppy)2Ir(PyBiz) shows a green photoluminescence emission with a maximum wavelength at 527 nm and quasi-reversible redox behaviors in acetonitrile solvent. Intense green ECL emissions that could be seen with the naked eyes were observed in annihilation and coreactant processes and the ECL spectra generated from three processes are identical to its photoluminescence spectrum, indicating that the same excited state is formed in these processes. The green ECL emission of (dfppy)2Ir(PyBiz) can be distinguished from the red emission of Ru(bpy)32 +, which provides the possibility of the identification of (dfppy)2Ir(PyBiz) and Ru(bpy)32 + in a single solution containing both compounds. The green ECL emission of (dfppy)2Ir(PyBiz) with high ECL efficiency makes it promising to develop ECL luminophore for multiple wavelength and potential use in multichannel analytical applications.

•The electrochemistry and ECL of benzoxazole derivatives were firstly reported.•The substituent effects on the electrochemical and ECL properties were studied.•All benzoxazole derivatives emit blue ECL emissions.•The substituents at 5-position of benzoxazole ring can modulate electrochemical properties.•The alkoxy chain length at 4′-position of biphenyl moiety has no effect on electrochemical properties.Benzoxazole derivatives, an important type of heterocyclic compounds, have attracted significant interest in photonics and electronics since they are highly efficient luminophores with high photo-stability. The electrochemistry and electrogenerated chemiluminescence (ECL) of benzoxazole derivatives were reported and the substituent effects on the electrochemical and ECL properties of benzoxazole derivatives were investigated in an acetonitrile:benzene (v:v = 1:1) solvent for the first time. The studied derivatives contain a 2-biphenyl benzoxazole moiety with the substituents (–CH3, –Cl, and –NO2 groups) at the 5-position of the benzoxazole ring and the alkoxy chains (CnH2n+1, n = 2–10) at the 4′-position of the biphenyl ring. The electrochemical and ECL behavior of benzoxazole derivatives were found to be dependent on the nature of the substituents at the 5-position of the benzoxazole ring but independent on the alkoxy chain length at 4′-position of biphenyl moiety. Under ion annihilation conditions, 5B–H (2-(4′-pentyloxy-biphenyl-4-yl)-benzoxazole), 5B–Me (2-(4′-pentyloxy- biphenyl-4-yl)-5-methylbenzoxazole), and 5B–Cl (2-(4′-pentyloxy-biphenyl-4-yl)-5-chlorobenzoxazole), display high ECL blue emission (ECL efficiencies are 0.51, 0.54, and 0.78 for 5B–H, 5B–Me, and 5B–Cl, respectively), while 5B-NO2 (2-(4′-pentyloxy-biphenyl-4-yl)-5-nitrobenzoxazole) does low ECL emission. In the presence of benzoyl peroxide, four derivatives exhibit strong ECL blue emissions. The results provide important electrochemical and ECL information of benzoxazole derivatives as well as their structure–properties relationships for future research.

A simple and sensitive electrochemical method was developed for the determination of trypsin by employing a specific heptapeptide (CRRRRRR) as a substrate. The positively charged heptapeptide substrate is self-assembled on the surface of a gold electrode through the thiol group of the cysteine (C) at the end of the peptide, which prevents the electrochemical probe [the ruthenium(III) hexammine complex] to access the electrode. The substrate peptide is hydrolyzed by trypsin, and this causes the fragments to leave the electrode and, consequently, the electrochemical signal to increase. The results show that the increase in the square wave voltammetric current of the ruthenium probe is linearly related to the activity of trypsin in the range from 0.0047 to 0.052 U·mL−1 with a detection limit of 0.0012 U·mL−1. This work demonstrates that the enzymatic cleavage of the substrate can be directly converted to an electrical signal to provide a simple and sensitive method for the determination of trypsin.

