Biological Recognition Element Research Articles

Carbon nanotubes functionalized electrospun nanofibers formed 3D electrode enables highly strong ECL of peroxydisulfate and its application in immunoassay

A new biosensing platform based on electrospun carbon nanotubes nanofibers (CNTs@PNFs) composite, which enabled strong electrochemiluminescent emission of peroxydisulfate, was firstly developed for immunoassay with favorable analytical performances, and then was utilized to evaluate the interaction between antibody and antigen in vitro. Moreover, the obvious ECL image of peroxydisulfate on the prepared sensing platform was firstly recorded in this report. In order to expand the application of peroxydisulfate ECL, the specific recognization biomolecules, α-fetoprotein (AFP) antibody was bound to the functionalized film via electrostatic interaction for fabricating label-free ECL immunosensor to detect α-AFP. Based on the ECL change resulting from the specific immunoreaction between antigen and antibody, the quantitative analysis for AFP with wide dynamic response in the range from 0.1pgmL−1 to 160ngmL−1 was realized. And the limit of detection was estimated to be 0.09pgmL−1. Therefore, the flexible sensing platform not only acted as the sensitized sensing element, but also offered a suitable carrier for immobilization of biological recognition elements with low-toxicity and eco-friendliness, which opened a promising approach to developing further electrospun nanofiber based amplified ECL biosensor with favorable analytical performances.

A novel sulfate-reducing bacteria detection method based on inhibition of cysteine protease activity

Sulfate-reducing bacteria (SRB) have been extensively studied in corrosion and environmental science. However, fast enumeration of SRB population is still a difficult task. This work presents a novel specific SRB detection method based on inhibition of cysteine protease activity. The hydrolytic activity of cysteine protease was inhibited by taking advantage of sulfide, the characteristic metabolic product of SRB, to attack active cysteine thiol group in cysteine protease catalytic sites. The active thiol S-sulfhydration process could be used for SRB detection, since the amount of sulfide accumulated in culture medium was highly related with initial bacterial concentration. The working conditions of cysteine protease have been optimized to obtain better detection capability, and the SRB detection performances have been evaluated in this work. The proposed SRB detection method based on inhibition of cysteine protease activity avoided the use of biological recognition elements. In addition, compared with the widely used most probable number (MPN) method which would take up to at least 15days to accomplish whole detection process, the method based on inhibition of papain activity could detect SRB in 2days, with a detection limit of 5.21×102cfumL−1. The detection time for SRB population quantitative analysis was greatly shortened.

Cytochrome C Mutants with Enhanced Capabilities in Multi Layer Arrangements

In the field of biosensors it is important to increase the surface concentration of the selected biological recognition elements for a higher sensitivity. Based on cytochrome c, a small heme containing electron carrier protein from the respiratory chain, fully redox active multi layer assemblies have been developed by using the layer by layer technique, where every immobilized protein contributes to the electron transfer with the electrode. This provides the basis for integrating catalytically active enzymes into the layers, giving the opportunity of tuning the sensitivity by the number of layers deposited. This was shown already e.g. for bilirubin oxidase (BOD) and sulfite oxidase (SOX). However, one limiting factor for the effective coupling of proteins in those multi layer systems is the electron self-exchange rate of the cytochrome c molecules. In order to follow the applied potential but also the enhanced conversion at the embedded enzymes, a rather high exchange rate between the immobilized proteins is necessary.Based on the hypothesis, that the electron transfer happens via hopping between the heme-centers which are embedded in a protein-pocket that is mainly positively charged at neutral pH, we tried to engineer human cytochrome c mutants with increased electron self exchange rates. By replacing some of the positively charged surface lysines around the heme pocket with uncharged alanines via site directed mutagenesis, repulsion between cytochrome c molecules should be decreased and ease the charge transfer.Six different mutants have been designed, expressed and evaluated. The electrochemical properties of the cytochrome c mutants have been analyzed by cyclic voltammetry at self assembling mono layers (SAMs) of 11-mercapto-1-undecanol (MU) and 11-mercapto-1-undecanoic acid (MUA) on gold. In a second step cytochrome c mutant multi layers have been prepared and analyzed with respect to different rates of the potential sweep in order to elucidate how the systems can follow the driving force.

