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Ratiometric electrochemiluminescence (ECL) sensors can efficiently remove environmental interference to attain precise detection. Nonetheless, two eligible luminophores or coreactants were usually needed, increasing the complexity and restricting their practical application. In this study, a single luminophore of luminol with a single coreactant of H

In the electrochemiluminescence (ECL) field, most reported luminophores were focused on high-triggering potential and short wavelength, which was adverse for the ECL theory study and application at low potentials. Perylene diimide derivatives could emit near-infrared (NIR) ECL at low-triggering potential; however, they are always highly aggregated into a microrod structure and stacked together, which largely limited their application in biological fields such as bio-sensing and bio-imaging. To overcome these obstacles, we designed a novel perylene diimide molecule, namely N,N′-dicaproate sodium-3,4,9,10-perylenedicarboximide (PDI-COONa). This molecule self-assembled into a two-dimensional network nanostructure, which largely decreased the aggregation degree of PDI molecules and provided solid bases for designing lowly-aggregated PDI molecules. Also, the formed nanoluminophore produced strong emission at −0.26 V with an NIR wavelength 700 nm, which should be due to the excited J-type PDI-COO− dimers. Moreover, this network nanoluminophore well-dispersed on graphene oxide (GO) as an ECL nanomaterial to label secondary antibodies and fabricate a sandwiched immunosensor for alpha-fetoprotein (AFP) detection between 0 and −0.6 V. This immunosensor showed a wider linear response for AFP ranging from 0.1 fg mL−1 to 1 μg mL−1 with a low detection limit 0.0353 fg mL−1 compared with other immunosensors based on PDI microrod-modified GO ECL materials. The fabricated immunosensor also showed good feasibility in human serum samples.

A unique ratiometric MALDI-MS strategy is proposed for the convenient and reliable quantitation of alkaline phosphatase based on the homogeneous enzymatic cleavage of a coded phosphopeptide (CPP)-triggered double-signal output. The dynamic range can be tuned by simply adjusting the primary concentration of CPP. The proposed strategy is also capable of being challenged by real human serum, and thus it may offer a wonderful approach for the convenient identification and quantitation of various enzyme activities in clinical diagnosis.

A novel ratiometric electrochemical biosensing strategy based on T7 exonuclease (T7 Exo)-assisted homogenous target recycling coupling hairpin assembly triggered dual-signal output was proposed for the accurate and sensitive detection of microRNA-141 (miRNA-141). Concretely, in the presence of target miRNA, abundant signal transduction probes were released via the T7 Exo-assisted homogenous target recycling amplification, which could be captured by the specially designed ferrocene-labeled hairpin probe (Fc-H1) on -electrode interface and triggered the nonenzymatic catalytic hairpin assembly (Fc-H1 + MB-H2) to realize the cascade signal amplification and dual-signal output. Through such a conformational change process, the electrochemical signal of Fc (IFc) and MB (IMB) is proportionally and substantially decreased and increased. Therefore, the signal ratio of IMB/IFc can be employed to accurately reflect the true level of original miRNA. Benefiting from the efficient integration of the T7 Exo-assisted target recycle, nonenzymatic hairpin assembly and dual-signal output mode, the proposed sensor could realize the amplified detection of miRNA-141 effectively with a wide detection range from 1 fM to 100 pM, and a detection limit of 200 aM. Furthermore, it exhibits outstanding sequence specificity to discriminate mismatched RNA, acceptable reproducibility and feasibility for real sample. This strategy effectively integrated the advantages of multiple amplification and ratiometric output modes, which could provide an accurate and efficient method in biosensing and clinical diagnosis.

Most biosensors for folate receptor (FR) detection based on folic acid (FA) recognition usually contain FA-linked single-strand DNA (FA-ssDNA) and nuclease to promote sensitivity, which increases expenses and involves complicated assay processes. A few electrochemiluminescence (ECL) sensors that do not use FA-ssDNA and nuclease directly graft FA onto an ECL nanomaterial through covalent bonding for FR detection. In this study, we used FA-ssDNA to non-covalently graft FA onto π-conjugated ECL nanomaterial graphene oxide (GO)/perylene–aniline for fabricating ultrasensitive FR sensors without nuclease. 3,4,9,10-Perylenetetracarboxylic dianhydride (PTCDA) and aniline (An) self-assembled into π-conjugated nanorods, which were then loaded onto GO. This material was reported to produce 673 nm-dominated ECL with the co-reactant K2S2O8, and was used as an ECL platform. FA-modified Poly-dA-ssDNA (FA-Poly-dA-ssDNA) molecules, consisting of 20 bases, were attached to the surface of GO/PTCDA-An to capture FR. A significant decrease of ECL intensity was observed due to the steric hindrance of FR. The proposed sensors exhibited high detection sensitivity with a linear range from 1 fg mL−1 to 1 ng mL−1 and a detection limit of 0.636 fg mL−1. The sensors also showed good potential in real sample detection. Without introducing nuclease and complicated chemical reactions, this work provides a new sensing strategy for protein detection based on molecular recognition, which is extremely important in clinical diagnosis.

