Carbon Nanomaterials are excellent electrode materials due to their extraordinary conductivity, prolific structures, and morphologies. Herein, a novel nanocarbon-based material (Au@NCNC) was synthesized by embedding gold nanoparticles (AuNPs) inside the pores of three-dimensional hierarchical nitrogen-doped carbon nanocages (NCNC) through an in situ chemical deposition method. The resultant Au@NCNC was employed as an electrochemical catalyst for the oxygen reduction reaction (ORR) and as an electrode material for supercapacitors. The conductivity and hydrophilicity of Au@NCNC were much more improved than those of pristine NCNC. Meanwhile, the bubble adhesive force on the Au@NCNC film was much lower underwater than that of NCNC, which provided easy accessibility to the active sites of reactants, such as hydrated O2. Therefore, the deposition of AuNPs inside pores of NCNC facilitated the transfer of electrons and diffusion of ions, promoting the electrocatalytic performance of Au@NCNC. As a result, Au@NCNC exhibited high performance toward ORR, which manifested in high numbers of electron transfer (3.7–3.9), high kinetic current density, enhanced electrocatalytic stability, and remarkable methanol durability. Moreover, Au@NCNC displayed high specific capacitance, good rate capability, and cycling stability with ∼97% of its initial capacitance retained at the high current density of 10 A g–1 after 5000 cycles. This could be attributed to the synergetic effect of ultrafine gold nanoparticles, the hierarchical porous structure, and the hydrophilic surface of NCNC as well. This work offers an excellent alternative for Pt-based catalysts in fuel cells, ORR, and supercapacitive electrode materials by enhancing the conductivity and surface hydrophilicity of electrocatalysts.Keywords: 3D nitrogen-doped carbon nanocages; capacitances; electrocatalytic oxygen reduction reaction; gold nanoparticles; hydrophilic;

The label-free localized surface plasmon resonance (LSPR) detection technique has been identified as a powerful means for in situ investigation of biological processes and localized chemical reactions at single particle level with high spatial and temporal resolution. Herein, a core–satellites assembled nanostructure of Au50@Au13 was designed for in situ detection and intracellular imaging of telomerase activity by combining plasmonic resonance Rayleigh scattering spectroscopy with dark-field microscope (DFM). The Au50@Au13 was fabricated by using 50 nm gold nanoparticles (Au50) as core and 13 nm gold nanoparticles (Au13) as satellites, both of them were functionalized with single chain DNA and gathered proximity through the highly specific DNA hybridization with a nicked hairpin DNA (O1) containing a telomerase substrate (TS) primer as linker. In the presence of telomerase, the telomeric repeated sequence of (TTAGGG)n extended at the 3′-end of O1 would hybridized with its complementary sequences at 5′-ends. This led the telomerase extension product of O1 be folded to form a rigid hairpin structure. As a result, the Au50@Au13 was disassembled with the releasing of O1 and Au13-S from Au50-L, which dramatically decreased the plasmon coupling effect. The remarkable LSPR spectral shift was observed accompanied by a detectable color change from orange to green with the increase of telomerase activity at single particle level with a detection limit of 1.3 × 10–13 IU. The ability of Au50@Au13 for in situ imaging intracellular telomerase activity, distinguishing cancer cells from normal cells, in situ monitoring the variation of cellular telomerase activity after treated with drugs were also demonstrated.

The low solubility of gases in aqueous solution is the major kinetic limitation of reactions that involve gases. To address this challenge, we report a nanochannel reactor with joint gas–solid–liquid interfaces and controlled wettability. As a proof of concept, a porous anodic alumina (PAA) nanochannel membrane with different wettability is used for glucose oxidase (GOx) immobilization, which contacts with glucose aqueous solution on one side, while the other side gets in touch with the gas phase directly. Interestingly, it is observed that the O2 could participate in the enzymatic reaction directly from gas phase through the proposed nanochannels, and a hydrophobic interface is more favorable for the enzymatic reaction due to the rearrangement of GOx structure as well as the high gas adhesion. As a result, the catalytic efficiency of GOx in the proposed interface is increased up to 80-fold compared with that of the free state in traditional aqueous air-saturated electrolyte. This triphase interface with controlled wettability can be generally applied to immobilize enzymes or catalysts with gas substrates for high efficiency.

An artificial metabolon with high conversion efficiency was constructed by confining a bi-enzyme into porous aluminum oxide nanochannels, which accelerated enzymatic reactions by minimizing the diffusion loss of intermediate species.

A three-dimensional hemin-functionalized graphene hydrogel (Hem/GH) was prepared by a facile self-assembly approach. The as-prepared Hem/GH showed good mechanical strength with a storage modulus of 609–642 kPa and a high adsorption capacity to organic dye contaminants (341 mg g–1 for rhodamine B). Moreover, Hem/GH could be used as a photosensitizer for the photocatalytic degradation of organic dyes and displayed superior photodegradation activity of methylene blue (MB). This result was better than that of counterparts such as graphene hydrogel (GH) and commercial catalyst P25. The excellent cycling performance of the Hem/GH was well maintained even after multiple cycles on adsorption process and photocatalytic reaction. Interestingly, after the photodegradation of MB, a light-induced pH change of the solution from alkaline pH 8.99 to acidic pH 3.82 was observed, and 10 wt % total organic carbon remained. The liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) analysis confirmed the generation of acidic degradation products. The photocatalytic mechanism was further investigated by trapping experiments, which revealed that the MB degradation was driven mainly by the participation of O2•– radicals in the photocatalytic reaction. As an extended application, visually intuitive observation showed the as-prepared Hem/GH also had strong antibacterial properties. These results suggest that Hem/GH could be potentially used for practical application due to its high adsorption ability, excellent photocatalytic activity, and strong antibacterial properties.Keywords: adsorption ability; antibacterial properties; cycling performance; hydrogel; photocatalytic activity; self-assembly;

A light-driven approach combined with a macroporous reactor for the enzymatic biocatalytic reaction has been developed by confining the enzyme/photosensitizer nanohybrids in a macroporous material, which exhibits high bio-conversion efficiency due to the fast diffusion and collision between the enzyme/photosensitizer nanohybrid and the substrate in the reactor.

•Multifunctional nanoprobe exhibits good dispersion, low cytotoxicity and excellent biocompatibility.•The nanoprobe targets cancer cells, providing for simultaneous fluorescence and magnetic resonance imaging.•The nanoprobe is used for real-time imaging in early liver cancer diagnosis.Multifunctional nanoprobes with distinctive magnetic and fluorescent properties are highly useful in accurate and early cancer diagnosis. In this study, nanoparticles of Fe3O4 core with fluorescent SiO2 shell (MFS) are synthesized by a facile improved Stöber method. These nanoparticles owning a significant core-shell structure exhibit good dispersion, stable fluorescence, low cytotoxicity and excellent biocompatibility. TLS11a aptamer (Apt1), a specific membrane protein for human liver cancer cells which could be internalized into cells, is conjugated to the MFS nanoparticles through the formation of amide bond working as a target-specific moiety. The attached TLS11a aptamers on nanoparticles are very stable and can't be hydrolyzed by DNA hydrolytic enzyme in vivo. Both fluorescence and magnetic resonance imaging show significant uptake of aptamer conjugated nanoprobe by HepG2 cells compared to 4T1, SGC-7901 and MCF-7 cells. In addition, with the increasing concentration of the nanoprobe, T2-weighted MRI images of the as-treated HepG2 cells are significantly negatively enhanced, indicating that a high magnetic field gradient is generated by MFS-Apt1 which has been specifically captured by HepG2 cells. The relaxivity of nanoprobe is calculated to be 11.5 mg−1s−1. The MR imaging of tumor-bearing nude mouse is also confirmed. The proposed multifunctional nanoprobe with the size of sub-100 nm has the potential to provide real-time imaging in early liver cancer cell diagnosis.

•CysC-specific nanobody to CysC is isolated from phage display nanobody library.•A photoelectrochemical immunosensor for CysC develops by Nb modified TNA.•An excellent sensitivity and good selectivity of CysC sensing was obtained.Cystatin C (CysC) is a sensitive marker for the estimation of the glomerular filtration rate and the clinical diagnosis of different diseases. In this paper, CysC-specific nanobodies (Nbs) were isolated from a phage display nanobody library. A simple and sensitive photoelectrochemical immunosensor based on TiO2 nanotube arrays (TNAs) was proposed for the sensitive detection of CysC. The TiO2 nanotube arrays deposited by electrochemical anodization displayed a high and stable photocurrent response under irradiation. After coupling CysC-specific nanobody to TNA (Nb/TNA), the proposed immunosensor for CysC can be utilized for tracking the photocurrent change of Nb/TNA caused by immunoreactions between CysC and the immobilized CysC-specific Nb. This allowed for the determination of CysC with a calibration range from 0.72 pM to 7.19 nM. The variation of the photocurrent was in a linear relationship with the logarithm of the CysC concentration in the range of 0.72 pM–3.6 nM. The immunosensor had a correlation coefficient of 0.97 and a detection limit of 0.14 pM at a signal-to-noise ratio of 3. The proposed immunosensor showed satisfactory intra- and inter-assay accuracy, high selectivity and good stability. As a result, this proposed strategy would offer a novel and simple approach for the detection of immunoreactions, provide new insights in popularizing the diagnosis of CysC, and extend the application of TiO2 nanotubes.

•An electrochemical strategy for sensing DNA MTase activity was proposed.•Signal amplification was achieved by mimic-hybridization chain reaction.•High specificity was obtained based on synergistic effect of Hap II and EXO III.•The strategy has great potential be applied in methylation-based disease diagnosis.A mimic-hybridization chain reaction (mimic-HCR) amplified strategy was proposed for sensitive electrochemically detection of DNA methylation and methyltransferase (MTase) activity In the presence of methylated DNA, DNA-gold nanoparticles (DNA-AuNPs) were captured on the electrode by sandwich-type assembly. It then triggered mimic-HCR of two hairpin probes to produce many long double-helix chains for numerous hexaammineruthenium (III) chloride ([Ru(NH3)6]3+, RuHex) inserting. As a result, the signal for electrochemically detection of DNA MTase activity could be amplified. If DNA was non-methylated, however, the sandwich-type assembly would not form because the short double-stranded DNAs (dsDNA) on the Au electrode could be cleaved and digested by restriction endonuclease HpaII (HapII) and exonuclease III (Exo III), resulting in the signal decrement. Based on this, an electrochemical approach for detection of M.SssI MTase activity with high sensitivity was developed. The linear range for M.SssI MTase activity was from 0.05 U mL−1 to 10 U mL−1, with a detection limit down to 0.03 U mL−1. Moreover, this detecting strategy held great promise as an easy-to-use and highly sensitive method for other MTase activity and inhibition detection by exchanging the corresponding DNA sequence.

•The CYP2D6/QDs hybrid served as a photocatalyst for light-driven drug metabolism.•MOSF was used for confining the CYP2D6/QDs nanohybrid.•A much stable and large photocurrent was achieved by confining CYP2D6 into MOSF.•The MOSF-based photoelectrochemical system showed high enzymatic reactivity.In this work, a simple and accurate in vitro drug metabolism strategy was proposed by using macroporous ordered siliceous foam (MOSF) as a nanoreactor for confining the nanohybrid of CdTe quantum dots (QDs) and cytochrome P450 2D6 (CYP2D6). After CYP2D6 was coupled to QDs through the carbodiimide coupling chemistry, the resulting nanohybrid of CYP2D6/QDs exhibited fluorescence emission at 645 nm, which could be used as a photocatalyst for drug metabolism. The energy level of CYP2D6 was located between the conduction band (CB) and valence band (VB) of QDs, which provided the possibility of the electron transferring from the CB of QDs to CYP2D6. Tramadol was chosen as the model substrate. Under the irradiation of a white-light, the photocurrent increased with the addition of tramadol in a wide linear range from 4.0 μM to 100 μM. The apparent Michaelis–Menten constant was measured to be 3.6 μM. The high performance liquid chromatography coupled with mass spectrometry (HPLC–MS) confirmed the production of metabolite of o-desmethyltramadol. Such nanoreactor provided a suitable environment to confine a mass of enzyme, as well as to maintain their catalytic activities for highly effective drug metabolic reactions. This showed promising potential for immobilizing various enzymes and other biomolecules in biomimetic metabolism study.

•Gold nanoparticle coated chitosan/reduced graphene oxide nanocomposites for cytochrome P450s immobilization.•The immobilized CYP2D6 and CYP1A1 displayed direct electrochemistry and electrochemically-driven drug metabolism.•The bioconversion of tramadol or benzo[a]pyrene were achievied by an electrochemistry-driven way.•The metabolic inbibition of the quinidine and alpha-naphthoflavone to the enzymatic activity of CYP2D6 and CYP1A1 were evaluated.In the present work, gold nanoparticles coated chitosan/reduced graphene oxide (Au-CS-RG) was prepared for cytochrome P450 2D6 (CYP2D6) and cytochrome P450 1A1 (CYP1A1) immobilization, investigation of the direct electrochemistry and electrochemically-driven drug metabolism. The immobilized CYP2D6 and CYP1A1 displayed respectively a pair of redox peaks with a formal potential of − 492 ± 4 and − 504 ± 6 mV. The response showed a surface-controlled electrode process with an average electron transfer rate constant of 5.19 ± 0.3 s− 1 for CYP2D6 and 3.24 ± 0.4 s− 1 for CYP1A1 determined in the scan rate of 100 mV/s. When the Au-CS-RG was treated with polyacrylic acid (PAA), the resulting nanocomposites (PAA-Au-CS-RG) changed the surface charge of Au-CS-RG from positive to negative and increased the size of the coated gold nanoparticles from ~ 15 to ~ 25 nm. This led to negative-shift of the formal potential to − 513 ± 6 mV for CYP2D6 and − 509 ± 5 mV for CYP1A1, while the electron transfer rate constant decreased to 4.10 ± 0.3 s− 1 for CYP2D6 and 2.78 ± 0.2 s− 1 for CYP1A1, respectively. The immobilized CYP2D6 and CYP1A1 in both cases showed excellent electrochemically-driven drug metabolism. The LC-MS analysis demonstrated the bioconversion from tramadol or benzo[a]pyrene to o-demethyl-tramadol or 7.8-diol benzo[a]pyrene by the electrochemically-driven way, respectively. The metabolic inhibition of the quinidine and alpha-naphthoflavone to the enzymatic activity of CYP2D6 and CYP1A1 were also evaluated.