Modular Peptide-Based Hybrid Nanoprobes for Bio-Imaging and Bio-Sensing

ABSTRACTThe self-organization of functional proteins directly onto solid materials is attractive to a wide range of biomaterials and systems that need to accommodate a biological recognition element. In such systems, inorganic binding peptides may be an essential component due to their high affinity and selective binding features onto different types of solid surfaces. This study demonstrates a peptide-enabled self-assembly technique for designing well-defined protein arrays over a metal surface. To illustrate this concept, we designed a fusion protein that simultaneously displays a red fluorescence protein (DsRed-monomer), which is highly selective for copper ions, and a gold binding peptide AuBP. The peptide tag, AuBP, self-directs the organization of DsRed-monomer protein onto a gold surface and forms arrays built upon an efficient control of the organic/inorganic interface at the molecular level. The peptide-assisted design offers a modular approach for fabrication of fluorescent-based protein arrays with copper ion sensing ability.

Functional nucleic acid entrapment in sol–gel derived materials

Functional nucleic acids (FNAs) are single-stranded DNA or RNA molecules, typically generated through in vitro selection, that have the ability to act as receptors for target molecules (aptamers) or perform catalysis of a chemical reaction (deoxyribozymes and ribozymes). Fluorescence-signaling aptamers and deoxyribozymes have recently emerged as promising biological recognition and signaling elements, although little has been done to evaluate their potential for solid-phase assays, particularly with species made of RNA due to their lack of chemical stability and susceptibility to nuclease attack. Herein, we present a detailed overview of the methods utilized for solid-phase immobilization of FNAs using a sol–gel entrapment method that can provide protection from nuclease degradation and impart long-term chemical stability to the FNA reporter systems, while maintaining their signaling capabilities. This article will also provide a brief review of the results of such entrapment studies involving fluorescence-signaling versions of a DNA aptamer, selected RNA-cleaving deoxyribozymes, and two different RNA aptamers in a series of sol–gel derived composites, ranging from highly polar silica to hydrophobic methylsilsesquioxane-based materials. Given the ability to produce sol–gel derived materials in a variety of configurations, particularly as thin film coatings on electrodes, optical fibers, and other devices, this entrapment method should provide a useful platform for numerous solid-phase FNA-based biosensing applications.

Stabilizing redox polymer films by electrochemically induced crosslinking

Electrochemically induced crosslinking is suggested to stabilize electrodeposition polymer/enzyme films selectively on an electrode surface. 4 different protected diamine or dithiol based bi-functional crosslinkers have been synthesized, which can be activated by a pH-shift invoked by electrochemical water oxidation or proton reduction. Deprotection occurs either simultaneously or sequentially to the deposition of specifically designed redox electrodeposition polymers. The stability of the resulting polymer films was substantially enhanced as evaluated using continuous potentiodynamic cycling alternated by difference pulse voltammetry. Electrochemically induced crosslinking is compatible with biological recognition elements using Trametes hirsuta laccase or glucose oxidase entrapped within specifically adapted Os-complex modified or phenothiazine-modified redox polymers.

Pyoverdine secreted by Pseudomonas aeruginosa as a biological recognition element for the fluorescent detection of furazolidone

Methods for the rapid and sensitive detection of furazolidone, a pesticide used for the treatment of infections of animals and human beings, have been urgently recommended for its large residual, strong carcinogenicity and genotoxicity in the environment. In this study, a method for the detection of furazolidone based on the rapid fluorescence quenching of pyoverdine by furazolidone was developed. Pyoverdine secreted by a Pseudomonas aeruginosa strain PA1 was purified through affinity chromatography and its fluorescent property was characterized. The fluorescence of pyoverdine could be quenched by furazolidone with specificity, and based on this phenomenon a fluorescent method for furazolidone detection was established. Fluorescence of pyoverdine was quenched by furazolidone probably due to the electron transfer from pyoverdine to furazolidone. The optimal pH for the detection was 7.2 in 50mM 3-(N-Morpholino) propanesulfonic acid solution, and the whole detection process could be completed within a few seconds. The linear range of the detection was 2–160µM and the limit of detection (LOD) was 0.5µM. This study developed a novel fluorescent method for furazolidone detection, and the rapid and specific fluorescent biosensor can be potentially applied for furazolidone detection in the aquatic samples.