A sensitive electrogenerated chemiluminescence peptide-based (ECL-PB) biosensing method for the determination of protein was developed by employing peptide-integrating Ru(bpy)32+(bpy = 2,2′-bipyridine)-functionalized gold nanoparticles (Ru(bpy)32+-AuNPs-peptide) as nanoprobe. Cardiac troponin I (cTnI), a reliable clinical biomarker for the detection of cardiac injury, was chosen as target protein, while a specific binding peptide (CFYSHSFHENWPS) was used as molecular recognition element. AuNPs were firstly functionalized with Ru(bpy)32+ through electrostatic interactions between citrate-capped AuNPs and Ru(bpy)32+ to form Ru(bpy)32+-AuNPs aggregates and then functionalized with peptide through Au-S bounds to form Ru(bpy)32+-AuNPs-peptide nanoprobe. AuNPs not only can capture numerous signal-generating molecules, resulting in high ECL intensity but also can capture a significant amount of the peptide, providing poly binding motif. The specific capture peptide was self-assembled on the surface of a gold electrode and then incubated with the target cTnI and Ru(bpy)32+-AuNPs-peptide successively. A sandwich-type peptide/cTnI/Ru(bpy)32+-AuNPs-peptide conjugate was formed on the surface of the electrode and an ECL signal was obtained in the presence of tri-n-propylamine. The novel biosensing method facilitates the sensitive detection of cTnI in the range from 3.0 × 10−12 g mL−1 to 7.0 × 10−11 g mL−1 with a low detection limit of 0.5 pg mL−1. This work provides a promising strategy for the determination of proteins with simplicity, high sensitivity, and selectivity.

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)32+) 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)32+ 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.

A sensitive electrogenerated chemiluminescence (ECL) peptide-based biosensor was fabricated for the determination of troponin I (TnI) by employing gold nanoparticles as amplification platform. Two specific peptides including peptide1 with a sequence of CFYSHSFHENWPS and peptide2 with a sequence of FYSHSFHENWPSK were employed as capture peptide and report peptide, respectively. The peptide2 was labeled with ruthenium bis(2,2′-bipyridine) (2,2′-bipyridine-4,4′-dicarboxylic acid)-N-hydroxysuccinimide ester (Ru(bpy)2(dcbpy)NHS) at NH2-containing lysine via acylation reaction and utilized as the ECL probe. Gold nanoparticles were electrodeposited onto gold electrode and used as an amplification platform. The peptide-based biosensor was fabricated by self-assembling peptide1 onto the surface of gold nanoparticles-modified gold electrode through a thiol-containing cysteine at the end of the peptide1. When the biosensor reacted with target TnI, and then incubated with the ECL probe, a strong ECL response was electrochemically generated. The ECL intensity is directly proportional to the logarithm of the concentration of TnI in the range from 1 to 300 pg/mL. The biosensor employing gold nanoparticles as amplification platform shows high sensitivity for the detection of TnI with a detection limit of 0.4 pg/mL (S/N = 3). Moreover, the biosensor is successfully applied to analysis of TnI in human serum sample. This work demonstrates that the combination of a highly binding peptide with nanoparticle amplification is a great promising approach for the design of ECL biosensor.

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.

•Double electrochemical covalent coupling method for the fabrication of immunosensor is reported.•Electro-click chemistry and diazonium chemistry are used.•An alkyne functionalized protein is used as the molecular recognition.•The possibility of electrochemical addressable immobilization of proteins is explored.•The feasibility for fabricating the amperometric immunosensor array is explored.A double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor was developed. As a proof-of-concept, a designed alkyne functionalized human IgG was used as a capture antibody and a HRP-labeled rabbit anti-goat IgG was used as signal antibody for the determination of the anti-human IgG using the sandwich model. The immunosensor was fabricated by electrochemically grafting a phenylazide on the surface of a glassy carbon electrode, and then, by coupling the alkyne functionalized human IgG with the phenylazide group through an electro-click chemistry in the presence of Cu(II). The amperometric measurement for the determination of the anti-human IgG was performed after the fabricated immunosensor was incubated with the target anti-human IgG and then with the HRP-labeled anti-goat IgG at −0.25 V in 0.10 M PBS (pH 7.0) containing 0.1 mM hydroquinone and 2.0 mM H2O2. The results showed that the increased current was linear with the logarithm of the concentration of the anti-human IgG in the range from 1.0 × 10−10 g mL−1 to 1.0 × 10−8 g mL−1 with a detection limit of 3 × 10−11 g mL−1. Furthermore, the feasibility of the double electrochemical covalent coupling method proposed in this work for fabricating the amperometric immunosensor array was explored. This work demonstrates that the double electrochemical covalent coupling method is a promising approach for the fabrication of the immunosensor and immunosensor array.

Herein, we demonstrate the digital electrogenerated chemiluminescence biosensor for the determination of multiple proteins based on Boolean logic gate. A reagentless, sensitive, disposable and multiplexed ECL sensing platform for one-spot simultaneous determination of multiple proteins is described. This system is designed for OR operation using two proteins as stimulus inputs.