•RCA was used for chronocoulometric detection of DNA MTase activity.•DNA probe density on the electrode surface were detected.•The detection limit of 0.09 U/mL is achieved.•The method was applied in complex matrix such as human serum samples.In this paper, a rolling chain amplification (RCA) strategy was proposed for chronocoulometric detection of DNA methyltransferase (MTase) activity. Briefly, after the double DNA helix structure was assembled on the surface of gold electrode, it was first methylated by M. SssI MTase and then RCA was realized in the presence of E. coli and phi29 DNA polymerase. Successively, numerous hexaammineruthenium (III) chloride ([Ru(NH3)6)3+, RuHex) were adsorbed on replicons by electrostatic interaction and generated a large electrochemical readout, the signal was “on”. On the contrary, in the absence of M. SssI MTase, the methylated CpG site in the unmethylated double DNA helix structure could be specifically recognized and cleaved by HpaII, resulting in a disconnection of RCA from the electrode. This led seldom RuHex to be absorbed onto the surface of electrode, the signal was “off”. Based on the proposed strategy, the activity of M. SssI MTase was assayed in the range of 0.5–60 U/mL with a detection limit of 0.09 U/mL (S/N=3). In addition, the inhibition of procaine and epicatechin on M. SssI MTase activity was evaluated. When the proposed method was applied in complex matrix such as human serum samples, acceptable accuracy, precision and high sensitivity were achieved. Therefore, the proposed method was a potential useful mean for clinical diagnosis and drug development.

•A multianalyte chemiluminescence imaging immunoassay strategy is developed.•Different isoforms of prostate specific antigen in serum can be sensitively detected.•The strategy possessed high throughput and acceptable reproducibility and accuracy.•Soybean peroxidase was used to label f-PSA or t-PSA monoclonal antibody.•SPTZ and MORP were used to significantly enhance the CL intensity.A multianalyte chemiluminescence (CL) imaging immunoassay strategy for sensitive detection of different isoforms of prostate specific antigen (PSA) was developed. The microtiter plates were fabricated by simultaneously immobilizing of free-PSA (f-PSA) and total-PSA (t-PSA) capture antibody on nitrocellulose (NC) membrane. Each of the array were spotted in replicates of six spots within a spacing of 2 mm. 16 or 48 detection wells were integrated on a single NC membrane and each well could be used as a microreactor and microanalysis chamber. Under a sandwiched immunoassay, the CL signals on each sensing site were collected by a charge-coupled device (CCD), presenting an array-based chemiluminescence imaging. Soybean peroxidase (SBP) was used to label f-PSA or t-PSA monoclonal antibody. With the amplification effects of two enhancers, 3-(10′-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP), the CL intensity could significantly enhanced, which improved the sensing sensitivity and detection limit. Under the optimal conditions, the linear response to the analyte concentration ranged from 0.01–36.7 ng/mL and 0.02–125 ng/mL for f-PSA and t-PSA, respectively. The results for the detection of forty serum samples from prostate cancer patients and cancer-free patients showed good agreement with the clinical data, suggesting that the proposed assay had acceptable accuracy. The proposed CL imaging immunoassay possess high throughput and acceptable reproducibility, stability and accuracy, which made it great potential to available to distinguish different isoforms of PSA in serum samples.

A novel nanocomposite of zinc porphyrin functionalized graphene quantum dots (GQDs/ZnPor) was prepared and used as a photocatalyst for the degradation of an organic pollutant under visible-light irradiation. In order to synthesise the nanocomposites, large graphene sheets were first cleaved into small pieces of graphene oxide by a mixture of concentrated H2SO4 and HNO3. Then, the GQDs/ZnPor was synthesized by a simple hydrothermal route with ZnPor and the as-synthesized graphene oxide as precursors. The resultant GQDs/ZnPor nanocomposites were characterized by transmission electron microscopy (TEM) and optical measurements. The photocatalytic activity of GQDs/ZnPor was evaluated by the degradation of methylene blue (MB) under visible-light irradiation. Enhancement of the photocatalytic activity of GQDs/ZnPor compared with pure zinc porphyrin was achieved. The photocatalytic degradation mechanism of GQDs/ZnPor was also illustrated by photoluminescence measurements and free radical and hole scavenging experiments. The proposed GQDs/ZnPor has potential application for decomposing organic pollutants in water.

We report a general method for the fabrication of three-dimensional (3D) macroporous graphene/conducting polymer modified electrode and nitrogen-doped graphene modified electrode. This method involves three consecutive steps. First, the 3D macroporous graphene (3D MG) electrode was fabricated electrochemically by reducing graphene oxide dispersion on different conducting substrates and used hydrogen bubbles as the dynamic template. The morphology and pore size of 3D MG could be governed by the use of surfactants and the dynamics of bubble generation and departure. Second, 3D macroporous graphene/polypyrrole (MGPPy) composites were constructed via directly electropolymerizing pyrrole monomer onto the networks of 3D MG. Due to the benefit of the good conductivity of 3D MG and pseudocapacitance of PPy, the composites manifest outstanding area specific capacitance of 196 mF cm–2 at a current density of 1 mA cm–2. The symmetric supercapacitor device assembled by the composite materials had a good capacity property. Finally, the nitrogen-doped MGPPy (N-MGPPy or MGPPy-X) with 3D macroporous nanostructure and well-regulated nitrogen doping was prepared via thermal treatment of the composites. The resultant N-MGPPy electrode was explored as a good electrocatalyst for the oxygen reduction reaction (ORR) with the current density value of 5.56 mA cm–2 (−0.132 V vs Ag/AgCl). Moreover, the fuel tolerance and durability under the electrochemical environment of the N-MGPPy catalyst were found to be superior to the Pt/C catalyst.Keywords: elcctrochemical reduced graphene oxide; graphene modified electrode; graphene/polypyrrole composites; hydrogen bubble template; nitrogen-doped; oxygen reduction reaction; supercapacitors

Surface patterns of well-defined nanostructures play important roles in fabrication of optoelectronic devices and applications in catalysis and biology. In this paper, the diporphyrin honeycomb film, composed of titanium dioxide, protoporphyrin IX, and hemin (TiO2/PPIX/Hem), was synthesized using a dewetting technique with the well-defined polystyrene (PS) monolayer as a template. The TiO2/PPIX/Hem honeycomb film exhibited a higher photoelectrochemical response than that of TiO2 or TiO2/PPIX, which implied a high photoelectric conversion efficiency and a synergistic effect between the two kinds of porphyrins. The TiO2/PPIX/Hem honeycomb film was also a good photosensitizer due to its ability to generate singlet oxygen (1O2) under irradiation by visible light. This led to the use of diporphyrin TiO2/PPIX/Hem honeycomb film for the photocatalytic inactivation of bacteria. In addition, the photocatalytic activities of other metal-diporphyrin-based honeycomb films, such as TiO2/MnPPIX/Hem, TiO2/CoPPIX/Hem, TiO2/NiPPIX/Hem, TiO2/CuPPIX/Hem, and TiO2/ZnPPIX/Hem, were investigated. The result demonstrated that the photoelectric properties of diporphyrin-based film could be effectively enhanced by further coupling of porphyrin with metal ions. Such enhanced performance of diporphyrin compounds opened a new way for potential applications in various photoelectrochemical devices and medical fields.Keywords: antibacterial activity; diporphyrin; photoelectrochemistry; singlet oxygen; synergistic effect; template synthesis;

Acute renal failure (ARF) represents a very important and potentially devastating disorder in clinical nephrology. Neutrophil gelatinase-associated lipocalin (NGAL) is an early biomarker for ARF in a wide range of different disease processes, which is frequently detected in clinical diagnosis. Herein, we present a label-free and sensitive photoelectrochemical (PEC) immunosensor for NGAL by utilizing a biotinylated anti-NGAL Nanobody (Nb) orientedly immobilized to streptavidin-coated cobalt 2,9,16,23-tetraaminophthalocyanine (CoPc)-sensitized TiO2 electrode. The Nb was biotinylated at the C-terminus, which is situated at the opposite site of the antigen binding region. Using highly oriented Nb as receptor molecules, a label-free PEC immunosensor for NGAL was developed by monitoring the changes in the photocurrent signals of the electrode resulting from immunoreaction. Immobilization of Nb to streptavidin-coated CoPc-sensitized TiO2 electrode surface provides high binding capacity to NGAL; thus, it can lead to a high sensitivity. The limit of detection (LOD) of the proposed immunosensor has been significantly lowered to 0.6 pg mL–1. This proposed immunosensor reveals high specificity to detect NGAL, with acceptable intra-assay precision and excellent stability. In addition, the present work provides a new approach to design Nb-based PEC immunosensor and increases versatility of Nbs.

Inspired by the high enzymatic reaction efficiency of the natural multi-enzyme complexes, construction of artificial multi-enzyme complexes for mimicking of the natural metabolic pathways in vitro has attracted significant interest from enzyme engineers. Herein, we have successfully assembled a cytochrome P450 (CYP) bienzyme complex on the Au nanoparticle/chitosan/reduced graphene oxide nanocomposite sheets (Au/CS/RGO) to explore the drug cascade metabolism using an electrochemically-driven approach. When model bienzymes, CYP1A2 and CYP3A4 isozymes, were sequentially assembled on Au/CS/RGO, one pair of well-defined redox peaks at -0.531 and -0.474 V (vs. SCE) was observed due to the overlap of the redox peaks of CYP1A2 and CYP3A4, confirming a good electrochemical activity of the CYP bienzyme complex. With an electrochemically-driven approach, the CYP bienzyme complex displayed synergic functions, whereby the intermediate of 2-oxo-clopidogrel generated from target substrate clopidogrel by CYP1A2 could be promptly converted into the final metabolite of clopidogrel carboxylic acid by CYP3A4. This sequential conversion could be demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Furthermore, the as-constructed CYP bienzyme complex could be also used in-situ to monitor the clopidogrel concentration with sensitivity of 143.4 μA mM−1 and detection limit of 0.63 μM. Therefore, the successful construction of the CYP bienzyme complex would offer a platform for studying the cascade enzymatic reaction, and provide a potential application in efficient biosensors for toxicity analysis and bioreactors for chemical synthesis.

•We review the innovative advances in polymer-based signal amplification.•Conceptual connectivity between different amplified methodologies is illustrated.•Examples explain the mechanisms of polymers/polymerizations-based amplification.•Several elegant applications are summarized that illustrate underlying concept.Demand is increasing for ultrasensitive bioassays for disease diagnosis, environmental monitoring and other research areas. This requires novel signal amplification strategies to maximize the signal output. In this review, we focus on a series of significant signal amplification strategies based on polymeric nanocomposites and polymerization. Some common polymers are used as carriers to increase the local concentration of signal probes and/or biomolecules on their surfaces or in their interiors. Some polymers with special fluorescence and optical properties can efficiently transfer the excitation energy from a single site to the whole polymer backbone. This results in superior fluorescence signal amplification due to the resulting collective effort (integration of signal). Recent polymerization-based signal amplification strategies that employ atom transfer radical polymerization (ATRP) and photo-initiated polymerization are also summarized. Several distinctive applications of polymers in ultrasensitive bioanalysis are highlighted.

MicroRNA (miRNA) is found to be up-regulated in many kinds of cancer and therefore is classified as an oncomiR. Herein, we design a multifunctional fluorescent nanoprobe (FSiNP-AS/MB) with the AS1411 aptamer and a molecular beacon (MB) co-immobilized on the surface of the fluorescent dye-doped silica nanoparticles (FSiNPs) for target-cell-specific delivery and intracellular miRNA imaging. The FSiNPs were prepared by a facile reverse microemulsion method from tetraethoxysilane and silane derivatized coumarin that was previously synthesized by click chemistry. The as-prepared FSiNPs possess uniform size distribution, good optical stability and biocompatibility. In addition, there is a remarkable affinity interaction between the AS1411 aptamer and the nucleolin protein on the cancer cell surface. Thus, a target-cell-specific delivery system by the FSiNP-AS/MB is proposed for effectively transferring a MB into the cancer cells to recognize the target miRNA. Using miRNA-21 in MCF-7 cells (a human breast cancer cell line) as a model, the proposed multifunctional nanosystems not only allow target-cell-specific delivery with the binding affinity of AS1411, but also can track simultaneously the transfected cells and detect intracellular miRNA in situ. The proposed multifunctional nanosystems are a promising platform for a highly sensitive luminescent nonviral vector in biomedical and clinical research.