In electrochemiluminescence (ECL) field, screening luminophores, especially potential-resolved luminophores with the same parent chromophore, is essential for the development of fundamental theory ...

A DNA immobilization-free ECL aptasensor was developed for the detection of 8-hydroxy-2'-deoxygunosine based on the diffusion mediated ECL quenching effect. This ECL aptasensor exhibited a high sensitivity and low detection limit by combining homogeneous DNA reaction with dual signal amplifications: target-induced multi-DNA release and Exo I-assisted target recycling.

Most electrochemiluminescence (ECL) studies focus on the single emission of luminophores, which severely limits the development of the multi-color ECL fundamental theory and applications. Herein, we prepared a multi-color ECL supramolecular nanomaterial self-assembled by 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) and aniline (An) through hydrogen bonding. This PTCDA-An supramolecular nanomaterial simultaneously produced multi-color emissions peaking at 486, 692 and 760 nm with K2S2O8 as a coreactant. These multi-color emissions were assigned to the excited PTCDA monomer (486 nm), H-dimer (692 nm) and J-dimer (760 nm). The simultaneously increased dual-color ECL intensity significantly enhanced the total ECL intensity of PTCDA-An. Furthermore, this highly efficient ECL nanomaterial was used as an ECL platform to construct a label-free immunosensor for tumor marker carcinoembryonic antigen (CEA) detection. The total ECL intensity of the immunosensor sensitively decreased due to the simultaneously decreased ECL of multiple emissions. Also, this immunosensor exhibited a wide linear range from 1 pg mL-1 to 10 μg mL-1 with a low detection limit of 0.23 pg mL-1. The multi-color ECL from the same luminophore PTCDA in this work also provides a new perspective for multi-color ECL biomaterial design.

In this work, an ultrasensitive aptasensor for the detection of Mucin 1 (MUC1) was presented based on the target-induced catalytic hairpin assembly combined with excellent mimic peroxidase performance of PtPd bimetallic nanoparticles (PtPdNPs). Traditionally, the cyclic reuse of target protein was achieved by protein conversion with enzyme cleavage or polymerization, which is costly and complex. However, in this work, it can be performed by simple strand displacement. In addition, PtPdNPs, a mimic peroxidase, was used a probe to catalyze the oxidation of tetramethylbenzidine (TMB) by H2O2, leading to the electrochemical signal amplification. With this ingenious design, the prepared aptasensor for MUC1 detection showed a favorable linear response from 100 fg mL−1 to 1 ng mL−1 and a relatively low detection limit of 16 fg mL−1. The proposed biosensor possessed acceptable stability, selectivity and reproducibility for MUC1 assay. Additionally, the fabricated aptasensor has been successfully applied to detect MUC1 in serum samples with satisfactory results. This new strategy supplied one efficient approach to improve signal amplification, which also open an avenue for sensitivity enhancement in targets detection.

In the cathodic electrochemiluminescence (ECL) field, most reported luminophores produced ECL emission at high potentials (more than −1.3 V vs. Ag/AgCl), which was adverse for both fundamental studies and practical application. It was important to screen novel ECL luminophores and coreactants for the development of ECL. In this work, N,N′-dimethyl-3,4,9,10-perylenedicarboximide (PDI-CH3) is reported to produce ECL at −0.47 V using K2S2O8 as a coreactant in an aqueous system. In addition, the ECL wavelength was 689 nm, which was interpreted with the emission of excited PDI-CH3 dimers. Finally, this low-triggering-potential ECL system was used to construct sandwiched immunosensors to detect carcinoembryonic antigen (CEA) with the potential range from 0 to −0.8 V. In this immunosensor, PDI-CH3 and gold nanoparticles (AuNPs) reduced by citrate were grafted onto graphite oxide (GO) to label secondary antibodies (Ab2). This immunosensor could sensitively detect CEA with the linear response range between 1 fg mL−1 and 1 μg mL−1 and detection limit 0.29 fg mL−1. In addition, this immunosensor showed good feasibility in various cancer serum samples.

A sensitive and enzyme-free electrochemical aptasensor was constructed for the sensing of 8-hydroxy-2′-deoxyguanosine (8-OH-dG). In the process of constructing the aptasensor, triple signal amplification strategies were introduced to enhance the sensitivity. First, every aptamer/pDNA complex immobilized on magnetic beads could release three kinds of pDNAs when 8-OH-dG was introduced, which caused three-fold magnification of the target. Second, the released three kinds of pDNAs initiated catalyzed hairpin assembly between two hairpin DNAs (HP1 and HP2) on a gold electrode. Meanwhile, the three kinds of pDNAs were released again by a strand displacement reaction to obtain the next catalyzed hairpin assembly. Third, the emerging toehold of HP2 further induced a hybridization chain reaction (HCR) between two hairpin DNAs (HP3 and HP4), forming a long double-stranded DNA concatemer on the surface of the electrode. Finally, [Ru(NH3)6]3+, an electroactive cation, was adsorbed onto the long dsDNA concatemer by electrostatic interactions and consequently, an electrochemical signal was generated. Under this triple signal amplification, a low detection limit down to 24.34 fM has been obtained for 8-OH-dG determination, which is superior to those of most previously reported methods.