A label-free electrochemical impedance spectroscopy (EIS) biosensor for the sensitive determination and discrimination of alpha-fetoprotein (AFP) was developed by employing wheat-germ agglutinin (WGA) lectin as molecular recognition element. The EIS biosensor was fabricated by adsorbing carboxyl-functionalized single-wall carbon nanotubes (SWNTs) onto a screen-printed carbon electrode (SPCE) and subsequently covalently coupling WGA onto the surface of the SWNTs-modified electrode. Upon binding of AFP to the biosensor, the electron transfer resistance was increased and the increase in the electron transfer resistance was linearly proportional to the logarithm of the concentration of AFP in the range from 1 to 100 ng/L with a detection limit of 0.1 ng/L. It was found that the employment of SWNTs as immobilization platform could reduce the background and enhance the EIS response. Moreover, the lectin-based biosensor array fabricated with different lectins was used to evaluate the glycan expression of AFP N-glycan and discriminate AFP between healthy and cancer patients serum samples. This work demonstrates that the employment of carbon nanotubes as immobilization platform and lectin as molecular recognition element in biosensor array is a promising approach for the determination and discrimination of glycoproteins for cancer diagnosis. The strategy proposed in this work could further be used for high-throughput, label-free profiling of the glycan expression of cancer-related glycoproteins and to develop methods for cancer diagnosis in the early stages.Highlights► An EIS biosensor for the determination of AFP was developed. ► Lectin was employed as the molecular recognition element. ► The detection limit for AFP was 0.1 ng/L. ► A biosensor array was used to evaluate the glycan expression of AFP N-glycan. ► A biosensor array was used to discriminate AFP between healthy and cancer serums.

A highly sensitive and attractive antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample was developed. The aptasensor was fabricated by co-assembling thiol-modified anti-thrombin binding aptamer, dithiothreitol and mercaptohexanol on the surface of gold electrode. The performance of aptasensor was characterized by atomic force microscopy, contact angle and electrochemical impedance spectroscopy. In the measurement of thrombin, the change in interfacial electron transfer resistance of aptasensor was monitored using a redox couple of Fe(CN)63−/4−. The increase in the electron transfer resistance was linearly proportional to the concentration of thrombin in the range from 1.0 to 20 ng/mL and a detection limit of 0.3 ng/mL thrombin was achieved. The fabricated aptasensor displayed attractive antifouling properties and allowed direct quantification of extrinsic thrombin down to 0.08 ng/mL in undiluted serum sample. This work provides a promising strategy for clinical application with impressive sensitivity and antifouling characteristics.Highlights► A simple, highly sensitive and attractive antifouling impedimetric aptasensor for determination of thrombin in undiluted serum sample was developed. ► A ternary monolayer was fabricated by co-assembling thiol-modified anti-thrombin binding aptamer, dithiothreitol and mercaptohexanol on gold electrode to form aptasensor. ► The fabricated aptasensor displayed attractive antifouling properties and it allowed the direct quantification of target thrombin down to 0.08 ng/mL in undiluted serum sample.

A novel electrochemical immunosensor array for the simultaneous detection of multiple tumor markers was developed by incorporating electrochemically addressing immobilization and one signal antibody strategy. As a proof-of-principle, an eight-electrode array including six carbon screen-printed working electrodes was used as a base array for the analysis of two important tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) and a horseradish peroxidase-labeled antibody was employed as a signal antibody. The immunosensor in the array was fabricated in sequence by covalently coupling the capture antibody onto the surface of the desired working electrode, which was firstly electrochemically addressably grafted with an aminophenyl group by reduction of in situ generated aminophenyl diazonium cation generated from p-phenylenediamine, using glutaraldehyde as cross-linker. This allowed the selective immobilization of the capture antibody at the desired position on a single array via an electrochemical operation. The immunoassay in sandwich mode was performed by specifically binding the targets, second antibodies and one signal antibody to the immunosensor array. The result showed that the steady current density was directly proportional to the concentration of target CEA/AFP in the range from 0.10 to 50 ng mL−1 with a detection limit of 0.03 ng mL−1 for CEA and 0.05 ng mL−1 for AFP (S/N = 3), respectively. This work demonstrates that the employment of an electrochemically addressing method for the fabrication of an immunosensor array and one signal antibody is a promising approach for the determination of multiple tumor markers in clinical samples.