The oxygen reduction reaction (ORR) is one of the crucial reactions in fuel cells and metal–air batteries. Heteroatom doped carbon spheres can serve as alternative low-cost non-metal electrocatalysts for ORR. Herein, we developed an effective route to the synthesis of uniform and electrochemically active B-doped hollow carbon nanospheres (BHCSs). BHCSs were synthesized via the carbonization of a boric phenolic resin supported by SiO2, followed by etching the SiO2 template. The content of B, B dopant species and specific surface area were adjusted by changing the content of the B precursor and the calcination temperature. Moreover, their influence on the performance of electrocatalytic activity was explored. It was found that, among these B-doping type materials (BC2O, BCO2, B4C and BC3), B–C bonds (B4C and BC3) played a crucial role on improving the electrocatalytic activity. Compared with the hollow carbon nanospheres (HCSs), a 70 mV positive shift of the onset potential and 1.7 times kinetic current density could be clearly observed with BHCSs. In addition, the BHCSs revealed better stability and methanol tolerance than commercial Pt/C (HiSPEC™ 3000, 20%). Thus, the as-prepared BHCSs, as inexpensive and efficient non-metal ORR catalysts, may have a promising application in direct methanol fuel cells.

The oxygen reduction reaction (ORR) plays an important role at the cathode of fuel cells in practical applications. Herein, a titanium dioxide/graphene supported hemin (TiO2/Gr/Hem) composite material with a flower-like superstructure was successfully prepared through a two-step solvothermal reaction. By a further heat-treatment at 300–900 °C, the electrocatalytic activity of the as-obtained catalysts was examined, and it was found that the pyrolysis at 700 °C gave rise to the best catalytic activity for the ORR in alkaline media. This heat-treatment temperature was found to be crucial in determining the activity and stability of catalysts, due to the enhanced structural defects, active sites, geometrical complexity, and larger fraction of the pyridinic nitrogen and pyrrolic nitrogen groups. The titanium dioxide/graphene (TiO2/Gr) and graphene/hemin (Gr/Hem) were also studied and compared, and it was revealed that the catalytic activity of TiO2/Gr/Hem catalysts for ORR can be further enhanced. In addition, the chemically bonded element iron in the heat-treated TiO2/Gr/Hem catalysts showed an inhibition effect for ORR and Ti–C–N materials garnered high catalytic activity compared with Ti–C–N–Fe materials in alkaline media. The higher methanol tolerance and durability of the TiO2/Gr/Hem composite materials during ORR were also confirmed. These results reflected the critical influences of the pyrolysis temperature and the chemically bonded element dopants to be the key factor for ORR.

•Graphene–CdS nanocomposites were synthesized by one-pot reduction of oxide graphene and CdCl2.•Photoactive multilayer by alternative assembly of G–CdS and PEI showed excellent homogeneity.•Ru(NH3)63+RuNH363+ as electroactive mediator improved the photo-to-current conversion efficiency.•The aptasensor exhibited high sensitivity and efficiency for thrombin determination.A photoelectrochemical (PEC) aptasensor for highly sensitive and specific detection of thrombin was developed by using graphene–CdS nanocomposites multilayer as photoactive species and electroactive mediator hexaammineruthenium(III) chloride (Ru(NH3)63+RuNH363+) as signal enhancer. Graphene–CdS nanocomposites (G–CdS) were synthesized by one-pot reduction of oxide graphene and CdCl2 with thioacetamide. The photoactive multilayer was prepared by alternative assembly of the negatively charged 3-mercaptopropionic acid modified graphene–CdS nanocomposites (MPA-G–CdS) and the positively charged polyethylenimine (PEI) on ITO electrode. This layer-by-layer assembly method enhanced the stability and homogeneity of the photocurrent readout of G–CdS. Thrombin aptamer was covalently bound to the multilayer by using glutaraldehyde as cross-linking. Electroactive mediator Ru(NH3)63+RuNH363+ could interact with the DNA phosphate backbone and thus facilitated the electron transfer between G–CdS multilayer and electrode and enhanced the photocurrent. Hybridizing of a long complementary DNA with thrombin aptamer could increase the adsorption amount of Ru(NH3)63+RuNH363+, which in turn boosted the signal readout. In the presence of target thrombin, the affinity interaction between thrombin and its aptamer resulted in the long complementary DNA releasing from the G–CdS multilayer and decreasing of photocurrent signal. On the basis of G–CdS multilayer as the photoactive species, Ru(NH3)63+RuNH363+ as an electroactive mediator, and aptamer as a recognition module, a high sensitive PEC aptasensor for thrombin detection was proposed. The thrombin aptasensor displayed a linear range from 2.0 pM to 600.0 pM and a detection limit of 1.0 pM. The present strategy provided a promising ideology for the future development of PEC biosensor.

•An electrochemical impedance immunosensor for ApoB-100 based on a nanobody.•An immunized phage display library against ApoB-100 was generated.•The immunosensor exhibited an extremely low detection limit of 0.03 ng mL−1 for ApoB-100.•The immunosensor exhibited acceptable selectivity, reproducibility and stability.Nanobodies (Nbs), also known as the variable domain of the heavy-chain-only antibody (VHH), are single-domain antigen-binding fragments derived from heavy-chain antibodies that occur naturally in sera of camelids. Due to their unique properties of small size (15 kD), intrinsic stability, high affinity and specificity, Nbs are suitable for detecting clinical relevant antigens. Apolipoprotein B-100 (ApoB-100) is a highly predictive marker for coronary artery disease (CAD), which is frequently detected in clinical diagnosis. Herein, we successfully obtained anti-ApoB-100 Nbs for the first time and further fabricated a label-free and sensitive immunosensor for ApoB-100 based on isolated anti-ApoB-100 nanobody (Nb) using the electrochemical impedance spectroscopy (EIS) technique. We have generated an immunized phage display library against ApoB-100 and isolated four anti-ApoB-100 Nbs with high affinity and stability. The Nb with the highest affinity was biotinylated based on in vivo BirA system. Further, we developed a label-free electrochemical impedance immunosensor for ApoB-100 using this anti-ApoB-100 Nb. The attachment of ApoB-100 onto the anti-ApoB-100 Nb-immobilized sensing layer led to the increased electron-transfer resistance, which was proportional to ApoB-100 concentration in the range from 0.05 to 5 ng mL−1 with a detection limit of 0.03 ng mL−1. This proposed immunosensor revealed high specificity to detect ApoB-100, acceptable intra-assay precision and good stability, functioning as a feasible technique for CAD diagnosis.

•Detection of DNA and DNA methylation was based on GO and HpaII.•It avoids complex and expensive clone and sequencing procedures for bisulfite method.•The method is simple and the detection limit for DNA is 43 pM.•It has been used to detect methylated DNA induced by chemical reagents and Mtase.DNA methylation plays an important role in many biological events and is associated with various diseases. Most traditional methods for detection of DNA methylation are based on the complex and expensive bisulfite method. In this paper, we report a novel fluorescence method to detect DNA and DNA methylation based on graphene oxide (GO) and restriction endonuclease HpaII. The skillfully designed probe DNA labeled with 5-carboxyfluorescein (FAM) and optimized GO concentration keep the probe/target DNA still adsorbed on the GO. After the cleavage action of HpaII the labeled FAM is released from the GO surface and its fluorescence recovers, which could be used to detect DNA in the linear range of 50 pM–50 nM with a detection limit of 43 pM. DNA methylation induced by transmethylase (Mtase) or other chemical reagents prevents HpaII from recognizing and cleaving the specific site; as a result, fluorescence cannot recover. The fluorescence recovery efficiency is closely related to the DNA methylation level, which can be used to detect DNA methylation by comparing it with the fluorescence in the presence of intact target DNA. The method for detection of DNA and DNA methylation is simple, reliable and accurate.Fluorescence detection of DNA and DNA methylation based on GO and restriction endonuclease HpaII.

On the basis of the photo-induced electron transfer (PET) from CdTe quantum dots (QDs) to cytochrome P450 2C9 (CYP2C9), a light-controlled drug metabolism system was successfully designed by using CYP2C9 functionalized-CdTe QDs as photocatalysts.

A graphene nano-cage with regulatable space for the assembly of a cytochrome P450 1A2–UDP-glucuronosyltransferase 1A10 bienzyme complex has been constructed via a click reaction, and successfully used to study drug sequential metabolism using an electrochemically-driven method.

The development of synthetic nanopores and nanochannels that mimick ion channels in living organisms for biosensing applications has been, and still remains, a great challenge. Although the biological applications of nanopores and nanochannels have achieved considerable development as a result of nanotechnology advancements, there are few reports of a facile way to realize those applications. Herein, a nanochannel-based electrochemical platform was developed for the quantitative detection of biorelated small molecules such as potassium ions (K+) and adenosine triphosphate (ATP) in a facile way. For this purpose, K+ or ATP G-quadruplex aptamers were covalently assembled onto the inner wall of porous anodic alumina (PAA) nanochannels through a Schiff reaction between −CHO groups in the aptamer and amino groups on the inner wall of the PAA nanochannels under mild reaction conditions. Conformational switching of the aptamers confined in the nanochannels occurs in the presence of the target molecules, resulting in increased steric hindrance in the nanochannels. Changes in steric hindrance in the nanochannels were monitored by the anodic current of indicator molecules transported through the nanochannels. As a result, quantitative detection of K+ and ATP was realized with a concentration ranging from 0.005 to 1.0 mM for K+ and 0.05 to 10.0 mM for ATP. The proposed platform displayed significant selectivity, good reproducibility, and universality. Moreover, this platform showed its potential for use in the detection of other aptamer-based analytes, which could promote its development for use in biological detection and clinical diagnosis.

Evaluating the kinetics of biological reaction occurring in confined nanospaces is of great significance in studying the molecular biological processes in vivo. Herein, we developed a nanochannel-based electrochemical reactor and a kinetic model to investigate the immunological reaction in confined nanochannels simply by the electrochemical method. As a result, except for the reaction kinetic constant that was previously studied, more insightful kinetic information such as the moving speed of the antibody and the immunological reaction progress in nanochannels were successfully revealed in a quantitative way for the first time. This study would not only pave the investigation of molecular biological processes in confined nanospaces but also be promising to extend to other fields such as biological detection and clinical diagnosis.

Understanding the enzymatic reaction kinetics that occur within a confined space or interface is a significant challenge. Herein, a nanotube array enzymatic reactor (CYP2C9/Au/TNA) was constructed by electrostatically adsorbing enzyme on the inner wall of TiO2 nanotube arrays (TNAs). TNAs with different dimensions could be fabricated by the anodization of titanium foil through varying the anodization potential or time. The electrical conductivity of TNAs was improved by electrodepositing Au nanoparticles on the inner wall of TNAs. The cytochrome P450 2C9 enzyme (CYP2C9) was confined inside TNAs as a model. The enzymatic activity of CYP2C9 and tolbutamide metabolic yield could be effectively regulated by changing the nanotube diameter and length of TNAs. The enzymatic rate constant kcat and apparent Michaelis constant Kmapp were determined to be 9.89 s–1 and 4.8 μM at the tube inner diameter of about 64 nm and length of 1.08 μm. The highest metabolic yield of tolbutamide reached 14.6%. Furthermore, the designed nanotube array enzymatic reactor could be also used in situ to monitor the tolbutamide concentration with sensitivity of 28.8 μA mM–1 and detection limit of 0.52 μM. Therefore, the proposed nanotube array enzymatic reactor was a good vessel for studying enzyme biocatalysis and drug metabolism, and has potential applications including efficient biosensors and bioreactors for chemical synthesis.

MicroRNAs (miRNAs) has been identified as diagnostic and prognostic biomarkers and predictors of drug response for many diseases, including a broad range of cancers, heart disease, and neurological diseases. The noninvasive theranostics system for miRNAs is very important for diagnosis and therapy of the cellular disease. Herein, a target-cell-specific theranostics nanoprobe for target-cell-specific delivery, cancer cells and intracellular miRNA-21 imaging, and cancer cell growth inhibition was proposed. The nanoprobe (FS-AS/MB) was prepared by simultaneously coupling of the AS1411 aptamer and miRNA-21 molecular beacon (miR-21-MB) onto the surface of Ru(bpy)32+-encapsulated silica (FS) nanoparticles. The FS nanoparticles synthesized by a facile reverse microemulsion method showed nearly monodisperse spherical shape with a smooth surface, good colloidal stability, a fluorescence quantum yield of ∼21%, and low cytotoxicity. The antibiofouling polymer PEG grafted onto a silica shell reduced nonspecific uptake of cells. The ability of FS-AS/MB for target-specific cells delivery, simultaneous cancer cells, intracellular miRNA-21 imaging, and inhibition of miRNA-21 function and suppression of cell growth in vitro, were also demonstrated. The results of the present study suggested that the proposed nanoprobes would be a promising theranostics for different cancers by imaging and inhibiting other intracellular genes.

•This biosensor overcomes the limitations of the ELISA or sandwich-type assays.•The sensors work well when challenged with complex, contaminant-ridden samples.•Stability of the “triplex-stem” DNA is adjusted conveniently and efficiently by Ag+.•The DNA switch architecture reduces the cost and complexity of the synthesis.•The biosensor shows excellent reproducibility by simple heating in water bath.A reversible and regenerable electrochemical biosensor is fabricated for quantitative detection of antibody based on “triplex-stem” molecular switches. A hairpin-shaped oligonucleotide (hairpin DNA) labeled with ferrocene (Fc) at the 3′-end is fixed on the gold electrode serving as a signal transduction probe. Its hairpin structure leads Fc close to the surface of gold electrode and produces a strong current signal (on-state). A single-strand oligonucleotide modified with two digoxin molecules on the two arm segments (capture DNA) interact with hairpin DNA with the help of Ag+ ions. The “triplex-stem” DNA forms, which separates Fc from the electrode and reduces the electrochemical signal (off-state). Binding of digoxin antibody to digoxin releases capture DNA from the hairpin DNA, creating an effective “off-on” current signal switch. The stability of the “triplex-stem” structure of hairpin/capture DNA is critical to the signal switch and the sensitivity of the method, which can be adjusted conveniently and efficiently by changing Ag+ concentrations. Based on the “off-on” current signal switch, this biosensor is used to detect digoxin antibody sensitively in blood serum. The linear range is 1.0–500 pg with a correlation coefficient of 0.996, and the detection limit is 0.4 pg. Also, this biosensor shows excellent reversibility and reproducibility, which are significant requirements for practical biosensor applications.Fabricating a reversible and regenerable electrochemical biosensor for quantitative detection of antibody by using “triplex-stem” DNA molecular switch.