The present work reported a simple, lable-free and sensitive electrochemical method for the detection of protein kinase A (PKA) activity. This method was based on the specific recognition of aptamer and the aptamer-induced hybridization chain reaction (HCR) amplification strategy. The aptasensor was constructed by immobilizing capture probe on a gold electrode via an Au–S bond. When adenosine triphosphate (ATP) aptamer was introduced, its one terminus hybridized with capture probe and the other hybridized with the complementary region of an auxiliary probe, which other region triggered HCR between two hairpin DNA (H1 and H2) to form a long DNA concatamer. At last a large number of electroactive methyle blue (MB) molecules were assembled on the dsDNA concatamer, which generated a significantly amplified electrochemical signal. In the presence of ATP, the HCR would not be performed because the aptamer specifically bond to ATP and the electrochemical response would decrease. However, when ATP and PKA coexisted, the electrochemical response would recovery because that ATP had been translated into ADP by PKA. So the activity of PKA could be effectively monitored according to the change of electrochemical signal. Based on the HCR amplification strategy, the aptasensor showed a wide linear range (4 − 4 ×105 U L−1) and a low detection limit (1.5 U L−1) for the detection of PKA. Furthermore, the method was applied to study the inhibitory effect of H-89 on PKA activity. The developed aptasensor was also used to the analysis of drug-induced PKA activity in cell lysates, indicating the potential application of the developed method in the fields of clinical diagnostics and discovery of new targeted drugs.

A unique and versatile ratiometric electrochemical DNA biosensor was designed for the first time by integration of homogeneous exonuclease III-assisted target recycling amplification and one-step triggered dual-signal output. The presence of the target DNA would hybridize with the specifically designed ferrocene-labeled hairpin probe (Fc-P1) and initiate homogeneous exonuclease III-assisted target recycling amplification, which led to the decrease of the local concentration of Fc-P1. The target DNA dependent amount of remaining Fc-P1 could be conveniently monitored by hybridize the remaining Fc-P1 with the methylene blue-labeled hairpin probe ((MB-PP1) on the sensor electrode to trigger the MB tags away from while the Fc tags close to the sensing interface. Therefore, such recognition event can be translated into the homogeneous amplification coupled with one-step triggered dual-signal ratiometric electrochemical readout. The proposed biosensor achieves a detection limit of 12.8 fM within a linear range of 0.02 pM to 2 nM for target DNA species related to oral cancer over expressed 1, along with a favorable specificity. More importantly, the biosensor had been applied in detection of target DNA in artificial saliva sample and provided significant potential application in clinical analysis. This strategy possessed good capacity to integrate the nuclease amplification techniques with ratiometric output modes, and would open new opportunities for sensitive detection of other biorecognition events.

Liver cancer is one of the factors that threaten human life. Highly up-regulated in liver cancer (HULC), a kind of long non-coding RNA (lncRNA), is regarded as a favorable noninvasive biomarker for the early diagnosis of hepatocellular carcinoma (HCC). So a strategy focused on improving the sensitivity of HULC detection is urgently desired. In this work, a highly sensitive electrochemiluminescent (ECL) sensor was constructed using Au@Ag core-shell nanoparticles/graphene quantum dots (Au@Ag/GQDs) as a signal indicator to detect HULC. Compared with the individual gold or silver nanoparticles, Au@Ag core-shell nanoparticles exhibited large specific surface area, better catalysis and excellent electronic transmission capacity due to the synergistic effect between Au and Ag. Thus, the ECL performance of GQDs could be greatly promoted by Au@Ag core-shell nanoparticles. Under the optimal conditions, this proposed ECL sensor exhibited ultrahigh sensitivity for quantitative detection of HULC with a wide linear range from 1 fM to 5 nM and a low detection limit of 0.3 fM (S/N = 3). These results showed that the sensor was promising for the sensitive detection of lncRNA in clinical application.

It is important to design a nice electrochemiluminescence (ECL) biological nanomaterial for fabricating sensitive ECL immunosensor to detect tumor markers. Most reported ECL nanomaterial was decorated by a number of mono-luminophore. Here, we report a novel ECL nanomaterial assembled by dual luminophores perylenetetracarboxylic acid (PTCA) and carbon quantum dots (CQDs). In the ECL nanomaterial, graphene was chosen as nanocarrier. Significant ECL intensity increases are seen in the ECL nanomaterial, which was interpreted with the proposed synergistic promotion ECL meachanism of PTCA and CQDs. Furthermore, this ECL nanomaterial was used to label secondary antibody and fabricate a sandwiched carcinoembryonic antigen (CEA) immunosensor. The CEA immunosensor exhibits high sensitivity and the linear semilogarithmical range was from 0.001fgmL-1 to 1ngmL-1 with low detection limit 0.00026fgmL-1. And the CEA immunosensor is also suitable for various cancers' sample detection providing potential specific applications in diagnostics.