Photolithographic Strategy for Patterning Preformed, Chemically Modified, Porous Silicon Photonic Crystal Using Click Chemistry

Porous silicon (PSi) is an ideal platform for label-free biosensing, and the development of porous silicon patterning will open a pathway to the development of highly parallel PSi biochips for detecting multiple analytes. The optical response of PSi photonic crystal is determined by the changes in the effective bulk refractive index resulting from reactions/events occurring within the internal pore space. Therefore, introducing precise chemical functionalities in the pores of PSi is essential to ensure device selectivity. Here we describe the fabrication of PSi patterns that possess discrete chemical functionalities that are restricted to precise locations. The key difference to previous patterning protocols for PSi is that the entire porous material is first modified with a self-assembled monolayer of a α,ω-diyne adsorbate prior to patterning using a microfabricated titanium mask. The distal alkyne moieties in the monolayer are then amenable to further selective modification by the archetypal "click" reaction, the copper catalyzed alkyne-azide cycloaddition (CuAAC), using the titanium mask as a resist. This type of patterning is suitable for further immobilization of biological recognition elements, and presents a new platform for highly parallel PSi biosensor for multiple detections.

Amperometric sensing — Bioelectroanalysis

It seems quite timely that Analytical and Bioanalytical Chemistry (ABC) is devoting a special issue to sensing. Less obvious is that the issue is limited to electrochemical sensing, even less so to amperometric sensing, bioelectrosensing included. Actually, electroanalysis is not the most widely applied analytical technique. Optical methods definitely overwhelm the electrochemical ones in popularity; once mass spectrometry, chromatography, and the relevant hyphenated techniques, are taken into account together with the optical ones, only a restricted room is seemingly left to electrochemical methods of analysis. However, especially in the field of biosensors, this general description does not alway hold and electroanalytical techniques are presently dominating successful commercial applications of biosensors. A big change in electroanalysis has occurred in the last 30 years, caused by the invention of modified electrodes. The modification of electrode surfaces has also led to improved anchoring of highly specific biological recognition elements, allowing unprecedented performance in electrocatalytic biosensing. Electrode systems have attained increasing complexity and a large variety of electrode modifiers have been proposed, such as synthetic and natural macromolecules, different types of nanoparticles and conducting polymers or clays. Among the novel materials developed in the last 20 years, only a limited number have been tested and used in an electrochemical context, and an even much lower number have been considered for electroanalysis. This encourages analytical chemists to closely follow developments in the field of material chemistry, basic studies in electrochemistry and electrochemical applications to potentially transfer this knowledge to effective electroanalytical systems. Concomitantly with the development of novel electrode materials, optimization of the overall system structure has become more complex, requiring its characterization using a large number of spectroscopic, microscopic and electrochemical techniques. Consequently, interdisciplinarity has become a must for the effective development of novel modified electrodes for electroanalysis. Only combinations of analytical techniques provide sufficient insight into the in-depth functioning of modified electrodes for electroanalysis and the interplay between the different components ultimately composing the complex electrode architecture. Complete knowledge, in the widest sense, is necessary to achieve the best performance of an envisaged sensor as a whole. A lot of time has passed since the years in which ‘bare’ electrodes were used in molecular electrochemistry studies, which were subsequently transferred to electroanalysis. More or less complex electrode mechanisms were studied only by electrochemical techniques that allowed the electrode mechanism to be fully elucidated from a qualitative and even quantitative point of view. Such a ‘simple’, though rigorous approach is not possible with more complex modified electrode systems any more, owing to the enormous variety of possible structures, depending on a number of possible compositions and the conditions under which the system has been realized. A danger lies in the tendency to separate the field into two ‘different kinds’ of scientists doing electroanalysis: those strictly following an ‘it works!’ approach describing often useful effects which cannot be given an in-depth account, owing to the too great complexity of the system, Published in the topical collections Amperometric Sensing with guest editors Renato Seeber, Fabio Terzi, and Chiara Zanardi and Bioelectroanalysis with guest editors Nicolas Plumere, Magdalena Gebala, and Wolfgang Schuhmann. R. Seeber (*) : F. Terzi :C. Zanardi Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, Via G. Campi, 183, 41100 Modena, Italy e-mail: renato.seeber@unimore.it

Special Issue on Organic Electronic Bio-Devices

The aim of the present editorial is to briefly summarize the current scientific and technological accomplishments in the field of organic electronic biosensors as described in the articles published in this Special Issue. By definition, a biosensor is a robust analytical device that combines a biological recognition element (e.g., antibodies, enzymes, cells) with a transducer. Organic electronic bio-devices are considered as potentially reliable substitutes of conventional and rather expensive analytical techniques employed for several applications such as medical diagnosis, food safety and environment pollution monitoring. Some insights into the selection and immobilization of recognition elements, signal amplification, fabrication techniques and analytical performance of biosensing devices will be presented.