Bright blue luminescent graphene quantum dots (GQDs) with major graphitic structured nanocrystals and a photoluminescence (PL) quantum yield of 15.5% were synthesized and used to monitor DNA damage. The GQDs were prepared by ultraviolet irradiation without using a chemical agent. The as-prepared GQDs showed excitation-dependent PL and stable electrochemiluminescence (ECL) behaviors. Gold nanoparticles (AuNPs) were linked with a probe of single-stranded DNA (cp53 ssDNA) to form AuNPs-ssDNA. The ECL signal of the GQDs could be quenched by non-covalent binding of the AuNPs-ssDNA to the GQDs, due to the occurrence of an electrochemiluminescence resonance energy transfer between the GQDs and the AuNPs. When AuNPs-ssDNA was then hybridized with target p53 DNA to form AuNPs-dsDNA, the non-covalent interaction between the GQDs and the ds-DNA weakened and the ECL of the GQDs recovered. This engendered an ECL sensor for the detection of target p53 ssDNA, with a detection limit of 13 nM. The resultant ECL sensor could be used for DNA damage detection based on its different bonding ability to damaged target p53 ssDNA and cp53 ssDNA linked AuNPs. The presented method could be expanded to the development of other ECL biosensors, for the quantification of nucleic acids, single nucleotide polymorphisms or other aptamer-specific biomolecules.

A novel polymerization-based signal amplification strategy by using a dual-functional macroinitiator for ultrasensitive detection of protein was proposed. The macroinitiator composed of streptravidins, initiators, and poly(acrylic acid-co-acrylamide) was endowed with the ability of both molecular recognition and high initiation efficiency. After the immunoreaction and streptravidin–biotin specific identification, the macroinitiators were immobilized on the surface of the substrate and then triggered the generation of an activator by the electron transfer for atom transfer radical polymerization (AGET ATRP) of 2-hydroxyethyl methacrylate (HEMA). The obtained polymers altered the surface reflectivity and opacity at the location of the macroinitiator, which could be easily distinguished by the naked eye within 10 min of polymerization. The numerous hydroxyl groups in the growing polymer chains also led the surface hydrophily change after only 7 min of polymerization. The detection limit of human immunoglobulin G antigen (IgG) by contact angle measurement was 0.13 ng mL−1. The excellent performance of IgG clinical serum sample assay was further examined, showing great potential to detect other biological sample by this sensitive and easy strategy.

•The photocatalyst composing of CYP2D6 and CdTe QDs is prepared by carbodiimide coupling chemistry.•The photocatalyst exhibit excellent photocatalytic activity towards tramadol metabolism.•The PEC platform in-situ monitor the light-driven drug metabolism and enzyme inhibition.•The Kmapp was easily obtained with the proposed PEC system.•A mechanism for light-driven drug metabolism was proposed.In this work, nanohybrids of CdTe quantum dots (QDs) and cytochrome P450 2D6 (CYP2D6) prepared by covalent binding of CYP2D6 to QDs were used as photocatalysts for drug metabolism. As a proof-of-concept of the light-driven drug metabolism, tramadol was chosen as the substrate. As control, the high performance liquid chromatography coupled with mass spectrometry (HPLC–MS) was used to confirm the production of metabolites. Under irradiation with a white-light, the catalytic cycle of P450-mediated oxygenation reactions occurred with the formation of radical species on QDs, which immediately activated the proximity of the bound P450 enzyme and transferred electrons from the conduction band (CB) of CdTe QDs to CYP2D6. This light-driven catalytic reaction via nanohybrids was comparable to the native biological metabolism by CYP2D6 isozyme microsomes with CYP reductase (CPR) using the enzyme cofactor nicotinamide-adenine dinucleotide phosphate (NADPH) as electron donor. Furthermore, a photoelectrochemical platform was designed for in-situ monitoring the metabolic reactions. Upon addition of tramadol, the photocurrent increased due to the electron transfer from the CB of CdTe QDs to CYP2D6. The apparent Michaelis–Menten constant was thus easily measured to be 1.35 μM. This enzyme-based photoelectrochemical platform showed accepted stability and great potential for studies on drug metabolism in vitro.

An immunogold chromatographic assay was developed for quantitative determination of human chorionic gonadotropin (HCG) antigen. The monoclonal antibody to beta-HCG antigen (Mab II) conjugated gold nanoparticles (GNPs) were sprayed onto a conjugation pad for specific binding with the target protein to form an immunocomplex. The monoclonal antibody to alfa-HCG antigen (Mab I) was immobilized on the test line (T zone) of the nitrocellulose membrane (NC membrane) to capture the immunocomplex of gold nanoparticle labeled Mab II and HCG protein. Therefore, GNPs would aggregate on the test line of the NC membrane in the presence of HCG, which could be easily distinguished by the naked-eye. As for quantitative detection, the gray value of the red color in the T zone was proportional to the corresponding sample concentration. The gray value versus logarithm concentration curve presented a good linear relationship in the range of 10–600 ng mL−1. The duration of the assay was within 15 min and no professional large-scale analytical instrument was necessary for quantification. When applied in human serum analysis, the strips could reach the requirements of the clinic tests.

As an emerging class of metal nanoclusters, oligonucleotide-stabilized silver nanoclusters (DNA–Ag NCs) show a number of applications in biosensing and bionanotechnology. Herein, we develop a label-free DNA sensor based on DNA–Ag NCs and exonuclease III (Exo III)-catalyzed target recycling amplification. The fluorescence of single-strand DNA-stabilized Ag NCs can be enhanced through hybridization with the guanine-rich DNA. With the addition of target DNA, the fluorescence intensity decreases comparable with that of DNA duplex-stabilized Ag NCs, which is attributed to the competitive hybridization reaction. With the addition of Exo III, the fluorescence intensity decreases more obviously. The calibration range for target DNA is 0.3 to 30 nM, and the detection limit is 0.2 nM. The sensor offers 100-fold improvement in detection sensitivity compared with that obtained without Exo III. The proposed strategy also shows excellent selectivity, which can differentiate between perfectly matched and mismatched target DNA. Therefore, the strategy presents a promising platform for DNA detection with high sensitivity and selectivity.

Cytochrome P450 enzymes (cyt P450s) with an active center of iron protoheme are involved in most clinical drugs metabolism process. Herein, an electrochemical platform for the investigation of drug metabolism in vitro was constructed by immobilizing cytochrome P450 2D6 (CYP2D6) with cyt P450 reductase (CPR) on graphene modified glass carbon electrode. Direct and reversible electron transfer of the immobilized CYP2D6 with the direct electron transfer constant of 0.47 s–1 and midpoint potential of -0.483 V was obtained. In the presence of substrate tramadol, the electrochemical-driven CYP2D6 mediated catalytic behavior toward the conversion of tramadol to o-demethyl-tramadol was confirmed. The Michaelis–Menten constant (Kmapp) and heterogeneous reaction rate constant during the metabolism of tramadol were calculated to be 23.85 μM and 1.96 cm s–1, respectively. The inhibition effect of quinidine on CYP2D6 catalyze-cycle was also investigated. Furthermore, this system was applied to studying the metabolism of other drugs.

•We establish a biocompatible system that enzyme is confined in PAA nanochannels.•The real-time electrochemical response to glucose is detected by our system.•Ionic strength, enzyme amount and pore diameter could affect the enzymatic reaction.•The steric and electrostatic effects on the reaction in nanochannel are investigated.The construction of nanodevices coupled with an integrated real-time detection system for evaluation of the function of biomolecules in biological processes, and enzymatic reaction kinetics occurring at the confined space or interface is a significant challenge. In this work, a nanochannel–enzyme system in which the enzymatic reaction could be investigated with an electrochemical method was constructed. The model system was established by covalently linking glucose oxidase (GOD) onto the inner wall of the nanochannels of the porous anodic alumina (PAA) membrane. An Au disc was attached at the end of the nanochannels of the PAA membrane as the working electrode for detection of H2O2 product of enzymatic reaction. The effects of ionic strength, amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics were illustrated. The GOD confined in nanochannels showed high stability and reactivity. Upon addition of glucose to the nanochannel–enzyme system, the current response had a calibration range span from 0.005 to 2 mM of glucose concentration. The apparent Michaelis–Menten constant (Kmapp) of GOD confined in nanochannel was 0.4 mM. The presented work provided a platform for real-time monitoring of the enzyme reaction kinetics confined in nanospaces. Such a nanochannel–enzyme system could also help design future biosensors and enzyme reactors with high sensitivity and efficiency.

Hemin-graphene nanosheets (H-GNs) can be controllably assembled by target DNA via a hybridization process. This results in a color change from dark blue-green to light blue-green. The degree of aggregation is dependent on DNA concentration and very sensitive to base mismatch. The formation of the blue-green color can be detected with bare eyes or a spectrometer. The method is simple, rapid, and works over the concentration range from 1.0 to 100 nM. The detection limit for target DNA is 0.2 nM. Excellent selectivity is also found in that a DNA with a single base mismatch can be discriminated. This was exploited to detect DNA damage as induced by styrene oxide, sodium arsenite, Fenton’s reagent, or UV radiation. We presume that this method represents a promising tool for evaluating genotoxicity.

•The bi-enzyme was easily and tightly immobilized on silica NPs.•SiO2-GOD/HRP was used to detect glucose by a visible method.•The method is sensitive and reliable.•It is possible to be applied in various fields by using SiO2-GOD/HRP to make pills.A novel glucose sensing strategy by using bi-enzyme coated monodispered silica nanoparticles (SiO2) was proposed. The monodispered SiO2 was synthesized according to our previously reported seed-growth methods. Glucose oxidase (GOD) and horseradish peroxidase (HRP) were simultaneously covalent immobilized on the surface of SiO2 nanoparticles through the cross-linker of glutaraldehyde. The immobilized bi-enzyme remained their bioactivities well for the substrate reaction. Thus, the resultant SiO2-GOD/HRP nanocomposites could be used as catalyst for enzymatic substrate reactions in the presence of 3,3′,5,5′-tetramethylbenzidine (TMB) as chromogenic reagent and glucose as substrate. The factors of affecting the catalytic activities of enzymes were optimized. Under optimal conditions, the absorbance at 450 nm in UV–visible spectra increased with the glucose concentration, which could be used for glucose detection with a linear range from 0.5 μM to 250 μM and a detection limit of 0.22 μM at a signal-to-noise ratio of 3σ. Considering the potential of making pills using this SiO2-GOD/HRP, the present strategy has good prospect in the clinic science and other fields in future.Graphical abstract

Due to great potential in nanobiotechnology, nanomachines, and smart materials, DNA-directed disassembly of gold nanoparticles (AuNPs) has been extensively explored. In a typical system, nonbase-paired regions (e.g., overhangs and gaps in the linker DNA and oligonucleotide spacers between thiol group and hybridization sequence) are indispensable portions in the disassembly of AuNPs based on DNA displacement reaction. Therefore, it is necessary to study the effect of nonbase-paired regions to improve the kinetics of disassembly of AuNPs. Herein, the disassembly rate of AuNPs based on DNA displacement reaction was investigated by using different length spacers and linker DNA containing various lengths of gaps or overhangs. Interestingly, it was revealed that among the gaps in the linker DNA could be most effectively used to improve the disassembly rate of the AuNPs. As a result, when we introduced gaps into linker DNA, the DNA displacement reaction of AuNPs was markedly shortened to less than 50 min, which was much faster than the previous methods. As a proof of the importance of our findings, a rapid AuNP-based colorimetric DNA biosensor has been successfully prepared. In addition, we showed that the signal of the biosensors could be further amplified using exonuclease III, resulting in a much lower detection limit in comparison with previous sensors similarly using AuNP aggregates as probes.

•Aptamer–cell affinity interaction was employed for selective collection and detection of MCF-7.•CdTe QDs and aptamer were coated on SiO2 NPs for bio-labeling.•Good sensitivity was achieved due to the signal amplification of SiO2 NPs.A novel strategy for selective collection and detection of breast cancer cells (MCF-7) based on aptamer–cell interaction was developed. Mucin 1 protein (MUC1) aptamer (Apt1) was covalently conjugated to magnetic beads to capture MCF-7 cell through affinity interaction between Apt1 and MUC1 protein that overexpressed on the surface of MCF-7 cells. Meanwhile, a nano-bio-probe was constructed by coupling of nucleolin aptamer AS1411 (Apt2) to CdTe quantum dots (QDs) which were homogeneously coated on the surfaces of monodispersed silica nanoparticles (SiO2 NPs). The nano-bio-probe displayed similar optical and electrochemical performances to free CdTe QDs, and remained high affinity to nucleolin overexpressed cells through the interaction between AS1411 and nucleolin protein. Photoluminescence (PL) and square-wave voltammetric (SWV) assays were used to quantitatively detect MCF-7 cells. Improved selectivity was obtained by using these two aptamers together as recognition elements simultaneously, compared to using any single aptamer. Based on the signal amplification of QDs coated silica nanoparticles (QDs/SiO2), the detection sensitivity was enhanced and a detection limit of 201 and 85 cells mL−1 by PL and SWV method were achieved, respectively. The proposed strategy could be extended to detect other cells, and showed potential applications in cell imaging and drug delivery.