Detection of tumor biomarker plays a crucial role in the early diagnosis and treatment of cancer. Herein, hollow polymeric nanospheres with a high loading efficiency of ferrocene molecules (Fc-HPNs) were prepared by self-assembly route between polyethylenimine-Fc (PEI-Fc) and poly acrylic acid (PAA) on SiO2 nanoparticles. Using Fc-HPNs as an efficient electrochemical tag and combining dual-enzyme assisted target amplification strategy, an ultrasensitive electrochemical biosensor for the detection of circulating tumor DNA (ctDNA) was developed. The electrochemical signals were greatly enhanced due to the high loading of Fc tags on the hollow polymeric nanospheres and the catalytic effect of ascorbic acid on the reduction reaction of Fc. Furthermore, the use of the magnetic electrode avoided the tedious and complex modification process of the traditional electrode, which made the fabrication of the electrochemical biosensor facile and time-saving. The developed biosensor exhibited a fine linear response to ctDNA concentrations from 10 fM to 10 nM with a low detection limit of 1.6 fM, which shows great potential in early-stage diagnosis and treatment of cancer.

A simple and novel one-step triggered ratiometric electrochemical biosensor was designed for lead (Pb2+) based on the highly specific lead-dependent DNAzyme and dual-signal output mode. The biosensor consists of a thiolated methylene blue-labeled DNA (MB-P1) as catalytic probe and “signal-on” readout, and a complementary strand modified with ferrocene (Fc-P2) as substrate probe and “signal-off” output. The presence of Pb2+ could activate the DNAzyme and cleave the sessile phosphodiester of the Fc-P2 into two fragments, which lead to the departure of Fc from the sensing interface along with the single stranded MB-P1 near the electrode surface. Therefore, the Pb2+ recognition event resulted in both the “signal-on” of MB and the “signal-off” of Fc for dual-signal ratiometric electrochemical readout. Combined with the efficient recognition capacity of the designed DNAzyme and the dual-signal amplification strategy, the proposed biosensor showed a wide detection range from 0.1 nM to 5 μM with a detection limit of 45.8 pM (S/N = 3). Meanwhile, this DNAzyme-based Pb2+ biosensor exhibits reasonable selectivity, fast analytical speed, acceptable fabrication reproducibility, and operational convenience. More importantly, the system is capable of detecting Pb2+ in biological fluid such as serum, suggesting promising applications of this biosensor in on-site and real-time clinical Pb2+ detection.

In the present work a highly sensitive and selective aptasensor was developed for the determination of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) based on the hybridization chain reaction (HCR) signal amplification. It was observed that the aptamer of 8-OH-dG could hybridize with the capture DNA immobilized on the gold electrode with a sticky tail left, which initiated the HCR and led to the formation of extended dsDNA structure on the electrode surface. Then the electroactive species ([Ru(NH3)6]3+, RuHex) intercalated into the dsDNA grooves to generate the amplified signal. However, in the presence of 8-OH-dG, the aptamer containing G-rich nucleic acid sequences would be induced to form a G-quadruplex structure, which made it impossible to continue the HCR. So the detection signal will significantly decrease. Under the optimal conditions, the peak current of RuHex was linear with the logarithm of 8-OH-dG concentration in the range from 10pM to 100μM with the detection limit of 2.5pM. By integrating the merits of enzyme-free amplification power of the HCR and the inherent high sensitivity of the electrochemical technique, the prepared aptasensor not only showed high sensitivity for the detection of 8-OH-dG, but also exhibited good selectivity against to the uric acid, an important interferent in the urine sample. Particularly, the aptasensor was applied to detect 8-OH-dG in urine samples with satisfactory results.

As a new type of nano-catalytic material, copper nanoclusters (Cu NCs) have received more and more attention in various fields such as biosensors and catalysis. In this work, an electrochemical stripping chemiluminescent (ESCL) aptamer sensor based on Cu NCs was constructed for the detection of carcinoembryonic antigen (CEA). The Cu NCs were generated using DNA duplex (the CEA aptamer and its complementary strand) as template. During the ESCL detection process, Cu NCs catalyzed the reduction of H2O2 and were gradually electrochemically oxidized to Cu2+, which both promoted the decomposition of H2O2 and thus greatly improved the ECL of luminol. CEA could specifically bind with its aptamer, so the DNA duplex was damaged, causing a decrease of Cu NCs and lower ECL signals. Under the optimized conditions, the sensor achieved a high sensitivity detection of CEA with a linear range of 0.2 fg mL−1 to 1 ng mL−1 and a detection limit of 66.67 ag mL−1 (S/N = 3). In addition, the sensor exhibited good specificity, stability and reproducibility, and the detection of actual samples obtained satisfactory results, indicating that the provided strategy has potential application prospects in clinical detection.

Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of recombinant SmM and three NM2s (NM2a, -2b, and -2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC50 values: 6.47 μM for SmM, 3.58 μM for NM2a, 2.30 μM for NM2b, and 1.57 μM for NM2c. To identify the blebbistatin-resistant myosin-2 mutant, we performed mutagenesis analysis of the conserved residues in the blebbistatin-binding site of SmM and NM2s. We found that the A456F mutation renders SmM and NM2s resistant to blebbistatin without greatly altering their motor activities or phosphorylation-dependent regul...

