Analytical and Bioanalytical Chemistry (v.408, #25)

Fiber-based monolithic columns for liquid chromatography by Michael Ladisch; Leyu Zhang (6871-6883).
Fiber-based monoliths for use in liquid chromatographic separations are defined by columns packed with aligned fibers, woven matrices, or contiguous fiber structures capable of achieving rapid separations of proteins, macromolecules, and low molecular weight components. A common denominator and motivating driver for this approach, first initiated 25 years ago, was reducing the cost of bioseparations in a manner that also reduced residence time of retained components while achieving a high ratio of mass to momentum transfer. This type of medium, when packed into a liquid chromatography column, minimized the fraction of stagnant liquid and resulted in a constant plate height for non-adsorbing species. The uncoupling of dispersion from eluent flow rate enabled the surface chemistry of the stationary phase to be considered separately from fluid transport phenomena and pointed to new ways to apply chemistry for the engineering of rapid bioseparations. This paper addresses developments and current research on fiber-based monoliths and explains how the various forms of this type of chromatographic stationary phase have potential to provide new tools for analytical and preparative scale separations. The different stationary phases are discussed, and a model that captures the observed constant plate height as a function of mobile phase velocity is reviewed. Methods that enable hydrodynamically stable fiber columns to be packed and operated over a range of mobile phase flow rates, together with the development of new fiber chemistries, are shown to provide columns that extend the versatility of liquid chromatography using monoliths, particularly at the preparative scale. Graphical Abstract Schematic representation of a sample mixture being separated by a rolled-stationary phase column, resulting separated peaks shown in the chromatogram
Keywords: Liquid chromatography; Rolled stationary phases; Press-fit devices; Fiber staple; Aligned fiber stationary phases; Protein separations

From single molecules to life: microscopy at the nanoscale by Bartosz Turkowyd; David Virant; Ulrike Endesfelder (6885-6911).
Super-resolution microscopy is the term commonly given to fluorescence microscopy techniques with resolutions that are not limited by the diffraction of light. Since their conception a little over a decade ago, these techniques have quickly become the method of choice for many biologists studying structures and processes of single cells at the nanoscale. In this review, we present the three main approaches used to tackle the diffraction barrier of ∼200 nm: stimulated-emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM). We first present a theoretical overview of the techniques and underlying physics, followed by a practical guide to all of the facets involved in designing a super-resolution experiment, including an approachable explanation of the photochemistry involved, labeling methods available, and sample preparation procedures. Finally, we highlight some of the most exciting recent applications of and developments in these techniques, and discuss the outlook for this field. Graphical Abstract Super-resolution microscopy techniques. Working principles of the common approaches stimulated-emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM).
Keywords: Super-resolution microscopy; Photophysics and photochemistry of fluorophores; Live cell imaging; Quantitative cell biology

Recent advances and progress in the detection of bisphenol A by Fengxia Sun; Lichao Kang; Xiaoli Xiang; Hongmin Li; Xiaoling Luo; Ruifeng Luo; Chunxia Lu; Xiayu Peng (6913-6927).
Bisphenol A (BPA) is an important industrial chemical used as a plasticizer in polycarbonate and epoxy resins in the plastic and paper industries. Because of its estrogenic properties, BPA has attracted increasing attention from many researchers. This review focuses primarily on analytical methods for BPA detection that have emerged in recent years. We present and discuss the advantages and disadvantages of sample preparation techniques (e.g., solvent extraction, solid-phase extraction, molecularly imprinted polymer solid-phase extraction, and micro-extraction techniques) and analytical methods (e.g., liquid chromatography, liquid chromatography−mass spectrometry, gas chromatography−mass spectrometry, capillary electrophoresis, immunoassay, and several novel sensors). We also discuss expected future developments for the detection of BPA. Graphical Abstract This review focuses primarily on the recent development in the detection of bisphenol A including sample pre-treatment and analytical methods
Keywords: Bisphenol A; Sample pretreatment; Mass spectrometry; Immunoassay; Sensors; Molecularly imprinted polymers

Greening the analytical methods used for analysis of pharmaceuticals has been receiving great interest aimed at eliminating or minimizing the amount of organic solvents consumed daily worldwide without loss in chromatographic performance. Traditional analytical LC techniques employed in pharmaceutical analysis consume tremendous amounts of hazardous solvents and consequently generate large amounts of waste. The monetary and ecological impact of using large amounts of solvents and waste disposal motivated the analytical community to search for alternatives to replace polluting analytical methodologies with clean ones. In this context, implementing the principles of green analytical chemistry (GAC) in analytical laboratories is highly desired. This review gives a comprehensive overview on different green LC pathways for implementing GAC principles in analytical laboratories and focuses on evaluating the greenness of LC analytical procedures. This review presents green LC approaches for eco-friendly analysis of pharmaceuticals in industrial, biological, and environmental matrices. Graphical Abstract Green pathways of liquid chromatography for more eco-friendly analysis of pharmaceuticals
Keywords: Pharmaceutical analysis; Green analytical chemistry; Eco-friendly analysis; Environment friendly LC; Green chromatography; GAC principles