Determination of sulfate-reducing bacteria with chemical conversion from ZnO nanorods arrays to ZnS arrays

This work presents a novel specific detection method for sulfate-reducing bacteria (SRB) based on sulfuration conversion from ZnO arrays to ZnS arrays. The sulfuration process was conducted in two steps. First, ZnO@ZnS intermediate nanocomposite arrays were formed by the sulfuration reaction between ZnO and sulfide, which is the characteristic enzymatic metabolism product of SRB. Next, unconverted ZnO cores were washed away in ammonia buffer solutions, leaving ZnS arrays on the surface of ITO substrates. The array conversion process can be used for SRB detection because the final state of ZnS arrays was highly related with the amount of accumulated sulfide, which was decided by the initial concentration of SRB. Electrochemical impedance spectroscopy has been used to optimize experimental conditions and detect SRB in aqueous solutions. This SRB detection method based on arrays conversion from ZnO to ZnS can avoid the use of biological recognition elements, which are expensive and easy to lose specific recognizing abilities. In addition, compared with the widely used MPN method which would take up to 15days to accomplish the detection process, the proposed method can shorten the detection time greatly.

Recent Advances in Bacteriophage Based Biosensors for Food-Borne Pathogen Detection

Foodborne diseases are a major health concern that can have severe impact on society and can add tremendous financial burden to our health care systems. Rapid early detection of food contamination is therefore relevant for the containment of food-borne pathogens. Conventional pathogen detection methods, such as microbiological and biochemical identification are time-consuming and laborious, while immunological or nucleic acid-based techniques require extensive sample preparation and are not amenable to miniaturization for on-site detection. Biosensors have shown tremendous promise to overcome these limitations and are being aggressively studied to provide rapid, reliable and sensitive detection platforms for such applications. Novel biological recognition elements are studied to improve the selectivity and facilitate integration on the transduction platform for sensitive detection. Bacteriophages are one such unique biological entity that show excellent host selectivity and have been actively used as recognition probes for pathogen detection. This review summarizes the extensive literature search on the application of bacteriophages (and recently their receptor binding proteins) as probes for sensitive and selective detection of foodborne pathogens, and critically outlines their advantages and disadvantages over other recognition elements.

Block Copolymer Modified Surfaces for Conjugation of Biomacromolecules with Control of Quantity and Activity

Polymer brush layers based on block copolymers of poly(oligo(ethylene glycol) methacrylate) (POEGMA) and poly(glycidyl methacrylate) (PGMA) were formed on silicon wafers by activators generated by electron transfer atom transfer radical polymerization (AGET ATRP). Different types of biomolecule can be conjugated to these brush layers by reaction of PGMA epoxide groups with amino groups in the biomolecule, while POEGMA, which resists nonspecific protein adsorption, provides an antifouling environment. Surfaces were characterized by water contact angle, ellipsometry, and Fourier transform infrared spectroscopy (FTIR) to confirm the modification reactions. Phase segregation of the copolymer blocks in the layers was observed by AFM. The effect of surface properties on protein conjugation was investigated using radiolabeling methods. It was shown that surfaces with POEGMA layers were protein resistant, while the quantity of protein conjugated to the diblock copolymer modified surfaces increased with increasing PGMA layer thickness. The activity of lysozyme conjugated on the surface could also be controlled by varying the thickness of the copolymer layer. When biotin was conjugated to the block copolymer grafts, the surface remained resistant to nonspecific protein adsorption but showed specific binding of avidin. These properties, that is, well-controlled quantity and activity of conjugated biomolecules and specificity of interaction with target biomolecules may be exploited for the improvement of signal-to-noise ratio in sensor applications. More generally, such surfaces may be useful as biological recognition elements of high specificity for functional biomaterials.

