Selenium and nitrogen co-doped carbon dots with highly efficient electrochemiluminescence for ultrasensitive detection of microRNA.

In this work, selenium and nitrogen co-doped carbon dots (SeN-CDs) possessing highly efficient electrochemiluminescence (ECL) and excellent biocompatibility were synthesized as a new emitter with S2O82- as a coreactant for constructing a biosensor to detect microRNA-221 (miRNA-221) sensitively. Notably, the SeN-CDs exhibited superior ECL performance compared with the N-doped CDs, in which selenium with excellent redox activity served as a coreaction accelerator for facilitating the electroreduction of S2O82- to significantly improve ECL efficiency. Furthermore, target-induced T7 exonuclease (T7 Exo)-assisted double cycle amplification strategy could convert traces of target miRNA-221 into large amounts of output DNA to capture three-dimensional (3D) nanostructures (DTN-Au NPs-DOX-Fc) loaded with large amounts of ECL signal quencher. The constructed biosensor could realize ultrasensitive detection of miRNA-221 and has a low detection limit reaching 2.3 aM, with a successful application to detect miRNA-221 in lysate of Hela and MHCC97-L cancer cell. This work explored a novel method to strengthen the ECL performance of CDs to construct an ECL biosensing platform with sensitive detecting of biomarkers and disease diagnosis.

Novel Ratiometric Electrochemiluminescence Biosensor Based on BP-CdTe QDs with Dual Emission for Detecting MicroRNA-126.

The electrochemiluminescence (ECL) ratiometric assay is usually based on two different ECL luminophores, and the choice of two suitable luminophores and shared co-reactant makes its construction challenging. The single-emitter-based ECL ratio mode could overcome the limitation of two luminophores and simplify the construction process, so it is an ideal choice. In this work, CdTe quantum dots (CdTe QDs) were modulated using black phosphorus (BP) nanosheet to simultaneously emit the cathodic and anodic ECL signals, and H2O2 and tripropylamine (TPrA) served as the cathodic and anodic co-reactants, respectively. MicroRNA-126 (miRNA-126) was selected as the template target to exploit the application of BP-CdTe QDs in the single-emitter-based ECL ratio detection. Through the target recycling triggering rolling-circle amplification (RCA) reaction, a large amount of glucose oxidase (GOx)-modified single strand 1 was introduced. GOx catalyzed glucose to produce H2O2 in situ, which acted as a dual-role moderator to quench the anodic ECL emission with TPrA as the co-reactant while enhancing the cathodic emission, thereby realizing the ratiometric detection of miRNA-126 with a low detection limit of 29 aM (S/N = 3). The dual-ECL-emitting BP-CdTe QDs with TPrA-H2O2 as dual co-reactant provide a superior ECL ratio platform involving enzyme catalytic reaction, expanding the application of single-emitter-based ratio sensing in the diverse biological analysis.

High-Fidelity and Simultaneous Sensing of Endogenous Mutant and Wild p53 Proteins for Precise Cancer Diagnosis and Drug Screening.

The simultaneous sensing of endogenous wild and mutant proteins plays a critical role in disease diagnosis and drug screening, and this remains a major current challenge. Here, we present a new and highly specific target-triggered dual proximity ligation assay (dPLA) strategy for sensitive and simultaneous sensing of wild and mutant p53 proteins from cancer cells. Two proximity DNA probes bind the target protein to form the primer/circular DNA template complexes with two nicks in the presence of the hairpin and ssDNA connector sequences via the strand displacement reaction. Only when the two nicks are simultaneously ligated can the rolling circle amplification be triggered with high fidelity for yielding substantially enhanced fluorescence. By encoding the hairpin sequence, two distinct fluorescence signals can be generated for simultaneous detection of the wild and mutant p53 proteins. Importantly, our method significantly reduces the possibility of nonspecific ligation reactions by using two ligation nicks, which minimizes the background noise. With this dPLA method, the regulation transition of intracellular mutant p53 to wild p53 proteins upon anticancer drug treatment has also been demonstrated, highlighting its usefulness for potential early disease diagnosis and drug screening with high fidelity.

Enhancing photoelectrochemical performance of ZnIn2S4 by phosphorus doping for sensitive detection of miRNA-155.

We report the synthesis of phosphorus-doped ZnIn2S4 (ZnIn2S4-xPx) materials through a solid/gas-phase reaction. ZnIn2S4-xPx shows enhanced photoelectrochemical (PEC) performance due to the improved photo-carrier separation efficiency achieved by substituting some of the sulfur with phosphorus. A PEC biosensor was further developed based on ZnIn2S4-xPx, which exhibits excellent analytical performance for miRNA-155.