Bioconjugation and functionalization of polymer surfaces are two major tasks in materials chemistry which are accomplished using a variety of coupling agents. Immobilization of biomolecules onto polymer surfaces and the construction of bioconjugates are essential requirements of many biochemical assays and chemical syntheses. Different linkers with a variety of functional groups are used for these purposes. Among them, the benzophenones, aryldiazirines, and arylazides represent the most commonly used photolinker to produce the desired chemical linkage upon their photo-irradiation. In this review, we describe the versatile applications of 4-fluoro-3-nitrophenyl azide, one of the oldest photolinkers used for photoaffinity labeling in the late 1960s. Surprisingly, this photolinker, historically known as 1-fluoro-2-nitro-4-azidobenzene (FNAB), has remained unexplored for a long time because of apprehension that FNAB forms ring-expanded dehydroazepine as a major product and hence cannot activate an inert polymer. The first evidence of photochemical activation of an inert surface by FNAB through nitrene insertion reaction was reported in 2001, and the FNAB-activated surface was found to conjugate a biomolecule without any catalyst, reagent, or modification. FNAB has distinct advantages over perfluorophenyl azide derivatives, which are contemporary nitrene-generating photolinkers, because of its simple, single-step preparation and ease of thermochemical and photochemical reactions with versatile polymers and biomolecules. Covering these aspects, the present review highlights the flexible chemistry of FNAB and its applications in the field of surface engineering, immobilization of biomolecules such as antibodies, enzymes, cells, carbohydrates, oligonucleotides, and DNA aptamers, and rapid diagnostics. Graphical Abstract An overview of the FNAB-engineered activated polymer surfaces for covalent ligation of versatile biomolecules
Keywords: 1-Fluoro-2-nitro-4-azidobenzene; Photolinker; Immobilization; Biomolecules; Polymer; Inert surface

Revealing the composition of organic materials in polychrome works of art: the role of mass spectrometry-based techniques by Cosima Damiana Calvano; Inez Dorothé van der Werf; Francesco Palmisano; Luigia Sabbatini (6957-6981).
The most recent advances in the identification and determination of organic constituents in paintings and other polychrome objects using mass spectrometry (MS)-based techniques are reviewed. The latest achievements in gas chromatography (GC)-MS and pyrolysis (Py-) GC-MS are mainly related to sample pretreatment protocols and to the employment of double-shot or laser desorption pyrolysis, respectively. MS techniques based on soft ionization methods such as matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) are discussed. So far, MALDI and ESI MS have been mainly used in the characterization of proteinaceous materials, but further applications are definitely emerging, e.g., in the fields of lipids, resins, and organic colorants analysis. Chemical imaging by time-of-flight secondary ion mass spectrometry (TOF SIMS), formerly applied to the detection and localization of lipid binders and inorganic materials, has been recently extended to proteins. Finally, the potential of niche techniques such as direct temperature resolved mass spectrometry (DTMS) and direct analysis in real time (DART) MS are outlined.
Keywords: Paint; GC-MS; MALDI/ESI; TOF SIMS; DTMS; DART

The evolution of instrumentation in terms of separation and detection has allowed a real improvement of the sensitivity and the analysis time. However, the analysis of ultra-traces of toxins such as ochratoxin A (OTA) from complex samples (foodstuffs, biological fluids…) still requires a step of purification and of preconcentration before chromatographic determination. In this context, extraction sorbents leading to a molecular recognition mechanism appear as powerful tools for the selective extraction of OTA and of its structural analogs in order to obtain more reliable and sensitive quantitative analyses of these compounds in complex media. Indeed, immunosorbents and oligosorbents that are based on the use of immobilized antibodies and of aptamers, respectively, and that are specific to OTA allow its selective clean-up from complex samples with high enrichment factors. Similar molecular recognition mechanisms can also be obtained by developing molecularly imprinted polymers, the synthesis of which leads to the formation of cavities that are specific to OTA, thus mimicking the recognition site of the biomolecules. Therefore, the principle, the advantages, the limits of these different types of extraction tools, and their complementary behaviors will be presented. The introduction of these selective tools in miniaturized devices will also be discussed.
Keywords: Ochratoxin A; Antibody; Aptamer; Molecularly imprinted polymer; Immunosorbent; Oligosorbent

Analytical electrochemiluminescence by Hua Cui; Francesco Paolucci; Neso Sojic; Guobao Xu (7001-7002).
is a Full Professor of Analytical Chemistry at the University of Science and Technology of China. She is also an editor of Analytical and Bioanalytical Chemistry. Her current research interests include nano-chemiluminescence and nano-electrochemiluminescence and their applications in interdisciplinary fields, including public health, food safety, and environmental monitoring. is a Professor at the Department of Chemistry of the University of Bologna. His most recent research interests include experimental electrochemistry and the use of electrochemiluminescence and scanning probe electrochemical microscopy for probing the electrochemical and redox properties of nanocarbons. is a Professor at the Institute of Molecular Sciences, University of Bordeaux – INP Bordeaux. His research interests include analytical electrochemistry and its coupling with luminescence processes. In the last few years, he has focused on electrogenerated chemiluminescence and on bioelectrochemistry. is a Professor at the State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. His main research interests include the development of new materials and devices for electrochemiluminescence biosensing and other electrochemical applications.

Generation of electrochemiluminescence at bipolar electrodes: concepts and applications by Laurent Bouffier; Stéphane Arbault; Alexander Kuhn; Neso Sojic (7003-7011).
Bipolar electrochemistry (BPE) is an unconventional technique where a conducting object is addressed electrochemically in an electrolyte without any wire connection with an external power supply. BPE has been known for decades but remained limited to only a couple of niche applications. However, it is now undergoing a true renewal of interest especially in the context of analytical chemistry. The bipolar electrode exhibits two distinct poles of opposite polarization with respect to the solution. This allows one to separate the localization of sensing elements versus reporting ones. Also, arrays of bipolar microelectrodes can be addressed simultaneously to perform parallel analyses. Among several reporting strategies, the combination of BPE with electro-chemiluminescence (ECL) is the most frequent choice owing to the very simple visual readout provided by ECL. This article reviews the field from the initial reports to the most recent ones, revealing numerous opportunities including novel analytical strategies for the detection of small molecular analytes and biorelevant molecules such as DNA, RNA, peptides, or other biomarkers. Graphical Abstract Principle of electrochemiluminescence generation at one extremity of a bipolar electrode
Keywords: Electrochemiluminescence; Spectroelectrochemistry; Bipolar electrochemistry; Biosensors; Wireless sensing