A novel homogeneous immunoassay based on Förster resonance energy transfer for sensitive detection of tumor, e.g., marker with carcinoembryonic antigen (CEA), was proposed. The assay was consisted of polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor. In presence of CEA, the bio-affinity between antigen and antibody made the QDs and AuNPs close enough, thus the photoluminescence (PL) quenching of CdTe QDs occurred. The PL properties could be transformed into the fluorometric variation, corresponding to the target antigen concentration, and could be easily monitored and analyzed with the home-made image analysis software. The fluorometric results indicated a linear detection range of 1–110 ng mL−1 for CEA, with a detection limit of 0.3 ng mL−1. The proposed assay configuration was attractive for carcinoma screening or single sample in point-of-care testing, and even field use. In spite of the limit of available model analyte, this approach could be easily extended to detection of a wide range of biomarkers.Graphical abstractA Förster resonance energy transfer system by using polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor for sensitive detection of tumor marker was proposed.Highlights► A homogeneous immunosensing strategy based on FRET for detection of tumor marker was proposed. ► Close of QDs and AuNPs allow the occurrence of quenching the photoluminescence of nano-bio-probes. ► Signal quenching was monitored by a self-developed image analyzer. ► The fluorometric assay format is attractive for widespread carcinoma screening and even field use.

A rapid sandwiched immunoassay of microcystin-LR (MC-LR) in water is proposed with flow injection chemiluminescence detection. The magnetic beads (MBs) were first modified with polyethyleneimine (PEI) by acylamide bond between the carboxyl group on the surface of MBs and the primary amine group in PEI, followed by immobilizing of anti-MC-LR (Ab1) onto PEI with glutaraldehyde as linkage. The resulting Ab1 modified MBs captured the target MC-LR in water, reacted with the horseradish peroxidase and anti-MC-LR co-immobilized silica nanoparticles, and were detected with flow injection chemiluminescence. When using PEI/MBs as the carrier of anti-MC-LR, the CL signal was greatly enhanced up to 9-fold compared to that using MBs without PEI modification. The CL signal was further amplified 13-fold when Si/Ab2 was used as the signal probe. Under the optimal conditions, the present immunoassay exhibited a wide quantitative range from 0.02 to 200 μg L−1 with a detection limit of 0.006 μg L−1, which was much lower than the WHO provisional guideline limit of 1.0 μg L−1 for MC-LR in drinking water. The relative standard deviation was 4.8% and the recoveries for the spiked samples ranged from 84% to 115%, which indicated acceptable precision and accuracy for MC-LR. The present method is easier to perform and less time-consuming (the entire analysis process lasted about 40 minutes) and has been applied to the detection of MC-LR in different water samples successfully.

Detection of DNA damage is significant for the evaluation of genotoxicity of new chemicals in the early stages of its development. An electrogenerated chemiluminescence (ECL) biosensor was fabricated to detect specific sequences of DNA by using CdTe@SiO2 as nanoprobes for signal amplification. This DNA biosensor was constructed by self-assembly of an aminated capture DNA on the glass carbon electrode. DNA detection was realized by outputting a remarkable ECL signal of the CdTe@SiO2 labeled probe DNA. When the target DNA was introduced into the system, it was complementary to the probe DNA at the one-half-segment and complementary to the capture DNA at the other half-segment, resulting in the formation of a stable duplex complex. As a result, the CdTe@SiO2 labeled probe was proximate to the electrode surface and the ECL was observed. This DNA biosensor was proved to have a low detection limit (0.03 nM) and a wide dynamic range (from 0.1 nM to 2 μM). Most importantly, the sensing system could differentiate the single base mismatched DNA from the complementary DNA. It was successfully applied to study the damage to DNA caused by several genotoxicity chemicals, which was rapid, simple, reliable and sensitive compared to the classical biological methods.

•Simple sonication procedure was used to prepare graphene–AuNPs nanocomposites.•The nanocomposites were used as biocompatible matrix for GOD immobilization.•Better direct electron transfer of GOD was obtained on graphene-based modified GCE.•Sensitive electrocatalytic response of GOD to glucose was showed on graphene-based modified GCE.Graphene–gold nanoparticle nanocomposites (GR–AuNPs) were synthesized and used as a matrix for glucose oxidase (GOD) immobilization in glucose sensing. Using layer-by-layer (LBL) method, a multilayered GOD sensor was constructed by alternatively immobilizing GR–AuNPs and GOD on electrodes. The immobilized GOD displayed a couple of stable and well-defined redox peaks with an electron transfer rate constant of 3.25 s−1 and a formal potential of −0.452 V in 0.1 mol L−1 pH 7.0 phosphate buffer solution (PBS). The apparent Michaelis–Menten constant for glucose was 0.038 mmol L−1. This method allowed to detect glucose with a sensitivity of 3.844 μA mmol L−1 cm−2, a linear range from 0.02 to 2.26 mmol L−1, and a detection limit of 4.10 μmol L−1 (S/N = 3). The results indicated that the proposed graphene–gold nanoparticle nanocomposites were good platforms for enzyme immobilization, thus facilitating the direct electron transfer and biosensing application.

Nitrogen-doped carbon hollow spheres (NCHSs) have been fabricated by carbonization of poly(o-phenylenediamine) hollow spheres. The NCHSs exhibited superior performances to glassy carbon and carbon nanotubes for the simultaneous determination of uric acid and dopamine in the presence of high concentration ascorbic acid.

•An electrochemically driven catalytic strategy was described.•Fast direct electron transfer between electrodes and D290nNOSoxy was observed.•D290nNOSoxy biocatalysis was successfully driven by electrodes in the presence of BH4 and oxygen.Nitric oxide synthase (NOS) plays a critical role in a number of key physiological and pathological processes. Investigation of electron-transfer reactions in NOS would contribute to a better understanding of the nitric oxide (NO) synthesis mechanism. Herein, we describe an electrochemically driven catalytic strategy, using a nanocomposite that consisted of the oxygenase domain of neuronal NOS (D290nNOSoxy), indium tin oxide (ITO) nanoparticles and polyvinyl alcohol (PVA). Fast direct electron transfer between electrodes and D290nNOSoxy was observed with the heterogeneous electron transfer rate constant (ket) of 154.8 ± 0.1 s− 1 at the scan rate of 5 V s− 1. Moreover, the substrate Nω-hydroxy-l-arginine (NHA) was used to prove the concept of electrochemically driven biocatalysis of D290nNOSoxy. In the presence of the oxygen cosubstrate and tetrahydrobiopterin (BH4) cofactor, the addition of NHA caused the decreases of both oxidation current at + 0.1 V and reduction current at potentials ranging from − 0.149 V to − 0.549 V vs Ag/AgCl. Thereafter, a series of control experiments such as in the absence of BH4 or D290nNOSoxy were performed. All the results demonstrated that D290nNOSoxy biocatalysis was successfully driven by electrodes in the presence of BH4 and oxygen. This novel bioelectronic system showed potential for further investigation of NOS and biosensor applications.

A colorimetric method for detection of DNA damage was developed by using hemin-graphene nanosheets (H-GNs). H-GNs were skillfully synthesized by adsorping of hemin on graphene through π–π interactions. The as-prepared H-GNs possessed both the ability of graphene to differentiate the damage DNA from intact DNA and the catalytic action of hemin. The damaged DNA made H-GNs coagulated to different degrees from the intact DNA because there were different amount of negative charge exposed on their surface, which made a great impact on the solubility of H-GNs. As a result, the corresponding centrifugal supernatant of H-GNs solution showed different color in the presence of 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2, which could be discriminated by naked eyes or by ultraviolet (UV)–visible spectrometer. Based on this, the damaged effects of styrene oxide (SO), NaAsO2 and UV radiation on DNA were studied. Results showed that SO exerted most serious damage effect on DNA although all of them damaged DNA seriously. The new method for detection of DNA damage showed good prospect in the evaluation of genotoxicity of new compounds, the maximum limit of pesticide residue, food additives, and so on, which is important in the fields of food science, pharmaceutical science and pesticide science.Graphical abstractHighlights► H-GNs can differentiate the damaged DNA from intact DNA. ► Damaged DNA could be detected by naked eyes or UV method. ► The method was simple and has good prospect in evaluation of genotoxicity of chemicals.

Because of the potential applications of biosensors in clini- cal diagnosis, biomedical research, environmental analysis, and food quality control, researchers are very interested in developing sensitive, selective, rapid, reliable, and low-cost versions of these devices. A classic biosensor directly transduces ligand–target binding events into a measurable physical readout. Because of the limited detection sensitivity and selectivity in earlier biosensors, researchers have developed a number of sensing/signal amplification strategies. Through the use of nanostructured or long chain polymeric materials to increase the upload of signal tags for amplification of the signal readout associated with the ligand–target binding events, researchers have achieved high sensitivity and exceptional selectivity.Very recently, target-triggered polymerization-assisted signal amplification strategies have been exploited as a new biosensing mechanism with many attractive features. This strategy couples a small initiator molecule to the DNA/protein detection probe prior to DNA hybridization or DNA/protein and protein/protein binding events. After ligand–target binding, the in-situ polymerization reaction is triggered. As a result, tens to hundreds of small monomer signal reporter molecules assemble into long chain polymers at the location where the initiator molecule was attached. The resulting polymer materials changed the optical and electrochemical properties at this location, which make the signal easily distinguishable from the background. The assay time ranged from minutes to hours and was determined by the degree of amplification needed.In this Account, we summarize a series of electrochemical and optical biosensors that employ target-triggered polymerization. We focus on the use of atom transfer radical polymerization (ATRP), as well as activator generated electron transfer for atom transfer radical polymerization (AGET ATRP) for in-situ formation of polymer materials for optically or electrochemically transducing DNA hybridization and protein-target binding. ATRP and AGET ATRP can tolerate a wide range of functional monomers. They also allow for the preparation of well-controlled polymers with narrow molecular weight distribution, which was predetermined by the concentration ratio of the consumed monomer to the introduced initiator.Because the reaction initiator can be attached to a variety of detection probes through well-established cross-linking reactions, this technique could be expanded as a universal strategy for the sensitive detection of DNA and proteins. We see enormous potential for this new sensing technology in the development of portable DNA/protein sensors for point-of-need applications.

This study described a facile and effective route for the synthesis of structurally uniform and electrochemically active nitrogen-doped hollow carbon spheres (NHCSs) via pyrolysis of hollow poly(o-phenylenediamine) (PoPD) spheres. The characters and the mechanism of the transformation between the as-prepared PoPD and the NHCSs were studied. Results showed that the ladder aromatic structure of the as-prepared PoPD was transformed into a polycyclic-type structure in which nitrogen atoms were successfully incorporated into the graphitic structures to replace the carbon atoms. The as-prepared NHCSs showed a much higher electrocatalytic current than multi-wall carbon nanotubes and nitrogen-doped carbon nanotubes for the oxygen reduction reaction through a four-electron pathway in alkaline solution. The enhanced catalytic activity of NHCSs for ORR arose from the doping of nitrogen in the form of quaternary nitrogen, along with pyridinic N and pyrrolic N. The NHCSs also presented high methanol tolerance and long-term operational stability. The results showed that the NHCSs had a promising application in direct methanol fuel cells and provided a new method to synthesize carbon-based metal-free electrocatalysts from organic polymers.

We describe herein an electrochemically driven drug metabolism strategy based on nanocomposites that integrate cyt P450 2C9 (CYP2C9) isozyme microsomes with cyt P450 reductase (CPR), indium tin oxide (ITO) nanoparticles and chitosan (CS). This novel bioelectronic system enables monitoring of the drug metabolism and enzyme inhibition.

Apoptosis is involved in the pathology of a variety of diseases. The measurement of apoptosis will help us to evaluate the onset of disease and the effect of therapeutic interventions. In addition, the increased demand for understanding the early stages of apoptosis is pushing the envelope for solutions in early instance real-time monitoring of death kinetics. Here we present a novel electrochemiluminescent cytosensing strategy to quantitate apoptotic cell numbers, screen some anticancer drugs, and evaluate their effects on hepatocarcinoma cell line (HepG2) cells by utilizing the human antiphosphatidyl serine antibody (APSA) conjugated Ru(bpy)32+-encapsulated silica nanoparticle (APSA-SiO2@Ru) as the detection probe. HepG2 cells were easily immobilized on the arginine-glycine-aspartic acid-serine (RGDS)-multiwalled carbon nanotubes (RGDS-MWCNTs) nanocomposite by the specific combination of RGD domains with integrin receptors on the cell surface. Then APSA-SiO2@Ru was introduced to the surface of apoptosis cells through the specific interaction between APSA and phosphatidylserine (PS) that distributed on the outer membrane of apoptotic cells. On the basis of the signal amplification of the APSA-SiO2@Ru nanoprobe, the cytosensor could respond as low as 800 cells mL–1, showing very high sensitivity. In addition, the dynamic alterations of surface PS expression on HepG2 cells in response to drugs and the cell heterogeneity were also demonstrated. The strategy presented a promising platform for highly sensitive cytosensing and convenient screening of some clinically available anticancer drugs.