A novel homogeneous electrogenerated chemiluminescence (ECL) peptide-based method for the determination of troponin I (TnI) was developed by using peptide (FYSHSFHENWPSK) as a molecular recognition element and ruthenium bis(2,2′-bipyridine) (2,2′-bipyridine-4,4′-dicarboxylic acid)-N-hydroxysuccinimide ester (Ru(bpy)2(dcbpy)NHS) as an ECL label. Ru(bpy)2(dcbpy)NHS was covalently labeled onto the peptide via acylation reaction and was utilized as an ECL probe. In the presence of TnI, a decrease in ECL signal was observed upon the binding event between peptide and target TnI. The ECL intensity versus the concentration of TnI was linear in the range from 7.8 × 10−10 g mL−1 to 7.8 × 10−8 g mL−1 with a detection limit of 1.2 × 10−10 g mL−1. The relative standard deviation for 2.6 × 10−9 g mL−1 was 3.8%. The proposed method has been applied to assay TnI in human control serum with satisfactory results. This work demonstrates that the combination of small peptide with a highly sensitive ECL technique for homogeneous ECL peptide-based bioassay is a great promising approach for simple, sensitive and selective determination of proteins.

A novel electrogenerated chemiluminescence peptide-based (ECL-PB) biosensor for highly sensitive and selective determination of prostate-specific antigen (PSA) was developed. A helix peptide (CHSSKLQK) was served as a molecular recognition element and ruthenium bis(2,2′-bipyridine) (2,2′-bipyridine-4,4′-dicarboxylic acid)-N-hydroxysuccinimide ester (Ru(bpy)2(dcbpy)NHS) was used as an ECL label. The helix peptide was labeled with the ECL label at NH2-containing lysine and utilized as ECL probe (Ru-peptide). The ECL-PB biosensor was fabricated by immobilizing the ECL probe onto a gold electrode surface via self-assembling technique through a thiol-containing cysteine at the end of the peptide. The principle of ECL measurement is based on the specific proteolytic cleavage event of Ru-peptide on the gold electrode surface in the presence of PSA, resulting in a decrease of ECL signal. The decreased ECL intensity was directly linear to the concentration of PSA in the range from 1.0×10−10 g/mL to 8.0×10−9 g/mL with a detection limit of 3.8×10−11 g/mL. This work demonstrates that the direct transduction of peptide cleavage events into an ECL signal provides a simple and sensitive method for detecting target protein.Highlights► An ECL peptide-based biosensor for prostate-specific antigen was fabricated. ► A helix peptide was employed as molecular recognition element. ► The detection limit for PSA was 0.038 ng/mL. ► The kinetic studies of peptide with PSA were performed. ► The method provides a promising strategy for detecting protein with simplicity.

An electrogenerated chemiluminescence (ECL) sensor for the determination of propranolol hydrochloride was fabricated by employing tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) as an ECL signal producer and graphene as a modified material. The ECL sensor was fabricated by adsorbing Ru(bpy)32+ onto a mixture of graphene and Nafion. The introduction of conductive graphene into Nafion was found to greatly enhance the ECL intensity of Ru(bpy)32+ and the ECL intensity of Ru(bpy)32+ decreased in the presence of propranolol hydrochloride. Based on this phenomenon, a sensitive and stable ECL method was developed for the determination of propranolol hydrochloride by employing a Ru(bpy)32+–graphene–Nafion modified electrode. Under the optimized conditions, the ECL intensity was linear with the concentration of propranolol hydrochloride over a range of 1.0 × 10−10 to 5.0 × 10−9 mol L−1 with a detection limit of 3 × 10−11 mol L−1. The ECL sensor exhibited long-term stability with a relative standard deviation of 4.1% for sixteen continuous determinations of 2.0 × 10−9 mol L−1propranolol hydrochloride. The developed method has been successfully applied to the determination of propranolol hydrochloride in pharmaceutical samples.