Recent development of the phosphorescent cyclometalated iridium(III) chelates has enabled, due to their advantageous electrochemical and photo-physical properties, important breakthroughs in many photonic applications. This particular class of 5d6 ion complexes has attracted increasing interest because of their potential application in electroluminescence devices with a nearly 100 % internal quantum efficiency for the conversion of electric energy to photons. Similar to electroluminescence, the cyclometalated iridium(III) chelates have been successfully applied in the electricity-to-light conversion by means of the electrochemiluminescence (ECL) processes. The already reported ECL systems utilizing the title compounds exhibit extremely large ECL efficiencies that allow one to envisage many potential application for them, especially in further development of ECL-based analytical techniques. This review, based on recently published papers, focuses on the ECL properties of this very exciting class of organometallic luminophores. The reported work, describing results from fundamental as well as application-oriented investigations, will be surveyed and briefly discussed. Graphical abstract Depending on the chemical nature of the cyclometalated irdium(III) chelate different colours of the emitted light can be produced during electrochemical excitation.
Keywords: Cyclometalated iridium(III) chelates; Electrogenerated chemiluminescence (ECL); Electroanalytical methods; Fluorescence/luminescence

Food quality and safety are closely related to human health. In the face of unceasing food safety incidents, various analytical techniques, such as mass spectrometry, chromatography, spectroscopy, and electrochemistry, have been applied in food analysis. High sensitivity usually requires expensive instruments and complicated procedures. Although these modern analytical techniques are sensitive enough to ensure food safety, sometimes their applications are limited because of the cost, usability, and speed of analysis. Electrochemiluminescence (ECL) is a powerful analytical technique that is attracting more and more attention because of its outstanding performance. In this review, the mechanisms of ECL and common ECL luminophores are briefly introduced. Then an overall review of the principles and applications of ECL sensors for food analysis is provided. ECL can be flexibly combined with various separation techniques. Novel materials (e.g., various nanomaterials) and strategies (e.g., immunoassay, aptasensors, and microfluidics) have been progressively introduced into the design of ECL sensors. By illustrating some selected representative works, we summarize the state of the art in the development of ECL sensors for toxins, heavy metals, pesticides, residual drugs, illegal additives, viruses, and bacterias. Compared with other methods, ECL can provide rapid, low-cost, and sensitive detection for various food contaminants in complex matrixes. However, there are also some limitations and challenges. Improvements suited to the characteristics of food analysis are still necessary.
Keywords: Electrochemiluminescence; Food analysis; Sensors; Contaminant

Effects of multi-walled carbon nanotubes on the electrogenerated chemiluminescence and fluorescence of CdTe quantum dots by Yiliyasi Wusimanjiang; Alexander Meyer; Liping Lu; Wujian Miao (7049-7057).
is a doctoral student of Dr. Wujian Miao in the Department of Chemistry and Biochemistry at the University of Southern Mississippi, USA. His PhD dissertation focuses on the electrogenerated chemiluminescence study of semiconductor nanoparticles towards the development of multiplexing detection of biomolecules. Yiliyasi received his BS degree in Chemical Engineering from Hunan University, China. is an MSc student in Biomedical Sciences at the University of Mississippi Medical Center, USA. He completed his undergraduate capstone course—a lab research project—under the supervision of Dr. Wujian Miao and graduated with a BS degree from the University of Southern Mississippi in May 2015. Alex is the recipient of the Chair Award of the spring 2015 Undergraduate Research Symposium. is an Associate Professor in the College of Environmental and Energy Engineering at Beijing University of Technology, China. She received her BS and MSc degrees in Chemistry from Anhui University, and PhD degree in electrochemistry from University of Science and Technology of China. As a visiting scholar, Dr. Lu spent a year in Professor Jacqueline Barton’s group at California Institute of Technology in 2010. Her research interests lie primarily in the area of electrochemical-based chemical sensors and biosensors. is an Associate Professor of Chemistry at the University of Southern Mississippi (USM), USA. His research interests lie in the field of electrochemistry, including chemical sensors and biosensors based on electrogenerated chemiluminescence, electrochemical quartz crystal microbalance, and scanning electrochemical microscopy. Dr. Miao received his PhD in electrochemistry under the supervision of Professor Alan M. Bond from Monash University, Australia, and his postdoctoral training from Dr. Allen J. Bard at the University of Texas at Austin, USA. He is the recipient of the National Science Foundation CAREER Award and the 2011 Innovation Award for Applied Research at USM. Dr. Miao is a Distinguished Visiting Professor of Beijing University of Technology, which is financially supported by the Beijing Overseas Talent Pooling Program (short-term) and selected by the Beijing Overseas Talent Center. Effects of multi-walled carbon nanotubes (CNTs) that were immobilized on glassy carbon electrode (GCE) on the electrogenerated chemiluminescence (ECL) of CdTe quantum dots (QDs) using tri-n-propylamine (TPrA) and 2-(dibutylamino)ethanol (DBAE) as the anodic coreactant are reported. Depending on the solution concentration of coreactant and QDs, the surface-confined CNTs could either quench or enhance the ECL intensity. Lowering the solution concentration of QDs was found to be beneficial for enhancing ECL. A V-shaped profile of ECL intensity ratio (at CNTs over bare GCE) versus coreactant concentration suggested that either low or high concentrations of coreactant were needed for effective ECL generation. The ECL quenching by CNTs was believed to follow the typical dynamic quenching mechanism, which was confirmed by fluorescent data that provided a Stern-Volmer and an estimated quenching constant of 11.7 g/L and 1.2 × 109 L/g•s, respectively, for the excited state CdTe* quenching by CNTs in solution. Furthermore, the ECL performance at CNTs was also affected by the type of the coreactant used, where up to 30 times enhancement in ECL was observed from the CdTe/DBAE system under the given experimental conditions. Graphical Abstract Illustration of anodic quantum dots ECL enhancement and quenching by multi-walled carbon nanotubes
Keywords: Electrogenerated chemiluminescence (ECL); Carbon nanotubes; CdTe quantum dots; Dynamic quenching; ECL coreactant