Commercial glucometer as signal transducer for simple evaluation of DNA methyltransferase activity and inhibitors screening.

DNA methyltransferase (MTase) plays an important role in many biological processes and has been recognized as a predictive cancer biomarker far before other signs of malignancy and a therapeutic target in cancer treatment. Thus simple and sensitive determination of DNA MTase activity is urgently required. The commercially available glucometer is considered as the most successful point-of-care (POC) sensor up to date, and researchers extend its application in monitoring different types of targets rather than only glucose. Here, we developed a simple strategy for the sensitive detection of the DNA MTase (using M.SssI as an example) activity by using a glucometer as the signal transducer. A S1/S2 hybrid probe was designed including a specific recognition sequence for both DNA MTase and restriction endonuclease, and a complementary sequence for biotin-S3. Firstly, the S1/S2 hybrid probe was self-assembled on the gold electrode and methylated by M.SssI MTase to form the methylated dsDNA. Then, HpaII endonuclease specifically cleaved the residue of the unmethylated dsDNA. Subsequently, biotin-S3 hybridized with the overhang sequence of the methylated dsDNA. Finally, the biotin tag was successively combined with streptavidin (STV) and biotin-invertase. The invertase efficiently catalyzed the hydrolysis of sucrose to generate abundant glucose, which led to an amplified response of glucometer. This strategy could detect DNA MTase activity as low as 0.3 U mL-1 with good selectivity against other two cytosine MTases (HaeIII MTase and AluI MTase), and be successfully applied for screening the DNA MTase inhibitors (5-azacytidine and 5-aza-2'-deoxycytidine), implying our proposed method holds great promising application in early cancer diagnosis and therapeutics.

In Situ Electrodeposited Synthesis of Electrochemiluminescent Ag Nanoclusters as Signal Probe for Ultrasensitive Detection of Cyclin-D1 from Cancer Cells.

Metal nanoclusters (NCs) as a new type of electrochemiluminescence (ECL) nanomaterials have attracted great attention, but their applications are limited due to relatively low luminescent efficiency and a complex preparation process. Herein, an ultrasensitive ECL biosensor for the detection of Cyclin-D1 (CCND1) was designed by utilizing in situ electrogenerated silver nanoclusters (AgNCs) as ECL emitters and Fe3O4-CeO2 nanocomposites as a coreaction accelerator. The ECL luminous efficiency of AgNCs on the electrode could be significantly enhanced with the use of the Fe3O4-CeO2 for accelerating the reduction of S2O82- to generate the strong oxidizing intermediate radical SO4•-. As a result, the assay for CCND1 detection achieved excellent sensitivity with a linear range from 50 fg/mL to 50 ng/mL and limit of detection down to 28 fg/mL. Impressively, the efficiency of Traditional Chinese Medicines (TCM), sophorae, toward MCF-7 cells was successfully investigated due to the overexpression of CCND1 in relation to the growth and metastasis of MCF-7 human breast cancer cells. In general, the proposed strategy provided an effective method for anticancer drug screening and expanded the application of metal NCs in ultrasensitive biodetection.

An amplified electrochemical proximity immunoassay for the total protein of Nosema bombycis based on the catalytic activity of Fe3O4NPs towards methylene blue.

A simple electrochemical proximity immunoassay (ECPA) system for the total protein of Nosema bombycis (TP N.b) detection has been developed on the basis of a new amplification strategy combined with target-induced proximity hybridization. The desirable ECPA system was achieved through following process: firstly, the methylene blue (MB) labeled hairpin DNA (MB-DNA) were immobilized on electrode through Au-S bonding. Then, the antibody labeled complementary single-stranded DNA probe (Ab1-S1) hybridized with MB-DNA to open its hairpin structure, which led to the labeled MB far away from electrode surface. After that, the presence of target biomarker (TP N.b) and antibody labeled single-stranded DNA (Ab2-S2) triggered the typical sandwich reaction and proximity hybridization, which resulted in the dissociation of Ab1-S1 from electrode and the transformation of the MB-DNA into a hairpin structure with MB approaching to electrode surface. In consequence, the hairpin-closed MB was electrocatalyzed by the modified magnetic nanoparticles (Fe3O4NPs), leading to an increased and amplified electrochemical signal for the quantitative detection of TP N.b. In the present work, Fe3O4NPs were acted as catalyst to electrocatalyze the reduction of electron mediator MB for signal amplification, which could not only overcome the drawbacks of protein enzyme in electrocatalytic signal amplification but also shorten the interaction distance between catalyst and substance. Under optimal condition, the proposed ECPA system exhibited a wide linear range from 0.001ngmL(-1) to 100ngmL(-)(1) with a detection limit (LOD) of 0.54pgmL(-1). Considering the desirable sensitivity and specificity, as well as the novel and simple features, this signal amplified ECPA system opened an opportunity for quantitative analysis of many other kinds of protein biomarker.