A novel dual-signal ratiometric electrochemical aptasensor for highly sensitive and selective detection of thrombin has been designed on the basis of signal-on and signal-off strategy. Ferrocene labeled hairpin probe (Fc-HP), thrombin aptamer and methyl blue labeled bio-bar-coded AuNPs (MB-P3-AuNPs) were rationally introduced for the construction of the assay platform, which combined the advantages of the recognition of aptamer, the amplification of bio-bar-coded nanoprobe, and the ratiometric signaling readout. In the presence of thrombin, the interaction between thrombin and the aptamer leads to the departure of MB-P3-AuNPs from the sensing interface, and the conformation of the single stranded Fc-HP to a hairpin structure to take the Fc confined near the electrode surface. Such conformational changes resulted in the oxidation current of Fc increased and that of MB decreased. Therefore, the recognition event of the target can be dual-signal ratiometric electrochemical readout in both the “signal-off” of MB and the “signal-on” of Fc. The proposed strategy showed a wide linear detection range from 0.003 to 30 nM with a detection limit of 1.1 pM. Moreover, it exhibits good performance of excellent selectivity, good stability, and acceptable fabrication reproducibility. By changing the recognition probe, this protocol could be easily expanded into the detection of other targets, showing promising potential applications in disease diagnostics and bioanalysis.

The tough challenges for the sensitive and accurate detection of circulating miRNAs (cmiRNAs) in the peripheral blood are low abundance, and high interference from environmental or background factors or other molecules. Here, we developed a double-signal mode based on metal–organic framework coupled cascaded nucleic acid circuits for the accurate and sensitive detection of serum circulating miRNAs.

In the field of clinical diagnosis, it is important to construct a potential-resolved multiplex electrochemiluminescence (ECL) biosensor for decreasing the false-positive rate and improving the diagnostic accuracy. However, the shortage of low-potential cathodic luminophores between -1 and 0 V (vs Ag/AgCl) severely limited the development of the biosensor. Herein, we synthesized a novel luminophore N,N-bis-(3-dimethyl aminopropyl)-3,4,9,10-perylene tetracarboxylic acid diimide (PDI), which gave dual emissions at -0.25/-0.26 V with K2S2O8 as a co-reactant in aqueous solution. The ECL was assigned to excited J-type PDI dimers. Then, PDI and luminol were used as luminophores to respectively combine with graphite oxide and gold nanoparticles and form potential-resolved ECL nanoprobes. Also, this potential-resolved ECL nanoprobes were respectively functionalized by secondary antibodies (Ab2) to construct a low-potential sandwiched ECL immunosensor for tumor markers carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) simultaneous determination during linear scanning potential range from -0.6 to 0.6 V. The prepared multiplex immunosensor exhibited sensitive ECL response for CEA at -0.6 V due to PDI and that for AFP at 0.6 V due to luminol, and both linear semilogarithmical ranges were from 0.1 pg to 1 ng mL-1. In addition, PDI with dual ECL peaks showed enticing prospect of built-in self-calibration for a precise quantitative and bioimaging analysis.

8-Hydroxy-2′-deoxyguanosine (8-OH-dG) is a principal stable marker of DNA oxidative damage. Sensitive and specific detection of 8-OH-dG is of great importance for early disease diagnosis. In this paper, we developed an electrochemiluminescence aptasensor for 8-OH-dG detection based on target induced multi-DNA release and nicking enzyme signaling amplification strategy. First, three kinds of short DNAs were aligned on the aptamers immobilized on the magnetic beads. In the presence of 8-OH-dG, the aptamer recognized and specifically bound with 8-OH-dG, leading to the release of three kinds of short DNAs and three-fold signal amplification. Then the released short DNAs hybridized with ferrocence (Fc) labeled hairpin DNA (Fc-HP) immobilized on the gold electrode to form a double strand DNA. Subsequently, nicking endonuclease (Nt.AlwI) recognized the asymmetric sequence in the dsDNA and cleaved the substrate strand (Fc-HP) into two parts, one fragments containing Fc would leave the surface of electrode. Based on the quenching effect of Fc on the electrochemiluminescence (ECL) of Ru(bpy)32+/TPA, a signal-on ECL aptasensor was developed. At the same time, three kinds of short DNAs were released again and reused to initiate the repeated cycles of hybridization-cleavage. Under double signal amplification, this aptasensor achieved a low detection of 25 fM and a wide linear range from 100 fM to 10 nM for 8-OH-dG. Besides, the amount of 8-OH-dG in urine samples derived from different people were determined with satisfactory results.