Electrogenerated chemiluminescence of tris(2,2'-bipyridine)ruthenium(II) using N-(3-aminopropyl)diethanolamine as coreactant by Shimeles Addisu Kitte; Chao Wang; Suping Li; Yuriy Zholudov; Liming Qi; Jianping Li; Guobao Xu (7059-7065).
Coreactant plays a critical role for the application of electrochemiluminescence (ECL). Herein, N-(3-aminopropyl)diethanolamine (APDEA) has been explored as a potential coreactant for enhancing tris(2,2'-bipyridyl)ruthenium(II) ECL. It is much more effective than tripropylamine at gold and platinum electrodes although it has one primary amine group besides a tertiary amine group. The presence of primary amine group and hydroxyl groups in APDEA promotes the oxidation rates of amine and thus remarkably increases ECL intensity. The ECL intensities of the Ru(bpy)3 2+/APDEA system are approximately 10 and 36 times stronger than that of Ru(bpy)3 2+/tripropylamine system and about 1.6 and 1.14 times stronger than that of Ru(bpy)3 2+/N-butyldiethanolamine system at Au and Pt electrodes, respectively. The ECL intensity of the Ru(bpy)3 2+/APDEA system is 2.42 times stronger than that of Ru(bpy)3 2+/N-butyldiethanolamine at glassy carbon electrodes.
Keywords: Tris(2,2'-bipyridyl)ruthenium(II); Electrochemiluminescence; Coreactant; N-(3-Aminopropyl)diethanolamine; Primary amine group

A simple and sensitive electrogenerated chemiluminescence biosensor was developed to monitor matrix metalloproteinase 2 (MMP-2) by employing a specific peptide (CGPLGVRGK) as a molecular recognition substrate. Bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) (Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2 (Ru1) was used as ECL-emitting species and covalently labeled onto the peptide through NH2-containing lysine on the peptide via acylation reaction to form Ru1-peptide as an ECL probe. An ECL peptide-based biosensor was fabricated by self-assembling the ECL probe onto the surface of gold electrode. MMP-2 can specifically cleave the Ru1-peptide on the electrode surface, which led the partly Ru1-peptide to leave the electrode surface and resulted in the decrease of the ECL intensity obtained from the resulted electrode in 0.1 M phosphate-buffered saline (pH 7.4) containing tri-n-propylamine. The decreased ECL intensity was piecewise linear to the concentration of MMP-2 in the range from 1 to 500 ng/mL. Moreover, the ECL biosensor is successfully applied to detection of MMP-2 secreted by living cell, such as HeLa cells. Additionally, the biosensor was also applied to the evaluation of matrix metalloproteinase inhibitors. The strategy presented here is promising for other disease-related matrix metalloproteinase assay and matrix metalloproteinase inhibitor profiling with sensitivity and simplicity. Graphical Abstract Detection of MMP-2 released from living cells by ECL peptide-based biosensor
Keywords: Electrogenerated͘͘͘ chemiluminescence; Biosensor; Peptide; Matrix metalloproteinase 2

The iridium(III) complex 1 operates as a versatile platform for molecular logic gates fulfilling multiple logic functions depending on the inputs and the interrogation channels. In the UV-vis channel, it works as an INH, OR, and NINH gate with F and H2PO4 as inputs when following the absorbance at 244, 262, and 457 nm, respectively. More importantly, the combination of photoluminescence (PL) intensity ratio (I 700/I 600) and transmittance at 457 nm resulted in the setup of a 1:2 demultiplexer with F and CO2 as inputs. Finally, a three-input (BF3, F, and H2PO4 ) full subtractor was realized by integrating the electrochemiluminescence (ECL) intensity at 642 nm and the absorbance at 262 nm. Graphical abstract Multichannel detection and the proper combination of inputs are the key for the elaboration of sophisticated logic gates
Keywords: Molecular logic; Photoluminescence; Electrochemiluminescence; Logic gate; Anions