A novel strategy for ultrasensitive detection of model protein based on the integration of tyramide signal amplification (TSA) and polymerization-assisted signal amplification was proposed. The surface-initiated atom transfer radical polymerization (SI-ATRP) of glycidyl methacrylate (GMA) was triggered by the initiator-coupled protein immobilized on the electrode surface through sandwiched immunoreactions. Growth of long chain polymeric materials provided numerous epoxy groups for subsequent coupling of horseradish peroxidase (HRP), which in turn significantly increased the loading of quantum dots (QDs) labeled tyramide in the presence of hydrogen peroxide. As a result, electrochemiluminescence (ECL) and square-wave voltammetric (SWV) measurements showed 9.4- and 10.5-fold increase in detection signal in comparison with the unamplified method, respectively. To demonstrate the feasibility of this approach, human immunoglobulin G antigen (IgG) as a model target protein was employed and the detection limits were 0.73 and 0.09 pg mL–1 for ECL and SWV, respectively. The results showed that sensitivity of the presented immunoassay significantly increased by one-order of magnitude and offered great application promises in providing a sensitive, specific, and potent method for biological detection.

Nitrogen-doped carbon nanotubes (NCNTs) were designed for the immobilization and biosensing of proteins. The glutaraldehyde functionalized chitosan (GCS) was mixed with NCNTs to generate nanocomposites for enzyme immobilization. Laccase (Lac) or glucose oxidase (GOD) in GCS/NCNT nanocomposites displayed excellent enzymatic activity towards the reduction of oxygen or the oxidation of glucose. This led to elaboration of a glucose/air biofuel cell with a maximum power density of 21 μW cm− 2 at 0.2 V.Nitrogen-doped carbon nanotubes (NCNTs) were designed for the immobilization of laccase and glucose oxidase for the elaboration of a glucose/air biofuel cell. The nitrogen doping accelerated the electron transfer from electrode surface to the protein, leading to the protein molecules in GCS/NCNTs that possessed higher electrocatalytic activity.Highlights► Negative charges from nitrogen doping led to better hydrophilicity of GCS/NCNT nanocomposite. ► Laccase in nanocomposites exhibited an excellent enzymatic activity to the reduction of oxygen. ► The nitrogen doping accelerated the electron transfer from electrode to protein. ► The GCS/NCNT nanocomposite based glucose/air biofuel cell yields a high power output. ► The GCS/NCNT nanocomposite was a good matrix to integrate protein and electrode.

A novel sensing strategy for sensitive detection of mucin 1 protein (MUC1) and MCF-7 cells based on electrochemiluminescence (ECL) resonance energy transfer (ERET) from bis(2,2′-bipyridine)-(5-aminophenanthroline)ruthenium(II) (Ru1) to graphene oxide (GO) was proposed. The MUC1 aptamer was covalently combined with Ru1 (Ru1-aptamer) using aqueous carbodiimide coupling chemistry. Due to the strong noncovalent interaction between the Ru1-aptamer and GO, the ECL of Ru1 was efficiently quenched because of the ERET. In the presence of a target MUC1 protein, the binding between the Ru1-aptamer and MUC1 disturbed the interaction between the Ru1-aptamer and GO. These interactions led to the release of the Ru1-aptamer from GO, and resulted in the restoration of Ru1 ECL. This was shown to detect MUC1 protein sensitively in a linear range from 64.9 to 1036.8 nM with a detection limit of 40 nM. With further application in the detection of MCF-7 cells, the presented method could respond at concentrations as low as 30 cancer cells per mL. By substituting the aptamer and the corresponding target, this method could be conveniently extended for the sensitive detection of other biomolecules.

Investigation of the catalytic activity and stability of enzymes in confined nano/microspace provides valuable contributions to the fundamental understanding of biological reactions taking place on a mesoscopic scale within confined spaces. In this paper, macroporous silica foam (MSF) is used as a nanoreactor to co-confine glucose oxidase (GOD) and horseradish peroxidase (HRP). Then, the enzymatic cascade reactions, which act in tandem inside nanoreactors, for oxidation of glucose and 3,3′,5,5′-tetramethylbenzidine (TMB) were studied. The catalytic kinetic parameters of apparent Michaelis constant (Kappm) and maximum rate (Vmax) were obtained from Lineweaver–Burk plot by UV-vis spectrometry. Results showed that the catalytic activity of the co-confined enzymes is reduced compared to that of free enzymes in solution at room temperature. The stabilities of co-confined enzymes in denaturing agents, such as guanidinium chloride (GdmCl) and urea, were higher than those of free enzymes in solution. When employing a co-confined bienzyme system as a biosensor for the detection of glucose, a wider linear range of glucose was obtained for the co-confined bienzyme system than for free enzymes in solution.

Cytochrome P450 enzymes (cyt P450s) are iron-heme proteins involved in the metabolism of both endogenous and exogenous compounds. Accurate and rapid in vitro mimicking of the natural metabolic pathways has attracted significant interest from enzyme engineers. Herein, we describe the electrochemically driven and dynamic enhancement of drug metabolism, which is based on the cyt P450 3A4 (CYP3A4) isozyme microsomes with cyt P450 reductase (CPR) immobilized on colloidal gold/graphene nanocomposites via electrostatic interactions. Direct and reversible electron transfer between the electrode and CYP3A4/CPR-microsomes was observed with a formal potential of −0.482 ± 0.003 V. The bioelectrocatalytic response of the P450-electrode has been validated by the catalysis of nifedipine. Through the application of the rotating disk electrode, the corresponding kinetic parameters, namely the Michaelis–Menten constant and the heterogeneous reaction rate constant, were calculated to be 1.30 μM and 7.12 cm s−1, respectively. The inhibition effect of ketoconazole on the oxidation of nifedipine was also investigated and the IC50 value was calculated to be 0.23 μM. The bioelectrocatalytic products were analyzed through high performance liquid chromatography-mass spectrometry (HPLC-MS) measurements. This new format of the cyt P450s system could probe the catalytic pathway with voltammetric kinetic analyses, and facilitated electronically-driven bioreactor and biosensor applications.

In this article, N-doped carbon hollow spheres (NCHS) were successfully synthesized through fastly and facilely thermally treating poly(o-phenylenediamine) (PoPD). The NCHS and chitosan composite (NCHS/Chi) provided a novel biosensing matrix for the immobilization of laccase due to its good conductivity, high stability and good biocompatibility. Based on this, an amperometric biosensor by entrapping laccase into the NCHS/Chi composite for industrial kraft lignin has been fabricated. Results displayed that the biosensor had high sensitivity and long-term stability in the presence of the mediator (2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS2−)) at pH 6.0. For practical applications, the methods used for the preparation of the biosensor should be simple, rapid, and reproducible.Highlights► We have developed a synthesis of N-doped carbon hollow spheres (NCHS). ► The NCHS with chitosan were an ideal composite matrix for laccase immobilization. ► The system showed high electrocatalytical oxidation for industrial lignin.

Interest in the fabrication of micro/nanoreactors for evaluation of the function of biomolecules in biological processes, enzymatic reaction kinetics occurring inside the nanospace is rapidly increasing. With a simple reverse-micelle microemulsion method, horseradish peroxidase (HRP), a model biomolecule, was herein skillfully confined in silica nanoshells (HRP@SiO2) and its biocatalytical behaviors were investigated in detail. Spectroscopic measurements showed that the entrapped HRP molecules retained their native structure and had high enzymatic activity toward 3,3′,5,5′-tetramethylbenzidine (TMB) with Michaelis constant (Km) of 3.02 × 10−5 mol L−1. The entrapped HRP displayed a good direct electron transfer behavior and sensitive electrocatalytic response toward the reduction of H2O2, which could be enhanced using thionine and o-phenylenediamine (o-PD) as electron mediators. When using thionine as mediator, the mass transport between the substrates in electrolyte and HRP confined in silica nanospheres through the mesoporous tunnels was slower than that of o-PD, which slowed down the electron transfer between heme in HRP in the confined nanospace and the electrode, and resulted in low sensitivity to H2O2 with thionine as mediator when compared to o-PD.Highlights► Evaluation of the basic behaviors of biomolecules in nanospace is very important for the development of life science. ► A facile strategy for fabrication of nanoreactor was conducted by skillful confining of HRP in silica nanoshells with a reverse-micelle microemulsion method. ► The entrapped HRP molecules retained their native structure and had high enzymatic activity toward TMB. ► The entrapped HRP displayed a good direct electron transfer behavior and sensitive electrocatalytic response toward the reduction of H2O2. ► The slower mass transport resulted in low sensitivity to H2O2 with thionine as mediator when compared to o-PD.

Atom transfer radical polymerization (ATRP) is a novel class of signal amplification method. In polymerization process, monomers accumulate and grow up to form long-chain polymers with abundant electroactive or photoactive groups for coupling other molecules, resulting in the distinct increase of the loading of signal molecules and the detection sensitivity. This article reviewed the mechanism of ATRP, and summarized the recent applications of ATRP in biosensing. Particularly, future studies and prospects were envisioned.

A novel label-free immunosensing strategy for sensitive detection of tumor necrosis factor-alpha antigen (TNF-α) via surface-initiated atom transfer radical polymerization (SI-ATRP) was proposed. In this strategy, the Au electrode was first modified by consecutive SI-ATRP of ferrocenylmethyl methacrylate (FMMA) and glycidyl methacrylate (GMA), and TNF-α antibody was coupled to the copolymer segment of GMA (PGMA) by aqueous carbodiimide coupling reaction. Subsequently, the target TNF-α antigen was captured onto the Au electrode surface through immunoreaction. The whole process was confirmed by scanning electron microscopy (SEM) and surface plasmon resonance (SPR) measurements. With introduction of redox polymer segment of FMMA (PFMMA) as electron-transfer mediator, the antigen-coupled Au electrode exhibited well electrochemical behavior, as revealed by cyclic voltammetry measurement. This provided a sensing platform for sensitive detection of TNF-α with a low detection limit of 3.9 pg mL−1. Furthermore, the “living” characteristics of the ATRP process can not only be readily controlled but also allow further surface functionalization of the electrodes, thus the proposed method presented a way for label-free and flexible detection of biomolecules.Graphical abstractHighlights► The successive ATRP of FMMA and GMA endow co-fixing both signal and target molecules. ► The loading of ferrocene moieties was highly increased for each bio-recognition event. ► A detection limit of approximate 4 pg mL−1 was obtained due to the signal amplification. ► The signal molecules were directly existed in the polymer brushes simplified the experiment procedure. ► The SI-ATRP provided a versatile mean for fabrication of immunosensor with desirable sensitivity.

Carbon nanotubes doped with N (NCNTs) enable 1.5 times faster electron-transfer kinetics for the oxidation of NO compared to pristine carbon nanotubes (CNTs), which may be due to the low adsorption energy for a NO on pyridinic NCNT(5,5) allowing NO to lose electrons readily and facilitate the following oxidation to nitrate.

A signal amplification strategy for sensitive detection of tumor necrosis factor-alpha (TNF-α) using quantum dots (QDs)-polymer-functionalized silica nanosphere as the label was proposed. In this approach, silica nanospheres with good monodispersity and uniform structure were employed as carriers for surface-initiated atom transfer radical polymerization of glycidyl methacrylate, which is readily available functional monomer that possessing easily transformable epoxy groups for subsequent CdTe QDs binding through ring-open reaction. Then, human anti rabbit TNF-α antibody (anti-TNF-α, Ab2, served as a model protein) was bonded to CdTe QDs-modified silica nanospheres coated with polymer to obtain QDs-polymer-functionalized silica nanosphere labels (Si/PGMA/QD/Ab2). The Si/PGMA/QD/Ab2 labels were attached onto a gold electrode surface through a subsequent “sandwich” immunoreaction. This reaction was confirmed by scanning electron microscopy (SEM) and fluorescence microscopic images. Enhanced sensitivity could be achieved by an increase of CdTe QD loading per immunoassay event, because of a large number of surface functional epoxy groups offered by the PGMA. As a result, the electrochemiluminescence (ECL) and square-wave voltammetry (SWV) measurements showed 10.0- and 5.5-fold increases in detection signals, respectively, in comparison with the unamplified method. The detection limits of 7.0 pg mL–1 and 3.0 pg mL–1 for TNF-α antibodies by ECL and SWV measurements, respectively, were achieved. The proposed strategy successfully demonstrated a simple, reproducible, specific, and potent method that can be expanded to detect other proteins and DNA.

A comprehensive review on the development of analytical methods, by coupling electrochemiluminescence (ECL) detection with capillary electrophoresis (CE) and microchip electrophoresis (ME), is presented. After the description of the basic mechanism of ECL, the addition mode of luminescence reagent in CE–ECL system has been discussed. The analytical applications of the CE–ECL technique in terms of different analytes are also given. Due to the importance of ME as a separation method for the present and future, the ME detection methods based on ECL are considered in a relatively detailed way. Finally, possible trends for CE/ME-ECL in the near future are discussed.The mechanism of Ru(bpy)32+ electrochemiluminescence, addition mode of Ru(bpy)32+, recent applications of capillary electrophoresis coupled with electrochemiluminescent detection in drug and other substrates analysis are reviewed.

We present a novel immunosensor by using polymerization-assisted signal amplification strategy coupled with electrochemical detection. A sandwich immunoassay process was used to immobilize a polymerization reaction center, the initiator-conjugated polyclonal prostate specific antigen (PSA) or polyclonal carcinoembryonic antigen (CEA) antibodies on the surface of the electrode. Activator generated electron transfer for atom transfer radical polymerization (AGET ATRP) subsequently triggered the local accumulation of glycidyl methacrylate (GMA) monomers. Growth of long chain polymers provided excess epoxy groups for electrochemical tags aminoferrocene (FcNH2) coupling, which in turn significantly increased the loading of the signal molecules and enhanced the electrochemical readouts. The detection limit was ∼0.14 pg mL−1 for PSA and ∼0.10 pg mL−1 for CEA in PBS buffers. The proposed immunosensor was highly sensitive, selective and has a good match to the clinical electrochemiluminescent method. This suggested that the polymerization-assisted immunosensing strategy could be used as an effective method to significantly enhance signal output of the sandwich immunoassays and acted as a promising platform for the clinical screening of cancer biomarkers.