Ultrasensitive Cytosensor Based on Self-Enhanced Electrochemiluminescent Ruthenium-Silica Composite Nanoparticles for Efficient Drug Screening with Cell Apoptosis Monitoring.

The self-enhanced electrochemiluminescence (ECL) with high sensitivity could be an effective method for anticancer drug screening with cell apoptosis monitoring. Here we reported an ultrasensitive ECL cytosensor for cell apoptosis monitoring by using self-enhanced electrochemiluminescent ruthenium-silica composite nanoparticles (Ru-N-SiNPs) labeled annexin V as signal probes. The Ru-N-SiNPs were first synthesized through simple hydrolysis of a novel precursor containing luminescent and intracoreactant groups in one molecule, which presented higher emission efficiency and enhanced ECL intensity due to the shorter electron-transfer path and less energy loss. Moreover, the as-proposed ECL cytosensor was successfully used to investigate efficiency of paclitaxel toward MDA-MB-231 breast cancer cell in the range from 1 nM to 200 nM with a detection limit of 0.3 nM and a correlation coefficient of 0.9917. The improved accuracy and excellent dynamic range revealed the potential applications in biomolecules diagnostics and cells detections, especially in living and complex systems.

Cleavage-based hybridization chain reaction for electrochemical detection of thrombin.

In the present work, we constructed a new label-free "inter-sandwich" electrochemical aptasensor for thrombin (TB) detection by employing a cleavage-based hybridization chain reaction (HCR). The designed single-stranded DNA (defined as binding DNA), which contained the thrombin aptamer binding sequence, a DNAzyme cleavage site and a signal reporter sequence, was first immobilized on the electrode. In the absence of a target TB, the designed DNAzymes could combine with the thrombin aptamer binding sequence via complementary base pairing, and then Cu(2+) could cleave the binding DNA. In the presence of a target TB, TB could combine with the thrombin aptamer binding sequence to predominantly form an aptamer-protein complex, which blocked the DNAzyme cleavage site and prevented the binding DNA from being cleaved by Cu(2+)-dependent DNAzyme. As a result, the signal reporter sequence could leave the electrode surface to trigger HCR with the help of two auxiliary DNA single-strands, A1 and A2. Then, the electron mediator hexaammineruthenium (III) chloride ([Ru(NH3)6](3+)) was embedded into the double-stranded DNA (dsDNA) to produce a strong electrochemical signal for the quantitative measurement of TB. For further amplification of the electrochemical signal, graphene reduced by dopamine (PDA-rGO) was introduced as a platform in this work. With this strategy, the aptasensor displayed a wide linearity in the range of 0.0001 nM to 50 nM with a low detection limit of 0.05 pM. Moreover, the resulting aptasensor exhibited good specificity and acceptable reproducibility and stability. Because of these factors, the fabrication protocol proposed in this work may be extended to clinical application.

Amplified electrochemiluminescence of luminol based on hybridization chain reaction and in situ generate co-reactant for highly sensitive immunoassay.

In this work, we described a simple and highly sensitive electrochemiluminescence (ECL) strategy for IgG detection. Firstly, L-cysteine functionalized reduced graphene oxide composite (L-cys-rGO) was decorated on the glassy carbon electrode (GCE) surface. Then anti-IgG was immobilized on the modified electrode surface through the interaction between the carboxylic groups of the L-cys-rGO and the amine groups in anti-IgG. And then biotinylated anti-IgG (bio-anti-IgG) was assembled onto the electrode surface based on the sandwich-type immunoreactions. By the conjunction of biotin and streptavidin (SA), SA was immobilized, which in turn, combined with the biotin labeled initiator strand (S1). In the presence of two single DNA strands of glucose oxidase labeled S2 (GOD-S2) and complementary strand (S3), S1 could trigger the hybridization chain reaction (HCR) among S1, GOD-S2 and S3. Herein, due to HCR, numerous GOD was efficiently immobilizated on the sensing surface and exhibited excellent catalysis towards glucose to in situ generate amounts of hydrogen peroxide (H2O2), which acted as luminol's co-reactant to significantly enhance the ECL signal. The proposed ECL immunosensor presented predominate stability and high sensibility for determination of IgG in the range from 0.1 pg mL(-1) to 100 ng mL(-1) with a detection limit of 33 fg mL(-1) (S/N=3). Additionally, the designed ECL immunosensor exhibited a promising application for other protein detection.