An ultra-trace voltammetric method was developed for the determination of single strand DNA (ss-DNA) related to the human immunodeficiency virus type 1 (HIV-1). It is based on the signal amplification of carbon nanotubes loaded with silver nanoparticles and placed on a gold microelectrode. The capture ss-DNA (a 21-mer) possessing a thiol group at the 3′ end was self-assembled onto the surface of the gold microelectrode. It was then hybridized with target HIV-1 ss-DNA (a 42-mer) and further hybridized with the electrochemical probe (a 18-mer ss-DNA) tagged with multiwall carbon nanotubes and loaded with silver nanoparticles. The resulting formation of a DNA sandwich conjugate led to a strong electrochemical oxidation signal that was linearly proportional to the concentration of HIV-1 ss-DNA in the range from 1.0 to 100 pM. The detection limit was 0.5 pM (at an S/N of 3). This was equivalent to 0.05 fmol of HIV-1 ss-DNA in a volume of 20 μL. The relative standard deviation was 4.0% at 1.0 pM (n = 11). Non-complementary ss-DNA of HIV-1 ss-DNA was effectively discriminated. This work demonstrates that the employment of the microelectrode and a sandwich hybridization model is promising in terms of sensitive and selective electrochemical detection of DNA.

A highly sensitive electrogenerated chemiluminesence (ECL) method for the determination of riboflavin was developed based on the enhancement of ECL intensity of lucigenin at room temperature ionic liquids (RTILs) modified gold electrode. RTILs modified gold electrode exhibited excellent electrochemical and ECL property to lucigenin system and the ECL intensity of lucigenin was greatly enhanced by riboflavin. The characterization of the RTILs modified electrode and the attractive performance of the sensitive ECL method for the determination of riboflavin were investigated. Under the optimized conditions, the ECL intensity was directly proportional to the concentration of riboflavin in the range from 5.0 × 10−10 g/mL to 1.0 × 10−8 g/mL with the detection limit of 1 × 10−10 g/mL. The method has been applied to the determination of riboflavin in the pharmaceutical preparations with satisfactory recovery from 96% to 101%. This work demonstrates that the incorporation of ECL method with RTILs modified electrode is a promising strategy for the determination of organic compounds with high sensitivity and good reproducibility.

A sensitive competitive flow injection chemiluminescence (CL-FIA) immunoassay for immunoglobulin G (IgG) was developed using gold nanoparticle as CL label. In the configuration, anti-IgG antibody was immobilized on a glass capillary column surface by 3-(aminopropyl)-triethoxysilane and glutaraldehyde to form immunoaffinity column. Analyte IgG and gold nanoparticle labeled IgG were passed through the immunoaffinity column mounted in a flow system and competed for the surface-confined anti-IgG antibody. CL emission was generated from the reaction between luminol and hydrogen peroxide in the presence of Au (III), generated from chemically oxidative dissolution of gold nanoparticle by an injection of 0.10 mol L−1 HCl–0.10 mol L−1 NaCl solution containing 0.10 mmol L−1 Br2. The concentration of analyte IgG was inversely related to the amount of bound gold nanoparticle labeled IgG and the CL intensity was linear with the concentration of analyte IgG from 1.0 ng mL−1 to 40 ng mL−1 with a detection limit of 5.2 × 10−10 g mL−1. The whole assay time including the injections and washing steps was only 30 min for one sample, which was competitive with CL immunoassays based on a gold nanoparticle label and magnetic separation. This work demonstrates that the CL immunoassay incorporation of nanoparticle label and flow injection is promising for clinical assay with sensitivity and high-speed.Graphical abstractHighlights► A sensitive competitive flow injection chemiluminescence immunoassay for immunoglobulin G was developed using gold nanoparticle as CL label. ► The sensitivity of CL immunoassay was much improved because of the release of a large number of Au (III) from each gold nanoparticle used as label. A lower detection limit of 5.2 × 10−10 g mL−1 was obtained for IgG. ► The developed competitive CL-FIA immunoassay of IgG is easily performed with high rapidity because of employing flow injection system. The whole assay time including the injections and washing steps was only 30 min for one sample.

A simple and sensitive biosensor was developed for the determination of human immunoglobulin G (IgG). Protein A was employed as molecular receptor and electrochemical impedance spectroscopy (EIS) was used as detection technique. The biosensor was obtained by self-assembling protein A on a gold electrode. The surface morphology of the self-assembled layer before and after interaction with IgG was studied by atomic force microscopy. Protein A bound specifically to the Fc portion of IgG, and this caused a change in the resistance of the interfacial electron transfer when using a ferrocyanide redox couple as a probe. The increase of the resistance of the electron transfer was linearly related to the concentration of IgG in the range from 10 ng.mL−1 to 1.0 μg.mL−1, with a detection limit of 5 ng.mL−1. The work demonstrates that protein A is a versatile matrix for the immobilization of antibodies.