Sandwich-type electrochemical biosensor for glycoproteins detection based on dual-amplification of boronic acid-gold nanoparticles and dopamine-gold nanoparticles

Glycoproteins play important roles in a wide variety of biological processes. The change in the concentration levels has been associated with many cancers, as well as other diseases. Thus, rapid, sensitive and selective determination of glycoproteins is much preferred. In this work, we reported a sandwich-type electrochemical biosensor based on dual-amplification of 4-mercaptophenylboronic acid (MBA)-capped gold nanoparticles (MBA-AuNPs) and dopamine (DA)-capped AuNPs (DA-AuNPs). Biological recognition elements such as synthetic receptor and aptamer immobilized onto gold electrodes were used to capture glycoproteins. The captured glycoproteins were then derivatized with MBA-AuNPs through the formation of tight covalent bonds between the boronic acids of MBA-AuNPs and diols of glycoproteins. Electroactive DA-AuNPs were attached by the anchored MBA-AuNPs via the interaction of boronic acids with DA tags, which facilities the amplified voltammetric detection of glycoproteins. With avidin and prostate specific antigen (PSA) as model analytes, we demonstrated the feasibility and sensitivity of the proposed method. The results indicated that sub-picomolar avidin/PSA can be readily measured. We believe that this strategy will be valuable for the electrochemical detection of other glycoproteins.

A new embedded biosensor platform based on micro-electrodes array (MEA) technology

Abstract This paper illustrates the application of a multi-response biosensor platform to the simultaneous analysis of several water and food components and contaminants, and demonstrates how this platform can represent a valid support to agrifood industry. This biosensor was based on micro-electrodes array (MEA) technology and employed an array of biological recognition elements intimately integrated to an electrochemical–optical transduction system. The modification of gold micro-electrodes transducer surface allowed the simultaneous determination of selected target analytes by means of amperometric and fluorescence signal variation of immobilized enzymes and micro-organisms able to recognize various food components, like sugars and phenols, and contaminants, like pesticides. The performance of the biosensor platform was evaluated against the traditional electrochemical and optical methods with laboratory set-up instrumentations showing good linearity, sensitivity and repeatability in the detection of glucose, lactose, catechol, atrazine, prometryn and diuron in standard and real samples, with linearity in the micromolar range and with high sensitive limits of detection. In particular, atrazine, prometryn and diuron were detected with a limit of detection of 0.5 nM, 0.31 nM and 0.48 nM, respectively; glucose and lactose were revealed with a limit of detection of 8.5 μM and 90 μM, respectively; catechol was identified with a limit of detection of 1 μM. Based on the comparative performance of laboratory set-up techniques and MEA biosensor, the multi-biosensor platform emerged as a suitable tool for large classes of compounds in fast, simple screening in agrifood quality.

Aptasensors for Small Molecule Detection

Aptamers are single-stranded oligonucleotides composed of RNA or DNA that are able to bind their corresponding targets via molecular recognition. Thus, aptamers can be thought of as nucleic acidbased alternatives to antibodies and have attracted attention as receptors in biosensors. Aptamers seem to be ideal biological recognition elements, since they enable the design of intelligent sensors based on their specific properties. Especially the fact that most aptamers undergo conformational changes during the binding of the target and their oligonucleotide nature can be used to rationally design novel sensing strategies. This review focuses on aptasensors for the detection of small molecules. In the first part, aptamers, their generation and their properties are briefly described. In the second part, different design strategies for aptasensors are reviewed, and examples for the detection of small molecules are given.

A disposable biosensor based on immobilization of laccase with silica spheres on the MWCNTs-doped screen-printed electrode