The low-potential electrochemiluminescence (ECL) sensors based on cathodic light emission of luminol have caused more and more concerns due to their good stability and reproducibility. In this work, highly porous platinum (Pt) nanostructures on ionic liquid functionalized graphene film (GR-IL/pPt) were prepared as platform to construct a label-free ECL sensor for the detection of carcinoembryonic antigen (CEA). Due to their good biocompatibility, excellent electrocatalytic activity and highly porous structure, the as-prepared GR-IL/pPt composites benefited amplified cathodic ECL signal of luminol and high loading density of the CEA antibody. After CEA was incubated with the CEA antibody, the cathodic ECL signal of luminol decreased thanks to the less conductive immunocomplex. The proposed ECL immunosensor realized high sensitivity for CEA detection with a wide linear range from 0.001 fg mL −1 to 1 ng mL −1 and an extremely low detection limit of 0.0003 fg mL −1 (S/N = 3). Moreover, the sensor showed good specificity, stability and reproducibility, indicating that the provided strategy had a promising potential in clinical detection.

Recently, tris(2,2′-bipyridine)ruthenium( ii ) (Ru(bpy)32+) has been widely used in electrochemiluminescence (ECL) biosensors. However, its ECL has to be triggered at high positive or negative voltage, leading to a series of undesired side effects containing electrical damage toward biomolecules and hydrogen/oxygen evolution reaction. In this work, a new low-potential-triggered Ru(bpy)32+ ECL system was reported using oxalate as coreactant with the aid of platinum nanoparticles (Pt NPs) electrodeposited on the surface of the graphene-ionic liquid composite. When cyclic scanning in a potential range from 0.3 to −0.2 V, the ECL signal appeared at onset potential of 0.05 V (vs. Ag/AgCl), and reached the maximum at about −0.15 V (vs. Ag/AgCl), which were quite lower than that in the typical Ru(bpy)32+-oxalate (C2O42−) ECL system. The possible mechanism was discussed in detail. Meanwhile, the low-potential-triggered ECL signals were strong and stable, which were further exploited to detect the carcinoembryonic antigen (CEA). The constructed ECL sensor exhibited a good linearity between the ECL signals and the logarithm of the concentrations of CEA in a range from 0.1 pg mL−1 to 100 ng mL−1 with the detection limit of 0.01 pg mL−1. In addition, the sensor showed a promising potential in clinical detection.

In this study, a novel sandwiched electrochemiluminescence (ECL) immunosensor for the detection of carcinoembryonic antigen (CEA) was developed. The nanocomposite of polydopamine and Ag nanoparticles (PDA-AgNPs) was prepared by the redox reaction between Ag+ and dopamine. This nanocomposite not only provided an effective matrix for the immobilization of primary antibody (Ab1) but also enhanced the conductivity of the electrode. Carbon quantum dots (CQDs) were immobilized on the poly(ethylenimine) functionalized graphene oxide (PEI-GO) through amido-bond. Then Au nanoparticles were decorated on the CQDs modified PEI-GO matrix, and the resulted complex AuNPs/CQDs-PEI-GO was introduced to link secondary antibody (Ab2). The CQDs can be connected to the electrode surface through the combination of CEA with Ab1 and Ab2, and then the amplified electrochemiluminescence signal of CQDs was obtained with the synergistic effect of AgNPs, polydopamine, AuNPs and PEI-GO. Under the optimal conditions, the ECL intensity was proportional to the logarithm value of CEA concentration in the linear range from 5pgmL-1 to 500ngmL-1 with a detection limit of 1.67pgmL-1 for CEA detection. The immunosensor was applied for the CEA detection in real samples with satisfactory results. The proposed ECL immunosensor showed good performance with high sensitivity, specificity, reproducibility, stability and will be potential in clinical detection.

A simple colorimetric sensing method for the detection of Hg2+ was developed by combining enzymatic catalysis with DNA-based hybridization chain reactions (HCRs). Firstly, thymine (T)-rich capture DNA (cDNA) was immobilized on a gold electrode via Au–S bonding. In the presence of Hg2+, thymine (T)-rich probe DNA (pDNA) hybridized with cDNA via T–Hg2+–T base pairs. Then the HCRs were realized using pDNA as an initiator and two biotin-labeled hairpin DNAs (H1 and H2) as fuel strands. Finally, numerous avidin-labeled horseradish peroxidase (HRP) enzymes were immobilized on long nicked ds-DNA strands, which can catalyze the H2O2-mediated oxidation of 3,3,5,5-tetramethylbenzidine dihydrochloride hydrate (TMB) to cause a dramatic color change. Under optimal conditions, the absorbance of TMB was linear with the logarithm of Hg2+ concentrations in the range of 1 fM to 1 pM, with a detection limit of 0.33 fM. This strategy exhibited good selectivity and high sensitivity, which might be a potential tool for the practical detection of Hg2+ in environmental monitoring.