We synthesized and studied a new class of A–(π-D)3 type donor–acceptor molecular graphene, triphenothiazinyl triazacoronenes, [2,3,6,7,10,11-hexamethoxy-4,8,12-tri-(10-alkyl-phenothiazine)-1,5,9-triazacoronene] (TPTZ-TAC derivatives). These molecules have been synthesized by employing three electron-rich triphenothiazine (PTZ) groups as electron donors, which were linked to an electron acceptor of an electron-deficient triazacoronene core (2,3,6,7,10,11-hexamethoxy-1,5,9-triazacoronene, TAC). These donor–acceptor molecular graphenes exhibited unique multiple fluorescence and electro-generated chemiluminescence (ECL) emissions that are dependent on the concentration of these molecules, attributed to strong π-stacking interactions. The electrochemical behaviour showed two closely spaced consecutive reversible one-electron oxidations occurring on the PTZ groups and a reversible one-electron reduction localizing on the TAC core. The absorption and fluorescence emission spectra reveal that the electronic properties are affected by the intramolecular charge transfer (ICT) interaction from the PTZ donors to TAC acceptors in the excited state. The effect of π-stacking interaction was noticed for the excimer emission at the lower energy region. The ICT properties of the TPTZ-TACs have been analyzed by concentration-dependence and solvatochromism of fluorescence spectral studies. Remarkably, multiple ECL emissions were produced from the TPTZ-TAC derivatives via a radical ion annihilation and coreactant process through the formation of a charge-transfer excimer state. This work demonstrates that the attachment of electron-rich PTZ groups as electron donors to an electron-deficient TAC core as an electron acceptor, is a promising route to improve the optoelectronic properties of the molecular graphene TAC core. The D–A molecule excimers will represent a new approach to red luminescence and a means to enhance the fluorescence efficiency.

A novel homogeneous electrogenerated chemiluminescence (ECL) peptide-based method for the determination of troponin I (TnI) was developed by using peptide (FYSHSFHENWPSK) as a molecular recognition element and ruthenium bis(2,2′-bipyridine) (2,2′-bipyridine-4,4′-dicarboxylic acid)-N-hydroxysuccinimide ester (Ru(bpy)2(dcbpy)NHS) as an ECL label. Ru(bpy)2(dcbpy)NHS was covalently labeled onto the peptide via acylation reaction and was utilized as an ECL probe. In the presence of TnI, a decrease in ECL signal was observed upon the binding event between peptide and target TnI. The ECL intensity versus the concentration of TnI was linear in the range from 7.8 × 10−10 g mL−1 to 7.8 × 10−8 g mL−1 with a detection limit of 1.2 × 10−10 g mL−1. The relative standard deviation for 2.6 × 10−9 g mL−1 was 3.8%. The proposed method has been applied to assay TnI in human control serum with satisfactory results. This work demonstrates that the combination of small peptide with a highly sensitive ECL technique for homogeneous ECL peptide-based bioassay is a great promising approach for simple, sensitive and selective determination of proteins.

Herein, we demonstrate the digital electrogenerated chemiluminescence biosensor for the determination of multiple proteins based on Boolean logic gate. A reagentless, sensitive, disposable and multiplexed ECL sensing platform for one-spot simultaneous determination of multiple proteins is described. This system is designed for OR operation using two proteins as stimulus inputs.

An electrogenerated chemiluminescence (ECL) sensor for the determination of propranolol hydrochloride was fabricated by employing tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) as an ECL signal producer and graphene as a modified material. The ECL sensor was fabricated by adsorbing Ru(bpy)32+ onto a mixture of graphene and Nafion. The introduction of conductive graphene into Nafion was found to greatly enhance the ECL intensity of Ru(bpy)32+ and the ECL intensity of Ru(bpy)32+ decreased in the presence of propranolol hydrochloride. Based on this phenomenon, a sensitive and stable ECL method was developed for the determination of propranolol hydrochloride by employing a Ru(bpy)32+–graphene–Nafion modified electrode. Under the optimized conditions, the ECL intensity was linear with the concentration of propranolol hydrochloride over a range of 1.0 × 10−10 to 5.0 × 10−9 mol L−1 with a detection limit of 3 × 10−11 mol L−1. The ECL sensor exhibited long-term stability with a relative standard deviation of 4.1% for sixteen continuous determinations of 2.0 × 10−9 mol L−1propranolol hydrochloride. The developed method has been successfully applied to the determination of propranolol hydrochloride in pharmaceutical samples.