BackgroundBiosensors have attracted increasing attention as reliable analytical instruments in in situ monitoring of public health and environmental pollution. For enzyme-based biosensors, the stabilization of enzymatic activity on the biological recognition element is of great importance. It is generally acknowledged that an effective immobilization technique is a key step to achieve the construction quality of biosensors.ResultsA novel disposable biosensor was constructed by immobilizing laccase (Lac) with silica spheres on the surface of multi-walled carbon nanotubes (MWCNTs)-doped screen-printed electrode (SPE). Then, it was characterized in morphology and electrochemical properties by scanning electron microscopy (SEM) and cyclic voltammetry (CV). The characterization results indicated that a high loading of Lac and a good electrocatalytic activity could be obtained, attributing to the porous structure, large specific area and good biocompatibility of silica spheres and MWCNTs. Furthermore, the electrochemical sensing properties of the constructed biosensor were investigated by choosing dopamine (DA) as the typical model of phenolic compounds. It was shown that the biosensor displays a good linearity in the range from 1.3 to 85.5 μM with a detection limit of 0.42 μM (S/N = 3), and the Michaelis-Menten constant (Kmapp) was calculated to be 3.78 μM.ConclusionThe immobilization of Lac was successfully achieved with silica spheres to construct a disposable biosensor on the MWCNTs-doped SPE (MWCNTs/SPE). This biosensor could determine DA based on a non-oxidative mechanism in a rapid, selective and sensitive way. Besides, the developed biosensor could retain high enzymatic activity and possess good stability without cross-linking reagents. The proposed immobilization approach and the constructed biosensor offer a great potential for the fabrication of the enzyme-based biosensors and the analysis of phenolic compounds.

Label-free electrochemical diagnosis of viral antigens with genetically engineered fusion protein.

We have developed a simple electrochemical biosensing strategy for the label-free diagnosis of hepatitis B virus (HBV) on a gold electrode surface. Gold-binding polypeptide (GBP) fused with single-chain antibody (ScFv) against HBV surface antigen (HBsAg), in forms of genetically engineered protein, was utilized. This GBP-ScFv fusion protein can directly bind onto the gold substrate with the strong binding affinity between the GBP and the gold surface, while the recognition site orients toward the sample for target binding at the same time. Furthermore, this one-step immobilization strategy greatly simplifies a fabrication process without any chemical modification as well as maintaining activity of biological recognition elements. This system allows specific immobilization of proteins and sensitive detection of targets, which were verified by surface plasmon resonance analysis and successfully applied to electrochemical cyclic voltammetry and impedance spectroscopy upto 0.14 ng/mL HBsAg.

Effective Surface Functionalization of Nanocrystalline Diamond Films by Direct Carboxylation for PDGF Detection via Aptasensor

An aptasensor was designed on a nanocrystalline diamond (NCD) surface that combined with biological recognition elements, PDGF-binding aptamers, which inherently possess high affinity to PDGF-BB proteins. Functional components such as carboxylic acids (-COOH) and amines (-NH2) were directly introduced onto the NCD surface and used as probing units for immobilization of PDGF-binding aptamers. The surface coverage of different components on the NCD was analyzed by X-ray photoelectron spectroscopy (XPS) measurements, and the effects of various functionalizations on the NCD biosensor performance were investigated via fluorescence observations. The coverages of carboxyl and amine groups achieved were 12 and 23%, respectively, for the directly aminated and carboxylated NCD; however, the lower density of carboxyl groups on the functionalized surface did not deteriorate the performance of the COOH-NCD biosensor. Fluorescence investigations demonstrated comparable performance in sensitivity and selectivity for PDGF protein detection on COOH-NCD and NH2-NCD biosensors. Multiple regeneration tests clearly showed that the COOH-NCD biosensor as well as the NH2-NCD biosensor retained a high performance without exhibiting any noticeable degradation.

Electrogenerated chemiluminescence biosensor incorporating ruthenium complex-labelled Concanavalin A as a probe for the detection of Escherichia coli

A novel electrogenerated chemiluminescence (ECL) biosensor for highly sensitive detection of Escherichia coli (E. coli) was first developed by employing Concanavalin A (Con A) as a biological recognition element and bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine ruthenium (II) (Ru1) complex as the detector. The ECL biosensor was fabricated by adsorbing carboxyl-functionalised single-wall carbon nanotubes (SWNTs) onto a paraffin-impregnated graphite electrode and further covalently coupling the Ru1–Con A probe onto the surface of the SWNT-modified electrode. Upon the binding of E. coli O157:H7 (as a model target), the biosensor showed a decreased ECL intensity in the presence of tri-n-propylamine (TPrA), which was in logarithmically direct proportion to the concentration of E. coli over a range from 5.0×102 to 5.0×105cells/mL. The detection limit of this sensor was 127cells/mL. Additionally, the ECL biosensor also showed satisfactory selectivity in discriminating gram-negative E. coli from gram-positive bacteria. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and rapid detection of other desired bacteria.