Microscopic imaging and tuning of electrogenerated chemiluminescence with boron-doped diamond nanoelectrode arrays by Milica Sentic; Francesca Virgilio; Alessandra Zanut; Dragan Manojlovic; Stéphane Arbault; Massimo Tormen; Neso Sojic; Paolo Ugo (7085-7094).
Nanoelectrode arrays (NEAs) are increasingly applied for a variety of electroanalytical applications; however, very few studies dealt with the use of NEAs as an electrochemical generator of electrogenerated chemiluminescence (ECL). In the present study, arrays of nanodisc and nanoband electrodes with different dimensions and inter-electrode distances were fabricated by e-beam lithography on a polycarbonate layer deposited on boron-doped diamond (BDD) substrates. In particular, NEAs with 16 different geometries were fabricated on the same BDD sample substrate obtaining a multiple nanoelectrode and ultramicroelectrode array platform (MNEAP). After electrochemical and morphological characterization, the MNEAP was used to capture simultaneously with a single image the characteristic behaviour of ECL emission from all the 16 arrays. Experiments were performed using Ru(bpy)3 2+ as the ECL luminophore and tri-n-propylamine (TPrA) as the co-reactant. With a relatively limited number of experiments, such an imaging procedure allowed to study the role that geometrical and mechanistic parameters play on ECL generation at NEAs. In particular, at high concentrations of TPrA, well-separated individual ECL spots or bands revealed an ECL signal which forms a pattern matching the nanofabricated structure. The analysis of the imaging data indicated that the thickness of the ECL-emitting zone at each nanoelectrode scales inversely with the co-reactant concentration, while significantly stronger ECL signals were detected for NEAs operating under overlap conditions.
Keywords: Nanoelectrode; Array; Electrogenerated chemiluminescence; Boron-doped diamond; Microscopy; Imaging

Ruthenium(II) complexes with carboxylic acid as a bioconjugatable group, i.e., [Ru(bathophenanthroline disulfonate)(2,2′-bipyridine)(4-methyl-4′-(3-carboxypropyl)-2,2′-bipyridine)]0, (C49H38N6O8S2Ru), and [Ru(bathophenanthroline disulfonate)2(4-methyl-4′-(3-carboxypropyl)-2,2′-bipyridine)]2− · 2Na+, (C63H44N6O14S4RuNa2) were characterized spectroscopically and electrochemically. As potential labels for electrochemiluminescence (ECL) immunoassays, the ECL intensities of the free labels in homogenous aqueous buffer solutions were compared under a condition that is similar to the one employed by a commercial clinical immunoassay system. The two labels were found to be more emissive and, thus, can be detected at 10- 12 pM compared with 5× 10−12 pM of the label currently used in the commercial ECL system. Furthermore, the improved ECL emission of the free labels in homogenous solutions was proven to be translated into more intense ECL signal in heterogeneous sandwich immunoassay and, thus, leading to a lower limit of detection in immunoassay. The data obtained from these ECL labels shed light on the further development of ECL-based clinical immunoassay technology. Graphical abstract Electrochemiluminescence immunoassays were carried out with three different ruthenium(II) complex labels. It was proved that the higher signal intensities found with the novel labels in homogeneous solutions were maintained in heterogeneous sandwich format.
Keywords: Electrochemiluminescence; Immunoassay; Ruthenium complexes; Biolabels

Telomerase is one of the most common markers of human malignant tumors, such as uterine, stomach, esophageal, breast, colorectal, laryngeal squamous cell, thyroid, bladder, and so on. It is necessary to develop some sensitive but convenient detection methods for telomerase activity determination. In this study, a label-free and ultrasensitive electrochemiluminescence (ECL) biosensor has been fabricated to detect the activity of telomerase extracted from HeLa cells. Thiolated telomerase substrate (TS) primer was immobilized on the gold electrode surface through gold-sulfur (Au-S) interaction and then elongated by telomerase specifically. Then, it was hybridized with complementary DNA to form double-stranded DNA (dsDNA) fragments on the electrode surface, and Ru(phen)3 2+ has been intercalated into the dsDNA grooves to act as the ECL probe. The enhanced ECL intensity has a linear relationship with the number of HeLa cells in the range of 5∼5000 and with a detection limit of 2 HeLa cells. The proposed ECL biosensor has high specificity to telomerase in the presence of common interferents. The relative standard deviations (RSDs) were <5 % at 100 HeLa cells. The proposed method provides a convenient approach for telomerase-related cancer screening or diagnosis.
Keywords: Electrochemiluminescence; Telomerase; Label free; HeLa cell

In this work, a simple, sensitive, and selective method for the determination of silver ions (Ag+) was proposed on the basis of the enhancement of Ag+ on the electrogenerated chemiluminescence (ECL) of peroxydisulfate (S2O8 2−), which can be attributed to the obvious electrocatalytic reduction of S2O8 2− by Ag+, giving rise to more abundant sulfate radical anion (SO4 •−). Under optimal conditions, a linear range from 4 to 500 nM (R = 0.997) with a detection limit of 2.5 nM (S/N = 3) was achieved for the detection of Ag+. The Ag+/S2O8 2− ECL system could not only provide an assay protocol for the determination of Ag+ but also offer an effective way to enhance the ECL of S2O8 2−, which may act as a promising platform for the detection of other species such as 4-chlorophenol. Graphical abstract ECL-potential curves of 0.1 M PBS (pH 8.5) containing (a) 500 nM AgNO3, (b) 50 mM K2S2O8, (c) 500 nM AgNO3 and 50 mM K2S2O8
Keywords: Electrogenerated chemiluminescence; Silver ion; Peroxydisulfate