In the present work, PDDA-functionalized graphene sheets (PDDA-GS) were prepared by reduction with hydrazine hydrate in situ in the presence of poly(diallyldimethylammonium chloride) (PDDA). The (PDDA-GS/PEDOT:PSS)n multilayer films were fabricated by a layer-by-layer self-assembly technique based on the electrostatic interaction between positively charged PDDA-GS and negatively charged poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS). The self-assembly process was characterized by ultraviolet-visible spectroscopy (UV-vis). Pt nanoparticles were electrodeposited in situ on the multilayer films. X-ray diffraction (XRD) analysis and field emission scanning electron microscopy (FE-SEM) were used to characterize the properties of Pt/[(PDDA-GS/PEDOT:PSS)n] multilayer films. Cyclic voltammetry and chronoamperometry were used to study the electrocatalytic activity of Pt/[(PDDA-GS/PEDOT:PSS)n] multilayer films towards methanol oxidation. The results indicated that Pt/[(PDDA-GS/PEDOT:PSS)n] showed high electrocatalytic activity toward methanol oxidation and good tolerance toward carbon monoxide (CO) poisoning. This novel support for Pt nanoparticle catalysts will be promising in the fabrication of fuel cell electrodes and other catalytic devices.

Carbon quantum dots (CQDs) decorated Ag nanoparticles (Ag@CQDs) were successfully synthesized and characterized by UV-Vis spectroscopy, fluorescence, FT-IR, and transmission electron microscopy (TEM), then utilized for the sensitive detection of chloride ions (Cl−). Compared to CQDs, the Ag@CQDs showed stronger electrochemiluminescence (ECL) in 0.1 M PBS (pH = 7.4) containing 0.1 M K2S2O8. In the presence of chloride (Cl−), a significant reduction in the ECL intensity of Ag@CQDs was noticed, which was used to develop a sensitive detection method of Cl−. The ECL quenching of Ag@CQDs was utilized to establish the method, of which the mechanism might be caused by the formation of AgCl on the Ag@CQDs composite surface. The prepared Cl− sensor showed a wide linear range (0.99–450 nM) and low detection limit (0.33 nM). Furthermore, the prepared sensor exhibited high sensitivity and good selectivity towards several ion inferences.

Ratiometric electrochemiluminescence (ECL) assays have attracted widespread attentions in biosensing owing to their precise measurements by eliminating the environmental interferences. However, they mostly needed two eligible luminophors, increasing the complexity of the systems and limiting their practical applications. Herein, using luminol as single luminophor, a dual-potential ratiometric ECL strategy was proposed to detect carcinoembryonic antigen (CEA). The luminol exhibited cathodic and anodic emissions on graphene-ionic liquid-platinum (GR-IL-Pt) composites and Ti3C2 MXenes-Au NPs hybrids, respectively. Then, a sandwich ECL sensor was fabricated using GR-IL-Pt composites as matrix to immobilize the primary antibodies of CEA and Ti3C2 MXenes-Au NPs hybrids as platform to load the secondary antibodies. With the presence of CEA, the ratio of anodic ECL to cathodic ECL (ECLanodic/ECLcathodic) increased obviously, realizing sensitive ratiometric detection of CEA. In addition, the ECLanodic/ECLcathodic was independent with the concentrations of H2O2, greatly improving the test reliability. The developed ECL sensor exhibited a sensitive detection toward CEA, performing a wide linearity in the range of 0.1 pg mL−1 - 10 ng mL−1 with a low detection limit of 34.58 fg mL-1 (S/N = 3). Furthermore, this strategy exhibited a good practicality to detect CEA in human serums, providing a promising strategy in ECL bioanalysis.

A versatile label-free electrochemical biosensor based on dual enzyme assisted multiple amplification strategy was developed for ultrasensitive detection of circulating tumor DNA (ctDNA). The biosensor consists of a triple-helix molecular switch (THMS) as molecular recognition and signal transduction probe, ribonuclease HII (RNase HII) and terminal deoxynucleotidyl transferase (TdT) as dual enzyme assisted multiple amplification accelerator. The presence of target ctDNA could open THMS and trigger RNase HII-assisted homogenous target recycling amplification to produce substantial signal transduction probe (STP). The released STP hybridized with the capture probe immobilized on a gold electrode, then TdT and assistant probe were further employed to fulfill TdT-mediated cascade extension and generate stable DNA dendritic nanostructures. The electroactive methyl blue (MB) was finally used as the signal reporter to realize the multiple electrochemical amplification ctDNA detection as the amount of MB is positively correlated with the target ctDNA. Combined with the efficient recognition capacity of the designed THMS and the excellent multiple amplification ability of RNase HII and TdT, the constructed sensing platform could detect KRAS G12DM with a wide detection range from 0.01 fM to 1 pM, and the limit of detection as low as 2.4 aM. Besides, the platform is capable of detecting ctDNA in biological fluid such as plasma. More importantly, by substituting the loop of THMS with different sequences, this strategy could be conveniently expanded into the detection of other ctDNA, showing promising potential applications in clinical cancer screening and prognosis.

A label-free electrochemical aptasensor for the determination of 8-hydroxy-2′-deoxyguanosine (8-OH-dG) was described here. This method was based on the conformational change of 8-OH-dG-aptamer (Apt) in the presence of 8-OH-dG. The G-rich Apt was self-assembled on the surface of gold electrode by Au-S bond. The assembly process of the aptasensor was characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The conformation of Apt changed from flexible single stranded structures to compact G-quadruplex in the presence of 8-OH-dG, which hindered the negatively charged [Fe(CN) 6 ] 4 − approach to the electrode. The switchable surface provided a sensing platform for the free-label detection of 8-OH-dG. Upon 8-OH-dG binding to the immobilized Apt, a significant decrease in the oxidation current of [Fe(CN) 6 ] 4 − was observed due to the Apt conformational change. The aptasensor showed a detection limit of 0.315 nM and a wide linear range between 1.75 nM and 3.675 μM for 8-OH-dG. The aptasensor displayed high stability as well as high specificity against uric acid, a strong interferent for the detection of 8-OH-dG.