Amperometric aptasensor for thrombin detection using enzyme-mediated direct electrochemistry and DNA-based signal amplification strategy.

In this work, a new electrochemical aptasensor based on direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) using exonuclease-catalyzed target recycling and hybridization chain reaction (HCR) for signal amplification was developed for highly sensitive detection of thrombin. The electrochemical signal was originated from HRP without the addition or labeling of redox probes. To construct the aptasensor, the capture probe was immobilized on gold nanoparticles (AuNPs) modified electrode for the following hybridization with the complementary thrombin binding aptamer. In the presence of thrombin, the formation of aptamer-thrombin complex would result in the dissociation of aptamer from the double-strand DNA (dsDNA). Subsequently, with the employment of exonuclease, aptamer was selectively digested and thrombin could be released for analyte recycling. The capture probe and two hairpin helper DNAs lead to the formation of extended dsDNA polymers through HCR on the electrode surface. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled HRP, resulting in significantly amplified electrochemical signal through the direct electrochemistry and electrocatalysis of HRP. The proposed strategy combined the amplification of analyte recycling and HCR, as well as the inherent electroactivity and catalytic activity of HRP, which exhibited high sensitivity for thrombin determination with an ultra-low detection limit of 1.2×10(-13) M. Moreover, the detection scheme could be easily extended to the detection of other biomolecules.

Supersandwich-type electrochemiluminescenct aptasensor based on Ru(phen)3(2+) functionalized hollow gold nanoparticles as signal-amplifying tags.

An electrochemiluminescent (ECL) aptasensor was fabricated and used for the amplified detection of thrombin (TB) based on DNA supersandwich structure. Herein, hollow gold nanospheres (HGNPs) were firstly employed as effective tag-carriers for the immobilization of detection aptamer (TBA 2) to form the HGNPs labeled TBA 2 (HGNPs-TBA 2). Subsequently, streptavidin (SA) was used to block the non-specific binding sites of HGNPs-TBA 2 as well as to supply binding sites, which could further introduce numerous initiator DNA strands (bio-S1) via biotin-streptavidin specific interaction. Next, bio-S1 could in situ trigger hybridization chain reaction (HCR) to create a long nicked double helices analogous (dsDNA) in the present of ssDNA S2 and ssDNA S3 (S3 is partially complementary to the S2) to obtain the DNA supersandwich structure. Furthermore, Ru(phen)3(2+), a well-known ECL luminophore, could be intercalated into the grooves of dsDNA (Ru-dsDNA) to form the Ru-dsDNA-SA-HGNPs-TBA 2 bioconjugate. As a result, the target of TB was sandwiched between Ru-dsDNA-SA-HGNPs-TBA 2 and TBA 1. In this strategy, numerous Ru(phen)3(2+) could be immobilized on the electrode based on the supersandwich structure, resulting in an increased ECL signal output. A supersandwich ECL assay for TB detection was developed with excellent sensitivity of a large concentration variation from 5fM to 50pM and a detection limit of 1.6fM.

DNAzyme-based highly sensitive electronic detection of lead via quantum dot-assembled amplification labels.

An electronic DNAzyme sensor for highly sensitive detection of Pb(2+) is demonstrated by coupling the significant signal enhancement of the layer-by-layer (LBL) assembled quantum dots (QDs) with Pb(2+) specific DNAzymes. The presence of Pb(2+) cleaves the DNAzymes and releases the biotin-modified fragments, which further hybridize with the complementary strands immobilized on the gold substrate. The streptavidin-coated, QD LBL assembled nanocomposites were captured on the gold substrate through biotin-streptavidin interactions. Subsequent electrochemical signals of the captured QDs upon acid dissolution provide quantitative information on the concentrations of Pb(2+) with a dynamic range from 1 to 1000 nM. Due to the dramatic signal amplification by the numerous QDs, subnanomolar level (0.6 nM) of Pb(2+) can be detected. The proposed sensor also shows good selectivity against other divalent metal ions and thus holds great potential for the construction of general DNAzyme-based sensing platform for the monitoring of other heavy metal ions.