Miniaturized analytical instrumentation for electrochemiluminescence assays: a spectrometer and a photodiode-based device by Marta M. P. S. Neves; Pablo Bobes-Limenes; Alejandro Pérez-Junquera; María Begoña González-García; David Hernández-Santos; Pablo Fanjul-Bolado (7121-7127).
Herein, a new miniaturized analytical instrumentation for electrochemiluminescence (ECL) assays is presented. A photodiode integrated in an ECL cell combined with a potentiostat/galvanostat, all integrated in a one-piece instrument (μSTAT ECL), was developed. In addition, a complementary micro-spectrometer integrated in a similar ECL cell for luminescence spectra recording is also proposed. Both cells are intended to be used with screen-printed electrodes and all the devices are portable and small sized. Their performance was corroborated with two innovative proofs-of-concept that centered on the luminol transduction chemistry: a first time reported ECL assay based on the enzymatic reaction between an indoxyl substrate and the enzyme alkaline phosphatase, and the electrochemiluminescence resonance energy transfer (ECL-RET) process triggered by the electro-oxidized luminol to the acceptor fluorescein. The photodiode system revealed to be more sensitive than the spectrometer device in collecting the light; however, with the latter, it is possible to discriminate different luminescent species according to their maximum wavelength emission, which is extremely useful for carrying out simple and simultaneous ECL multiplex analyzes. The spectrometer device works as an excellent accessory to couple with the μSTAT ECL instrument, complementing the experiments. Graphical abstract Schematic representation of the ECL-RET: from luminol–H2O2 system to fluorescein, the micro-spectrometer for the light collection and the 3D representation of the ECL-RET reaction.
Keywords: Electrochemiluminescence; Portable instrumentation; Screen-printed electrode; Miniaturized photodiode and spectrometer; Luminol; Alkaline phosphatase

Optimising electrogenerated chemiluminescence of quantum dots via co-reactant selection by Rebekah Russell; Alasdair J. Stewart; Lynn Dennany (7129-7136).
We demonstrate that for quantum dot (QD) based electrochemiluminescence (ECL), the commonly used co-reactant does not perform as effectively as potassium persulfate. By exploiting this small change in co-reactant, ECL intensity can be enhanced dramatically in a cathodic-based ECL system. However, TPA remains the preferential co-reactant-based system for anodic ECL. This phenomenon can be rationalised through the relative energy-level profiles of the QD to the co-reactant in conjunction with the applied potential range. This work highlights the importance of understanding the co-reactant pathway for optimising the application of ECL to bioanalytical analysis, in particular for near-infrared (NIR) QDs which can be utilised for analysis in blood. Graphical Abstract Optimising ECL Production Through Careful Selection of Co-Reactions Based on Energetics Involved
Keywords: Electroanalytical methods; Electrochemiluminescence; Quantum dots

Investigation of perfluorooctanoic acid induced DNA damage using electrogenerated chemiluminescence associated with charge transfer in DNA by Liping Lu; Linqing Guo; Meng Li; Tianfang Kang; Shuiyuan Cheng; Wujian Miao (7137-7145).
An electrogenerated chemiluminescence (ECL)-DNA sensor was designed and fabricated for the investigation of DNA damage by a potential environmental pollutant, perfluorooctanoic acid (PFOA). The ECL-DNA sensor consisted of a Au electrode that had a self-assembled monolayer of 15 base-pair double-stranded (ds) DNA oligonucleotides with covalently attached semiconductor CdSe quantum dots (QDs) at the distal end of the DNA. Characterization of the ECL-DNA sensor was conducted with X-ray photoelectron spectroscopy (XPS), electrochemical impedance spectroscopy (EIS), ECL, and cyclic voltammetry before and after the exposure of the sensor to PFOA. Consistent data revealed that the dsDNA on Au was severely damaged upon the incubation of the electrode in PFOA, causing significant increase in charge (or electron) transfer (CT) resistance within DNA strands. Consequently, the cathodic coreactant ECL responses of the Au/dsDNA-QDs electrode in the presence of K2S2O8 were markedly decreased. The strong interaction between DNA and PFOA via the hydrophobic interaction, especially the formation of F···H hydrogen bonds by insertion of the difluoro-methylene group of PFOA into the DNA base pairs, was believed to be responsible for the dissociation or loosening of dsDNA structure, which inhibited the CT through DNA. A linear relationship between the ECL signal of the sensor and the logarithmical concentration of PFOA displayed a dynamic range of 1.00 × 10−14–1.00 × 10−4 M, with a limit of detection of 1.00 × 10−15 M at a signal-to-noise ratio of 3. Graphical Abstract Illustration of ECL detection of PFOA on a Au/dsDNA-QDs ECL-DNA sensor
Keywords: Electrogenerated chemiluminescence (ECL); Biosensors; DNA damage; DNA charge transfer; Perfluorooctanoic acid (PFOA); Quantum dots

Electrochemiluminescence (ECL) immunosensor for detection of Francisella tularensis on screen-printed gold electrode array by Anna-Maria Spehar-Délèze; Sandra Julich; Rainer Gransee; Herbert Tomaso; Samuel B. Dulay; Ciara K. O’Sullivan (7147-7153).
An electrochemiluminescence (ECL) immunosensor for the rapid detection of the Francisella tularensis pathogen using whole antibodies or antibody fragments as capture biomolecule is described. A sandwich immunoassay was used with either lipopolysaccharide (LPS) or the whole inactivated bacterial cell (LVS) as a target, while Ru(bpy)3 2+-encapsulated silicate nanoparticles were linked to the secondary antibody and used as ECL labels. The assay was performed in a fluidic chip housed in a custom-built black box incorporating electronics, optics and fluidics. The obtained limit of detection for LPS was 0.4 ng/mL, while for the LVS it was 70 and 45 bacteria/mL when the capturing molecule was the whole antibody and the antibody F(ab) fragment, respectively.
Keywords: Electrochemiluminescence; Immunoassay; Antibody fragment; Biowarfare agent; Screen-printed gold array