Colloidal gold nanoparticles (AuNPs), with unique properties such as highly resonant particle plasmons, direct visualization of single nanoclusters by scattering of light, catalytic size enhancement by silver deposition, conductivity, and electrochemical properties, are very attractive materials for several applications in biotechnology. Furthermore, as excellent biological tags, AuNPs can be easily conjugated with biomolecules and retain the biochemical activity of the tagged biomolecules, making AuNPs ideal transducers for several biorecognition applications. The goal of this article is to review recent advances of using AuNPs as labels for signal amplification in biosensing applications. We focus on the signal amplification strategies of AuNPs in biosensing/biorecognition, more specifically, on the main optical and electrochemical detection methods that involve AuNP-based biosensing. Particular attention is given to recent advances and trends in sensing applications.

Simultaneous detection of multianalytes associated with a particular cancer is beneficial for disease diagnosis. Here, a facile immunosensing strategy was designed to allow simultaneous electrochemical detection of dual proteins, in a single run. CdSe and PbS water-soluble quantum dots (QDs) were prepared and coated on monodisperse silica nanoparticles as labels for proteins detection. Rabbit immunoglobulin G antigen (IgG) and carcinoembryonic antigen (CEA) were chosen as model proteins for analysis. After a typical sandwich immunoassay, CdSe and PbS QDs labels were introduced onto the Au substrates’ surface, which were then dissolved and could be simultaneously monitored by square-wave-voltammetric (SWV) stripping measurements. Under selected conditions, IgG and CEA could be assayed in the ranges of 0.05–40 ng mL−1 and 0.05–25 ng mL−1, respectively. The proposed method possessed high sensitivity, good precision, and satisfactory reproducibility and regeneration.

A novel colorimetric immunosensing strategy based on protein-modified gold nanoparticle probes combined with atom transfer radical polymerization (ATRP) technology was proposed. Gold nanoparticles (GNPs, ∼15 nm) were functionalized with antibodies through an acylamide-bond between the carboxylic group of 11-mercaptoundecanoic acid that previously self-assembled on the surface of GNPs and the amino group of the protein (here, goat anti-rabbit immunoglobulim G (anti-IgG) used as model). The surface functionalized GNPs were used for IgG capture, which introduced initiator coupled anti-IgG (Ab2*) onto the surface of GNPs through immunoreactions. Subsequently triggered polymer growth resulted in the surface graft of preformed polymer chains onto nanoparticles that altered the optical property of GNPs. A distinct color change occurred. This could be designed for IgG detection. The spectrum absorption and colorimetric detection gave a linear range of 0.5–25 ng mL−1 with a detection limit of 0.03 ng mL−1 for IgG. The proposed approach showed high sensitivity from both visual and absorbance measurements. In spite of the limitations of available IgG antibodies, this approach could be easily extended to the detection of other biomarkers.

A novel electrochemiluminescent immunosensor with polymerization-assisted signal amplification has been developed and enhanced sensitivity was achieved by increasing tertiary amine loading on polymers.

A versatile immunosensor using a CdTe quantum dots (QDs) coated silica nanosphere (Si/QD) as a label was proposed for ultrasensitive detection of a biomarker. In this approach, silica nanospheres with good monodispersity and uniform structure were employed as the carrier for immobilization of QDs and antibodies. Rabit IgG served as a model protein to demonstrate the performance of the immunosensor. Goat antirabbit IgG antibody was covalently bound to CdTe QDs on the surface of silica nanospheres. CdTe QDs coated with a silica nanosphere label (Si/QD/Ab2) were attached onto the gold electrode surface through a subsequent “sandwich” immunoreaction. This reaction was confirmed by scanning electron microscopic (SEM) and fluorescence microscopic images. Due to signal amplification from the high loading of CdTe QDs, 6.6- and 5.9-fold enhancements in electrochemiluminescent (ECL) and square-wave voltammetric (SWV) signals for IgG detection were achieved compared to the unamplified method. The detection limits for IgG were 1.3 and 0.6 pg mL−1 for ECL and SWV measurements, respectively. The resulting versatile immunosensor possesses high sensitivity, satisfactory reproducibility and regeneration, and good precision. This simple and specific strategy has vast potential to be used in other biological assays.

Here, we describe a new approach for electrochemiluminescence (ECL) assay with Ru(bpy)32+-encapsulated silica nanoparticle (SiO2@Ru) as labels. A water-in-oil (W/O) microemulsion method was employed for one-pot synthesis of SiO2@Ru nanoparticles. The as-synthesized SiO2@Ru nanoparticles have a narrow size distribution, which allows reproducible loading of Ru(bpy)32+ inside the silica shell and of α-fetoprotein antibody (anti-AFP), a model antibody, on the silica surface with glutaraldehyde as linkage. The silica shell effectively prevents leakage of Ru(bpy)32+ into the aqueous solution due to strong electrostatic interaction between the positively charged Ru(bpy)32+ and the negatively charged surface of silica. The porous structure of silica shell allowed the ion to move easily through the pore to exchange energy/electrons with the entrapped Ru(bpy)32+. The as-synthesized SiO2@Ru can be used as a label for ultrasensitive detection of biomarkers through a sandwiched immunoassay process. The calibration range of AFP concentration was 0.05–30 ng mL−1 with linear relation from 0.05 to 20 ng mL−1 and a detection limit of 0.035 ng mL−1 at 3σ. The resulting immunosensors possess high sensitivity and good analytical performance.

This study compares the electrocatalytic activity of nitrogen-doped carbon nanotubes (NCNTs) with multiwalled carbon nanotubes (MWCNTs). Results indicate that NCNTs possess a marked electrocatalytic activity toward oxygen reduction reaction (ORR) by an efficient four-electron process in the alkaline condition, while the process of MWCNTs is through a two-electron pathway. Meanwhile, NCNTs show a very attractive electrochemical performance for the redox reaction of hydrogen peroxide (H2O2) and could be employed as a H2O2 sensor at a low potential of +0.3 V. The sensitivity of the NCNT-based biosensor reaches 24.5 μA/mM, more than 87 times that of the MWCNT-based one. Moreover, NCNTs exhibit striking analytical stability and reproducibility, which enables a reliable and sensitive determination of glucose by monitoring H2O2 produced by an enzymatic reaction between glucose oxidase/glucose or choline oxidase/choline at +0.3 V without the help of the electron mediator. The NCNT-based glucose biosensor has a linear range from 2 to 140 μM with an extremely high sensitivity of 14.9 μA/mM, and the detection limit is estimated to be 1.2 μM at a signal-to-noise ratio of 3. The results indicate that the NCNTs are good nanostructured materials for potential application in biosensors.Keywords: choline; electrocatalytic; glucose; hydrogen peroxide; nitrogen-doped carbon nanotubes; oxygen reduction

A novel and ultrasensitive immunosensing strategy based on activators generated electron transfer for atom transfer radical polymerization (AGET ATRP) in combination with flow injection chemiluminescent (CL) and electrochemical detection was proposed. The initiator-conjugated polyclone PSA antibodies (Ab2*), prepared by coupling of N-hydroxysuccinmidyl bromoisobutyrate (initiator) with polyclone PSA antibodies (Ab2), were immobilized on the substrate surface through sandwiched immunoreactions to trigger polymerization. AGET ATRP is used for local accumulation of glycidyl methacrylate (GMA) monomers. Horseradish peroxidase (HRP) was chosen as signal species for its well-characterized chemiluminescent and electrochemical behavior, strong enzyme activity, good solubility and ease in coupling. Growth of long chain polymeric materials provided excess epoxy groups for HRP coupling, which in turn significantly increased the loading of signal molecules and enhanced the chemiluminescent and electrochemical readouts. With the proposed strategy, a detection limit of 4.0 and 1.3 pg mL−1 was obtained for flow injection chemiluminescent and electrocatalytic measurements, respectively. A more than 13- and 14-fold enhancement in the chemiluminescent intensity and electrocatalytic current was achieved comparing to the traditional sandwiched immunoassays using HRP-conjugated antibody directly. The proposed method exhibited an efficient amplification performance for immunosensing. This paved a new way for ultrasensitive detection of cancer biomarkers.

We have successfully constructed a novel gold film with open interconnected macroporous walls of nanoparticles by combining the hydrogen bubble dynamic template synthesis with galvanic replacement reaction. After modified by a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA), the three-dimensionally (3D) interconnected macroporous Au film has been used as a biocompatible substrate for the immobilization of cytochrome c. The morphology, structure and electrochemical features of the modified and unmodified macroporous Au films were characterized by field-emission scanning electron microscopy (FESEM), energy-dispersive X-ray (EDX), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results reveal that the resultant films had a large electroactive surface area for high protein loading, enhanced electron transfer of cytochrome c, retained electrochemical activity, good stability and repeatability. And the excellent electrochemical behaviors could be attributed to the hierarchical structure of the macroporous Au film constructed by nanoparticles.

The enhanced sensitivity for detection of biomarkers based on CdTequantum dot functionalized silica nanosphere labels can be achieved by an increase in CdTeQD loading per sandwiched immunoreaction.

A novel signal amplification strategy for electrochemical detection of DNA and proteins based on the amplification-by-polymerization concept is described. Specifically, a controlled radical polymerization reaction is triggered after the capture of target molecules on the electrode surface. Growth of long chain polymeric materials provides numerous sites for subsequent aminoferrocene coupling, which in turn significantly enhances electrochemical signal output. Activators generated electron transfer for atom transfer radical polymerization (AGET ATRP) is used in this study for its high efficiency in polymer grafting and better tolerance toward oxygen in air. 2-Hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA) are examined to provide excess hydroxyl or epoxy groups for aminoferrocene coupling. A limit of detection of 15 pM and 0.07 ng/mL is demonstrated for DNA and ovalbumin, respectively. More than 7-fold signal enhancement in ovalbumin detection has been achieved comparing to the unamplified method. In addition, a more than 5 orders of magnitude of dynamic range is achieved with a linear correlation coefficient (R2) of 0.997 for DNA, and a more than 3 orders of magnitude with R2 of 0.999 for ovalbumin. Together, the results show that the coupling of amplification-by-polymerization concept with electrochemical detection offers great promises in providing a sensitive and cost-effective solution for biosensing applications.

A novel strategy for sensitive detection of biomarkers using horseradish peroxidase (HRP)-functionalized silica nanoparticles as the label is presented. The enzyme-functionalized silica nanoparticles were fabricated by coimmobilization of HRP and α-fetoprotein antibody (anti-AFP, the secondary antibody, Ab2), a model protein, onto the surface of SiO2 nanoparticles using γ-glycidoxypropyltrimethoxysilane (GPMS) as the linkage. Through “sandwiched” immunoreaction, the enzyme-functionalized silica nanoparticle labels were brought close to the surface of gold substrates, as confirmed by the scanning electron microscopy (SEM) images. Enhanced detection sensitivity was achieved where the large surface area of SiO2 nanoparticle carriers increased the amount of HRP bound per sandwiched immunoreaction. The electrochemical and chemiluminescence measurement showed 29.5- and 61-fold increases in detection signals, respectively, in comparison with the traditional sandwich immunoassay. The improved particle synthesis using a “seed-particle growth” route yielded particles of narrow size distribution, which allowed consistent loading of HRP and anti-AFP on each microsphere and ensured subsequent immunosensing possessed high sensitivity and reproducibility. This strategy was successfully demonstrated as a simple, cost-effective, specific, and potent method to detect AFP in practical samples.

A novel immunosensing system for determination of human α-fetoprotein (AFP) was proposed by using a boronate immunoaffinity column as the glycated antigen collector in combination with flow injection chemiluminescence. The column was fabricated by filling boronic acid-modified sepharose gel into a glass tube. With a sugar–boronic acid interaction, the AFP antigen could be effectively immobilized on the sepharose gel matrix. After an off-line incubation, the mixture of the analyte AFP and horseradish peroxidase-labeled AFP antibody (HRP-anti-AFP) was injected into the column. The free HRP-anti-AFP was trapped by the immobilized antigen in the column and detected via chemiluminescence due to its sensitive effect on the reaction of luminol and hydrogen peroxide. A calibration curve with two linear ranges of 5–120 and 300–1000 ng mL−1 was obtained under the optimized conditions. The whole assay process including regeneration of the reactor can be completed in 36 min. The presented immunoassay exhibited a high sensitivity, a wide linear range, a low interference with other antigens and a good reproducibility. It is potentially used to detect the serum AFP level in clinical diagnosis.

This is to report a practical and convenient method for 3D macroporous Pt synthesis based on the monodispersion silica template. The silica nanospheres were fabricated using an improved Stöber method. The alternative method of tetraethoxysilane (TEOS) addition to the reaction system was found to be helpful in gaining silica spheres with high monodispersion. The diameter of the final silica spheres increased proportionally with adding TEOS. The silica template was built with the self-assembling method by dispersing a dilute suspension of SiO2 nanosphere onto the smooth Au support surface. After evaporating the solvent naturally, a three-dimensional template with some void spaces around the arranged nanospheres, on the Au slide, was obtained. The macroporous Pt was synthesized by electrodeposition of Pt into the void spaces of the template, which was confirmed by SEM images. The surface activities of the resultant macroporous Pt were investigated by comparing the hydrogen adsorption/desorption peaks in H2SO4 and the methanol oxidation activity. The resultant macroporous Pt displayed high activity level during Pt loading of 0.532 mg cm−2. The current work presents a useful method for nanoparticle synthesis, 3D template and macroporous material fabrication.