Aim: The goal of this study is to identify differentially methylated (DM) loci associated with long noncoding RNA (lncRNA)/mRNA expression in non-small-cell lung cancer (NSCLC). Materials & methods: Microarrays were used to interrogate genome-wide methylation and expression of lncRNA/mRNA in NSCLC. Results: We identified 113,644 DM loci between tumors and adjacent tissues. Among them, 26,310 DM loci were associated with 1685 differentially expressed genes, and 839 genes had significant correlations between methylation and expression, of which 26 hypermethylated loci in transcription start site 200 were correlated with low gene expression. We validated the correlations between methylation and expression in five genes (CDO1, C2orf40, SCARF1, ZFP106 and IFFO1) using pyrosequencing and quantitative polymerase chain reaction. We also found significant correlations between lncRNAs and mRNAs, and validated four of the correlations with quantitative polymerase chain reaction. Conclusion: Integrated analysis of genome-wide DNA methylation and lncRNA/mRNA expression allows us to identify new DM loci-correlated with gene expression in NSCLC.

In the present work we demonstrated an ultrasensitive detection platform for specific DNA based on nonlinear hybridization chain reaction (HCR) by triggering chain-branching growth of DNA dendrimers. HCR was initiated by target DNA (tDNA) and finally formed dendritic structure by self-assembly. The electrochemical signal was drastically enhanced by capturing multiple catalytic peroxidase with high-ordered growth. Electrochemical signals were obtained by measuring the reduction current of oxidized 3, 3', 5, 5'-tetramethylbenzidine sulfate (TMB), which was generated by HRP in the presence of H2O2. This method exhibited ultrahigh sensitivity to tDNA with detection limit of 0.4 fM. Furthermore, the biosensor was also capable of discriminating single-nucleotide difference among concomitant DNA sequences.

The motor function of smooth muscle myosin (SmM) is regulated by phosphorylation of the regulatory light chain (RLC) bound to the neck region of the SmM heavy chain. It is generally accepted that unphosphorylated RLC induces interactions between the two heads and between the head and the tail, thus inhibiting the motor activity of SmM, whereas phosphorylation of RLC interrupts those interactions, thus reversing the inhibition and restoring the motor activity to the maximal value. One assumption of this model is that single-headed SmM is fully active regardless of phosphorylation. To re-evaluate this model, we produced a number of SmM constructs with coiled coils of various lengths and examined their structure and regulation. With these constructs we identified the segment in the coiled-coil key for the formation of a stable double-headed structure. In agreement with the current model, we found that the actin-activated ATPase activity of unphosphorylated SmM increased with shortening of the coiled-coil. However, contrary to the current model, we found that the actin-activated ATPase activity of phosphorylated SmM decreased with shortening coiled-coil and only the stable double-headed SmM was fully activated by phosphorylation. These results indicate that single-headed SmM is neither fully active nor fully inhibited. Based on our findings, we propose that cooperation between the two heads is essential, not only for the inhibition of unphosphorylated SmM, but also for the activation of phosphorylated SmM.

It is generally accepted that dephosphorylation of regulatory light chain (RLC) induces the interactions between the two heads of smooth muscle myosin (Sm) and between the head and the tail, thus inhibiting the motor activity, and phosphorylation of RLC interrupts above interactions, thus reversing the inhibition and stimulating the motor activity to maximal value. Thus it is predicted that Sm subfragment-1 (S1) containing only one head without the tail is fully active. However, no solid evident so far supports this prediction, although early studies showed that S1 produced by limited protease treatment is partially active. Here we produced a number of Sm truncations with various length of the tail. The stability of double-headed structure of Sm is dependent on the length of the coiled-coil tail. The Sm truncations with coiled-coil longer than 214 amino acids form stable double-headed structure (stable HMM), those with coiled-coil shorter than 179 amino acids form unstable double headed structure (unstable HMM), and that without coiled-coil, i.e. S1, is completely single-headed. Phosphorylation of RLC regulates the motor activity of stable HMM completely, regulates that of unstable HMM partially, and does not regulate that of S1. Unexpectedly, the actin-activated ATPase activity of S1, either unphosphorylated or phosphorylated, is higher than that of unphosphorylated stable HMM but less than 10% of that of phosphorylated stable HMM. The actin-activated ATPase activities of unphosphorylated Sm truncations increase with the shortening of the coiled-coil tail, and that of phosphorylated Sm truncations decrease with the shortening of the coiled-coil tail. These results indicate that the stable double-headed structure is critical not only for the inhibited state of unphosphorylated Sm, but also for the fully activated state of phosphorylated Sm.