The cathodic electrochemiluminescence (ECL) behavior of meso-tetra(4-sulfonatophenyl)porphyrin (TSPP) with potassium peroxydisulfate (K2S2O8) as the coreactant in aqueous solution with strong and stable emission was exploited to determine Cu2+ down to nanomolar concentration. Two possible reaction mechanisms have been proposed to understand the generation of ECL by the TSPP/K2S2O8 system. The effects of the concentration of TSPP and K2S2O8, pH of the medium, and scan rate on the ECL intensity were studied in detail. The ECL intensity was efficiently quenched by trace amounts of Cu2+. This phenomenon was used to develop a new method, which can offer rapid, reliable, and selective detection of Cu2+. Under the optimum conditions, plots of the ECL of the TSPP/K2S2O8 system versus the concentration of Cu2+ are linear in the range of 5 to 160 nM with a detection limit of 1.56 nM (S/N = 3). The proposed ECL sensor was successfully applied for analysis of tap and river water samples. It is anticipated that TSPP could be a new class of promising luminescent agent for ECL sensors. Graphical Abstract A two-step cathodic elelctrochemiluminescence (ECL) behavior of TSPP/K2S2O8 system in the aqueous solution and Cu2+ determination using the same
Keywords: TSPP; K2S2O8 ; Electrochemiluminescence; Cu2+ ; Sensor

Here, we report highly enhanced electrochemiluminescence (ECL) of luminol in the presence of H2O2 on indium tin oxides (ITOs) modified with both of dendrimer-encapsulated Pt nanoparticles (Pt DENs) and chemically converted graphenes (CCGs). The ITO electrodes were electrochemically modified with size-monodisperse Pt DENs via electrooxidative grafting of the terminal amines of the dendrimers encapsulating Pt nanoparticles. The Pt DEN-modified ITOs were then decorated with CCG sheets via electrostatic attachments of graphene oxides (GOs) and subsequent chemical reduction of the GOs to the CCGs. The resulting CCG-Pt DEN/ITO electrodes exhibited highly catalyzed electrochemical oxidation of luminol/H2O2, leading to significantly enhanced ECL of the luminol/H2O2 system, i.e., ∼15-fold enhancement, compared to ECL emission from bare ITOs even at lower applied potentials, which allowed sensitive ECL-based analysis of H2O2 using the CCG-Pt DEN/ITOs. Graphical abstract We report the highly enhanced electrochemiluminescence of the luminol/H2O2 system on the indium tin oxide electrodes modified with both of Pt nanoparticles and chemically converted graphenes using amine-terminated dendrimers
Keywords: Electrochemiluminescence; Luminol; H2O2 ; Dendrimer-encapsulated Pt nanoparticle; Chemically converted graphene

Multiwalled carbon nanotubes/gold nanocomposites-based electrochemiluminescent sensor for sensitive determination of bisphenol A by Weiwei Guo; Amin Zhang; Xin Zhang; Chusen Huang; Dapeng Yang; Nengqin Jia (7173-7180).
An electrochemiluminescence (ECL) sensor for bisphenol A was proposed by using l-cysteine-functionalized multiwalled carbon nanotubes/gold nanocomposites-modified glassy carbon electrode (MWCNTs-Au/GCE) based on ECL of peroxydisulfate solution. The ECL behaviors of peroxydisulfate solution had been investigated at the chitosan/MWCNTs-Au/GCE, and bisphenol A was found to have quenching effects on the ECL of peroxydisulfate solution. Both Au nanoparticles (AuNPs) and multiwalled CNTs could promote the electron transfer and synergetically amplify the ECL signal of peroxydisulfate solution. Under the optimized conditions, the ECL signal intensity was linear with the concentration of bisphenol A in the concentration range between 0.25 and 100 μM (R = 0.9931) with a detection limit (S/N = 3) of 0.083 μM. The constructed ECL sensor has the advantages of simplicity, sensitivity, good selectivity, and reproducibility, exhibiting a great potential application in the determination of bisphenol A.
Keywords: Electrochemiluminescence; Sensors; Bisphenol A; Peroxydisulfate

Electrochemiluminescence of graphitic carbon nitride and its application in ultrasensitive detection of lead(II) ions by Yan Zhang; Lina Zhang; Qingkun Kong; Shenguang Ge; Mei Yan; Jinghua Yu (7181-7191).
Graphitic carbon nitride (g-C3N4) materials with a layered structure have unusual physicochemical properties. Herein it was shown that g-C3N4 quantum dots (QDs) obtained through a thermal-chemical etching route exhibited attractive upconversion and electrochemiluminescence (ECL) properties. After modification on nanoporous gold (NPG) with a sponge-like porous structure, g-C3N4 QDs were employed to fabricate an ECL sensor for the determination of Pb2+ using target - dependent DNAzyme as the recognition unit. Moreover, magnetic reduced graphene oxide nanosheets (rGO) attached with Fe3O4 nanoparticles (rGO-Fe3O4) were obtained via a one-pot in situ reduction approach, and used as carriers of DNAzyme. To make full use of the unique magnetic property the prepared rGO-Fe3O4, a flow injection ECL detecting cell was designed using indium tin oxide (ITO) glass as working electrode. Due to the unique separation and enrichment properties of magnetic Fe3O4-rGO materials as well as wire-like conductivity of NPG, high sensitivity and selectivity for the determination of Pb2+ in real water samples were achieved. This indicates that g-C3N4 has excellent anodic ECL performance in the presence of triethanolamine, and could be applied in real environmental samples analyses. Graphical Abstract Graphitic carbon nitride based electrochemiluminescence sensor for the sensitive monitor of lead(II) ions in real samples was constructed.
Keywords: Electrochemiluminescence; g-C3N4 QDs; rGO-Fe3O4