Using zinc powders as source material, ZnO nanorods (ZnONR) were fabricated on gold wire by a hydrothermal reaction without any other surfactant. The gold wire end was coated by a thin layer of Zn−Au alloy to improve the nucleation for growth of ZnO nanostructures and to further improve the performance of the biosensor, which was constructed by alternatively immobilizing poly(sodium 4-styrenesulfonate) (PSS) and horseradish peroxidase (HRP) on the ZnONR. Electrochemical measurement, ultraviolet−visible spectrum, ζ-potential, and scanning electron microscopic analysis demonstrated that PSS and HRP were stably adsorbed layer by layer on the ZnONR surface, and the HRP kept bioactivity for H2O2 detection without an electron transfer mediator. The multilayered HRP sensors exhibited a wide linear range and low detection limit. The sensitivity of the biosensor increased with the immobilized HRP layers from the lowest value of 36.28 μA mM−1 for a monolayer.

Directly using zinc powders as source material, ZnO nanorods were fabricated on gold wire by hydrothermal reaction without any other surfacant and stabilizing agent. The gold wire was skillfully treated to improve the nucleation for growth of ZnO nanostructures and to further improve the performance of the biosensor, which was construct by immobilizing tyrosinase (Tyr) on the ZnO nanorods for phenol detection. Electrochemical measurement, Fourier transform infrared and scanning electron microscopic analyses demonstrated that the Tyr was stably adsorbed on the ZnO nanorods surface with bioactivity for phenol oxidization. The biosensor reached 95% of steady-state current within 5s, and the sensitivity was as high as 103.08 μA/mM at Cphenol > 20 μM and was 40.76 μA/mM at Cphenol < 20 μM. The detection limit of 0.623 μM was obtained at a signal/noise ratio of 3.

A reusable amperometric immunosensor based on the reversible boronic acid–sugar interaction is proposed. The immunosensor was prepared by self-assembling a thiol-mixed monolayer comprised of conjugates of 3-aminophenylboronic acid with 11-mercaptoundecanoic acid (APBA-MUA) and 11-mercapto-1-undecanol (MU) on gold. The resulting boronic acid coating layer can specifically bind with the glycoproteinantibody, enzyme conjugated carcinoembryonic antibody (HRP-anti-CEA). Voltammetric and electrochemical impedance spectroscopic (EIS) studies and surface plasmon resonance (SPR) measurements show that the binding of HRP-anti-CEA to the APBA interface is reversible and the HRP-anti-CEA can be removed with an acidic buffer or a solution containing sorbitol. The bound enzyme-conjugated antibody can retain its enzymecatalytic activity to the reduction of hydrogen peroxide (H2O2) and its immunoactivity while binding with CEA to form an immunocomplex. After the formation of the immunocomplex, the access of the active center of HRP to thionine was partially inhibited. This leads to a linear decrease in the electrocatalytic response of HRP-anti-CEA-modified electrode over a CEA concentration range of 2.5 to 40.0 ng mL−1. After monitoring the immunoreaction signals, the immunocomplex can be easily removed from the APBA interface with a regeneration solution. This regenerated APBA interface can rebound with HRP-anti-CEA and be recognized by the antigen, through which a reusable immunosensor with an RSD of 7.1% for four cycles can be obtained. Under optimal conditions, the detection limit for the CEA immunoassay is 1.1 ng mL−1, at three times background noise. Serum CEA determination results, obtained with the proposed method, shows that the immunosensor has an acceptable accuracy

In this work, the adsorption of tyrosinase on ZnO nanorods and its electrocatalytic behaviors were investigated. The mushroom tyrosinase with low isoelectric point was expected to adhere on the positively charged surface of ZnO nanorods by electrostatic attraction in a neutral solution. Scanning electron microscope images and spectroscopic analysis demonstrated the adsorption of tyrosinase on ZnO nanorods and the adsorbed tyrosinase remain its bioactivity to a large extent. In the presence of tyrosinase, a roughly and cyathiform of nanosized ZnO films was obtained. This open, three-dimensioned ramiform structure made the Fe(CN)63-/4- move through and exchange the electron with GCE more easily, and thus accelerating the electron transfer between electroactive Fe(CN)63-/4- and GCE. The adsorbed tyrosinase could catalyze the oxidation of phenol and catechol. The linear concentration ranges were from 0.02 to 0.1 mM and 0.01 to 0.4 mM, for phenol and catechol, respectively. The apparent Michaelis-menten constant (KMapp), a reflection of the enzymatic affinity, was 0.24 mM for phenol and 1.75 mM for catechol, which suggests a large affinity to phenolic compound. The proposed methods presented a way for further studies of the immobilization and electrochemistry of proteins on nanostructured materials.

In the current study, we describe an improved system to study the two-electron delivery reaction pathway of cytochrome P450, family 2, subfamily B, polypeptide 6 (CYP2B6) in vitro. In particular, a biocompatible film containing colloidal gold nanoparticles and chitosan was used to encapsulate CYP2B6 on an electrode. The electrocatalytic behaviors of CYP2B6 toward common drugs in the absence of NADHP–cytochrome P450 reductase as electron donor were studied. In an anaerobic solution, direct and reversible electron transfer between the electroactive heme center of CYP2B6 and the electrode was observed with a formal potential of –0.454 ± 0.006 V at pH 7.4. In an air-saturated solution, an increase in the bioelectrocatalytic reduction current was observed after drug addition. The bioelectrocatalytic products were analyzed using high-performance liquid chromatography (HPLC) and electrospray ionization–mass spectrometry (ESI–MS). Both results confirmed that C-hydroxylation and heteroatom release were the main pathways for CYP2B6-mediated drug oxidation, similar to what occurred in vivo. The use of immobilized proteins in nanoparticle-containing films in drug biosensing was also demonstrated.

A novel reusable electrochemical immunosensor for α-fetoprotein (AFP) based on phenylboronic acid monolayer on gold was proposed. The sensor was fabricated by immobilizing of 3-aminophenylboronic acid (APBA) conjugated thiol-mixed monolayer on gold through 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) as linkage. AFP and enzyme-conjugated antibody were further trapped to the modified electrode surface through sugar–boronic acid and immunoaffinity interactions, respectively. The attached enzyme-conjugated antibody on the electrode surface could catalyze the reduction of hydrogen peroxide in the presence of thionine, which can be used to detect AFP in human serum by a competitive mechanism. Cyclic voltammetric, electrochemical impedance studies and photometric activity assays were used to probe the assembly and regeneration process of the immunosensor. The influences of the competitive ratio of antigen and antibody, pH value of the measuring solution, incubation temperature and time were explored for optimizing the analytical performance. The whole assay process including incubation, detection and regeneration of the electrode could be completed in 35 min. The detection of AFP in five serum samples provided from clinically diagnosed patients with liver cancer showed acceptable accuracy. The proposed immunosensor enabled fast, low-cost and would be valuable for clinical diagnosis.

A novel nanocomposite of zinc porphyrin functionalized graphene quantum dots (GQDs/ZnPor) was prepared and used as a photocatalyst for the degradation of an organic pollutant under visible-light irradiation. In order to synthesise the nanocomposites, large graphene sheets were first cleaved into small pieces of graphene oxide by a mixture of concentrated H2SO4 and HNO3. Then, the GQDs/ZnPor was synthesized by a simple hydrothermal route with ZnPor and the as-synthesized graphene oxide as precursors. The resultant GQDs/ZnPor nanocomposites were characterized by transmission electron microscopy (TEM) and optical measurements. The photocatalytic activity of GQDs/ZnPor was evaluated by the degradation of methylene blue (MB) under visible-light irradiation. Enhancement of the photocatalytic activity of GQDs/ZnPor compared with pure zinc porphyrin was achieved. The photocatalytic degradation mechanism of GQDs/ZnPor was also illustrated by photoluminescence measurements and free radical and hole scavenging experiments. The proposed GQDs/ZnPor has potential application for decomposing organic pollutants in water.

An artificial metabolon with high conversion efficiency was constructed by confining a bi-enzyme into porous aluminum oxide nanochannels, which accelerated enzymatic reactions by minimizing the diffusion loss of intermediate species.

In situ time-resolved identification of interfacial transient reaction species were captured using imaging mass spectrometry, leading to the discovery of more complex elementary electrode reactions and providing an unprecedented understanding of the reaction mechanism on the electrode surface and solid–electrolyte interface using dynamic molecular imaging.

An immunogold chromatographic assay was developed for quantitative determination of human chorionic gonadotropin (HCG) antigen. The monoclonal antibody to beta-HCG antigen (Mab II) conjugated gold nanoparticles (GNPs) were sprayed onto a conjugation pad for specific binding with the target protein to form an immunocomplex. The monoclonal antibody to alfa-HCG antigen (Mab I) was immobilized on the test line (T zone) of the nitrocellulose membrane (NC membrane) to capture the immunocomplex of gold nanoparticle labeled Mab II and HCG protein. Therefore, GNPs would aggregate on the test line of the NC membrane in the presence of HCG, which could be easily distinguished by the naked-eye. As for quantitative detection, the gray value of the red color in the T zone was proportional to the corresponding sample concentration. The gray value versus logarithm concentration curve presented a good linear relationship in the range of 10–600 ng mL−1. The duration of the assay was within 15 min and no professional large-scale analytical instrument was necessary for quantification. When applied in human serum analysis, the strips could reach the requirements of the clinic tests.

As an emerging class of metal nanoclusters, oligonucleotide-stabilized silver nanoclusters (DNA–Ag NCs) show a number of applications in biosensing and bionanotechnology. Herein, we develop a label-free DNA sensor based on DNA–Ag NCs and exonuclease III (Exo III)-catalyzed target recycling amplification. The fluorescence of single-strand DNA-stabilized Ag NCs can be enhanced through hybridization with the guanine-rich DNA. With the addition of target DNA, the fluorescence intensity decreases comparable with that of DNA duplex-stabilized Ag NCs, which is attributed to the competitive hybridization reaction. With the addition of Exo III, the fluorescence intensity decreases more obviously. The calibration range for target DNA is 0.3 to 30 nM, and the detection limit is 0.2 nM. The sensor offers 100-fold improvement in detection sensitivity compared with that obtained without Exo III. The proposed strategy also shows excellent selectivity, which can differentiate between perfectly matched and mismatched target DNA. Therefore, the strategy presents a promising platform for DNA detection with high sensitivity and selectivity.

Nitrogen-doped carbon hollow spheres (NCHSs) have been fabricated by carbonization of poly(o-phenylenediamine) hollow spheres. The NCHSs exhibited superior performances to glassy carbon and carbon nanotubes for the simultaneous determination of uric acid and dopamine in the presence of high concentration ascorbic acid.

A light-driven approach combined with a macroporous reactor for the enzymatic biocatalytic reaction has been developed by confining the enzyme/photosensitizer nanohybrids in a macroporous material, which exhibits high bio-conversion efficiency due to the fast diffusion and collision between the enzyme/photosensitizer nanohybrid and the substrate in the reactor.

On the basis of the photo-induced electron transfer (PET) from CdTe quantum dots (QDs) to cytochrome P450 2C9 (CYP2C9), a light-controlled drug metabolism system was successfully designed by using CYP2C9 functionalized-CdTe QDs as photocatalysts.

A graphene nano-cage with regulatable space for the assembly of a cytochrome P450 1A2–UDP-glucuronosyltransferase 1A10 bienzyme complex has been constructed via a click reaction, and successfully used to study drug sequential metabolism using an electrochemically-driven method.

We describe herein an electrochemically driven drug metabolism strategy based on nanocomposites that integrate cyt P450 2C9 (CYP2C9) isozyme microsomes with cyt P450 reductase (CPR), indium tin oxide (ITO) nanoparticles and chitosan (CS). This novel bioelectronic system enables monitoring of the drug metabolism and enzyme inhibition.

Carbon nanotubes doped with N (NCNTs) enable 1.5 times faster electron-transfer kinetics for the oxidation of NO compared to pristine carbon nanotubes (CNTs), which may be due to the low adsorption energy for a NO on pyridinic NCNT(5,5) allowing NO to lose electrons readily and facilitate the following oxidation to nitrate.

A novel electrochemiluminescent immunosensor with polymerization-assisted signal amplification has been developed and enhanced sensitivity was achieved by increasing tertiary amine loading on polymers.

Due to great potential in nanobiotechnology, nanomachines, and smart materials, DNA-directed disassembly of gold nanoparticles (AuNPs) has been extensively explored. In a typical system, nonbase-paired regions (e.g., overhangs and gaps in the linker DNA and oligonucleotide spacers between thiol group and hybridization sequence) are indispensable portions in the disassembly of AuNPs based on DNA displacement reaction. Therefore, it is necessary to study the effect of nonbase-paired regions to improve the kinetics of disassembly of AuNPs. Herein, the disassembly rate of AuNPs based on DNA displacement reaction was investigated by using different length spacers and linker DNA containing various lengths of gaps or overhangs. Interestingly, it was revealed that among the gaps in the linker DNA could be most effectively used to improve the disassembly rate of the AuNPs. As a result, when we introduced gaps into linker DNA, the DNA displacement reaction of AuNPs was markedly shortened to less than 50 min, which was much faster than the previous methods. As a proof of the importance of our findings, a rapid AuNP-based colorimetric DNA biosensor has been successfully prepared. In addition, we showed that the signal of the biosensors could be further amplified using exonuclease III, resulting in a much lower detection limit in comparison with previous sensors similarly using AuNP aggregates as probes.