A reagentless, disposable and multiplexed electronic biosensing platform: application to molecular logic gates.

The construction of a reagentless, sensitive, disposable and multiplexed electronic sensing platform for one-spot simultaneous determination of biomolecules with significant difference in size (proteins and small molecules) is described. The sensing surface is fabricated by the hybridization of two types of redox-tags conjugated aptamers with the corresponding complementary DNAs, which are self-assembled on a gold nanoparticle-modified screen printed carbon electrode. The presence of the target analytes leads to the release of the tagged signaling aptamers from the sensing surface, and the surface-remained tags exhibit well-resolved peaks, whose positions and sizes reflect the identities and concentrations of the target analytes, respectively. The application of the proposed sensing platform for molecular logic gate operations is also demonstrated.

Multi-walled carbon nanotubes and Ru(bpy)3(2+)/nano-Au nano-sphere as efficient matrixes for a novel solid-state electrochemiluminescence sensor.

An effective method for immobilization of Ru(bpy)(3)(2+) on glassy carbon electrode surface (GCE) is developed for the preparation of a novel electrochemiluminescence sensor. First of all, the positively charged Ru(bpy)(3)(2+) is modified on the surface of negatively charged gold nanoparticles (nano-Au) via the electrostatic interactions to obtain the Ru(bpy)(3)(2+)/nano-Au nano-sphere (abbreviate as Ru-AuNPs). Subsequently, the large amount of Ru-AuNPs are immobilized on the multi-wall carbon nanotubes (MWCNTs)-Nafion homogeneous composite coated GCE by dual interaction: firstly, the Nafion, a kind of typical cation-exchange membrane, can absorb the Ru-AuNPs as the enrichment of cation Ru(bpy)(3)(2+) on the Ru-AuNPs surface; secondly, the employment of carboxylic MWCNTs in the Nafion film can also chemosorb the Ru(bpy)(3)(2+) cation on the Ru-AuNPs surface to increase the carrier content. At the same time, the experiment confirms that the enhancement of the ECL intensity on the sensor is attributed to following reasons. One hand, the employment of MWCNTs in the Nafion film enlarged the electro-active surface areas to benefit the contact between the signal probe on the composite film and coreactant used as reinforcing agent. On the other hand, the nano-materials of MWCNTs and nano-Au also improve the conductivity of the assembled film to increase the quantity of excited state of Ru(bpy)(3)(2+) in the unit time under the electrochemical condition and finally cause better properties in luminescence. In the experiment, the influence of the coreactant tripropylamine (TPA) on proposed ECL sensor is investigated. The logarithm of ECL intensity is proportional to the logarithm of TPA concentration on the range of 4 x 10(-10)M to 2.8 x 10(-6)M and 2.8 x 10(-6)M to 0.71 x 10(-3)M. After optimizing these conditions, the ECL sensor with TPA as coreactant is employed to detect a kind of alkaloid medicine, Matrine, for evaluating the practical application in the medicine analysis. The present sensor with TPA as coreactant shows the good response to the medicine concentration of the Matrine from 2.0 x 10(-6)M to 6.0 x 10(-3)M, which is used to detect the Matrine concentration in the Matrine injection.

Organically nanoporous silica gel based on carbon paste electrode for potentiometric detection of trace Cr(III).

A new ion-selective electrode (ISE) for the detection of trace chromium(III) was designed by using 2-acetylpyridine and nanoporous silica gel (APNSG)-functionalized carbon paste electrode (CPE). The presence of APNSG acted as not only a paste binder, but also a reactive material. With 7.5 wt% APNSG proportions, the developed electrode exhibited wide dynamic range of 1.0 x 10(-8) to 1.0 x 10(-3) M toward Cr(III) with a detection limit of 8.0 x 10(-9) M and a Nernstian slope of 19.8 +/- 0.2 mV decade(-1). The as-prepared electrode displayed rapid response (approximately 55 s), long-time stability, and high sensitivity. Moreover, the potentiometric responses could be carried out with wide pH range of 1.5-5.0. In addition, the content of Cr(III) in food samples, e.g. coffee and tea leaves, has been assayed by the developed electrode, atomic absorption spectrophotometer (AAS) and atomic emission spectrometer (ICP-AES), respectively, and consistent results were obtained. Importantly, the response mechanism of the proposed electrode was investigated by using AC impedance and UV-vis spectroscopy.