A novel solid-state electrochemiluminescence sensor for detection of cytochrome c based on ceria nanoparticles decorated with reduced graphene oxide nanocomposite by Mohammad Reza Karimi Pur; Morteza Hosseini; Farnoush Faridbod; Amin Shiralizadeh Dezfuli; Mohammad Reza Ganjali (7193-7202).
A novel ultrasensitive sensing system for the rapid detection of cytochrome c (Cyt C) was developed on the basis of an electrochemiluminescence (ECL) method. A nanocomposite biosensor was made of reduced graphene oxide decorated with cerium oxide/tris(2,2-bipyridyl)ruthenium(II)/chitosan (CeO2NPs-RGO/ Ru(bpy)3 2+/CHIT) and used for this purpose. The ECL signal was produced by an electrochemical interaction between Ru(bpy)3 2+ and tripropyl amine (TPA) on the surface of the electrode. Addition of Cyt C to the solution decreases the ECL signal due to its affinity for TPA and inhibition of its reaction with Ru(bpy)3 2+. The effects of the amount of CeO2NPs-RGO, Ru(bpy)3 2+, TPA concentration as a co-reactant, and the pH of the electrolyte solution on the ECL signal intensity were studied and optimized. The results showed that the method was fast, reproducible, sensitive, and stable for the detection of Cyt C. The method has a linear range from 2.5 nM to 2 μM (R 2 = 0.995) with a detection limit of 0.7 nM. Finally, the proposed biosensor was used for the determination of Cyt C in human serum samples with RSDs of 1.8–3.6 %. The results demonstrate that this solid-state ECL quenching biosensor has high sensitivity, selectivity, and good stability. Graphical Abstract A novel solid-state electrochemiluminescence sensor for detection of cytochrome C based on Ceria Nanoparticles Decorated Reduced Graphene Oxide Nanocomposite
Keywords: Electrochemiluminescence; Cerium oxide; Reduced graphene; Biosensor

Faraday cage-type electrochemiluminescence immunosensor for ultrasensitive detection of Vibrio vulnificus based on multi-functionalized graphene oxide by Zhiyong Guo; Yuhong Sha; Yufang Hu; Zhongqing Yu; Yingying Tao; Yanjie Wu; Min Zeng; Sui Wang; Xing Li; Jun Zhou; Xiurong Su (7203-7211).
A novel Faraday cage-type electrochemiluminescence (ECL) immunosensor devoted to the detection of Vibrio vulnificus (VV) was fabricated. The sensing strategy was presented by a unique Faraday cage-type immunocomplex based on immunomagnetic beads (IMBs) and multi-functionalized graphene oxide (GO) labeled with (2,2′-bipyridine)(5-aminophenanthroline)ruthenium (Ru-NH2). The multi-functionalized GO could sit on the electrode surface directly due to the large surface area, abundant functional groups, and good electronic transport property. It ensures that more Ru-NH2 is entirely caged and become “effective,” thus improving sensitivity significantly, which resembles extending the outer Helmholtz plane (OHP) of the electrode. Under optimal conditions, the developed immunosensor achieves a limit of detection as low as 1 CFU/mL. Additionally, the proposed immunosensor with high sensitivity and selectivity can be used for the detection of real samples. The novel Faraday cage-type method has shown potential application for the diagnosis of VV and opens up a new avenue in ECL immunoassay. Graphical abstract Faraday cage-type immunoassay mode for ultrasensitive detection by extending OHP
Keywords: Faraday cage-type electrochemiluminescence immunosensor; Vibrio vulnificus ; Multi-functionalized graphene oxide; Outer Helmholtz plane

Electrochemiluminescent (ECL) [Ru(bpy)3]2+/PAMAM dendrimer reactions: coreactant effect and 5-fluorouracil/dendrimer complex formation by Aila Jimenez-Ruiz; Elia Grueso; Pilar Perez-Tejeda; Fernando Muriel-Delgado; Concepcion Torres-Marquez (7213-7224).
Electrogenerated chemiluminescence (ECL) reactions between tris(2,2′-bipyridine)ruthenium(II) and PAMAM dendrimers of the full (G1.0) and half (G1.5) generations were carried out in an aqueous medium at pH 6.1 and 10.0. In the absence of 5-fluoro-1H,3H-pyrimidine-2,4-dione (5-fluorouracil, 5-Fu) (coreactant effect study), the ECL efficiency trends as a function of [G1.0] and [G1.5] at pH 6.1 and 10.0 revealed that PAMAM dendrimers are about 100 (G1.5, pH 6.1), 60 (G1.5, pH 10.0), 26 (G1.0, pH 10.0) and 13 (G1.0, pH 6.1) times more efficient as ECL coreactants than oxalate anion is. Moreover, ECL reactions were done in the presence of several solutions of 5-Fu at a fixed concentration of the G1.0 and G1.5 dendrimers at pH 6.1 and 10.0 (binding study). The ECL efficiency trends as a function of [5-Fu] highlighted a dendrimer/5-Fu binding. Therefore, one of the most remarkable and novel findings of this work is the potential of PAMAM dendrimers to be used as both sensors and biosensors in an aqueous medium in the presence of a suitable sensitizer. Redox potentials of the [Ru(bpy)3]3+/2+ couple were also determined in the absence and presence of 5-Fu at both pHs. In the absence of 5-Fu the positive or negative shift of redox potentials showed the influence of the repulsive or attractive electrostatic long-range and short-range interactions between the charged dendrimer surface and the oxidized and reduced forms of the couple. In the presence of 5-Fu the trends of redox potentials highlighted the existence of a charged dendrimer/5-Fu species. Graphical Abstract ECL emission for the [Ru(bpy)3]2+/ G1.0 dendrimer reaction in the presence of the 5-Fu at pH 6.1
Keywords: Electrogenerated chemiluminescence; PAMAM dendrimers; Coreactant effect; Dendrimer/5-fluorouracil binding