Label-free and sensitive faradic impedance aptasensor for the determination of lysozyme based on target-induced aptamer displacement

A label-free and sensitive faradic impedance spectroscopy (FIS) aptasensor based on target-induced aptamer displacement was developed for the determination of lysozyme as a model system. The aptasensor was fabricated by self-assembling the partial complementary single strand DNA (pcDNA)–lysozyme binding aptamer (LBA) duplex on the surface of a gold electrode. To measure lysozyme, the change in interfacial electron transfer resistance of the aptasensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The introduction of target lysozyme induced the displacement of the LBA from the pcDNA–LBA duplex on the electrode into the solution, decreasing the electron transfer resistance of the aptasensor. The decrease in the FIS signal is linear with the concentration of lysozyme in the range from 0.2 nM to 4.0 nM, with a detection limit of 0.07 nM. The fabricated aptasensor shows a high sensitivity, good selectivity and satisfactory regeneration. This work demonstrates that a high sensitivity of the fabricated aptasensor can be obtained using a relatively short pcDNA. This work also demonstrates that the target-induced aptamer displacement strategy is promising in the design of an electrochemical aptasensor for the determination of lysozyme with good selectivity and high sensitivity.     

Colorimetric aptasensor for progesterone detection based on surfactant-induced aggregation of gold nanoparticles

This paper proposes an aptasensor for progesterone (P4) detection in human serum and urine based on the aggregating behavior of gold nanoparticles (AuNPs) controlled by the interactions among P4-binding aptamer, target P4 and cationic surfactant hexadecyltrimethylammonium bromide (CTAB). The aptamer can form an aptamer-P4 complex with P4, leaving CTAB free to aggregate AuNPs in this aptasensor. Thus, the sensing solution will turn from red (520 nm) to blue (650 nm) in the presence of P4 because P4 aptamers are used up firstly owing to the formation of an aptamer-P4 complex, leaving CTAB free to aggregate AuNPs. However, in the absence of P4, CTAB combines with aptamers so that AuNPs still remain dispersed. Therefore, this assay makes it possible to detect P4 not only by absorbance measurement but also through naked eyes. By monitoring the variation of absorbance and color, a CTAB-induced colorimetric assay for P4 detection was established with a detection limit of 0.89 nM. Besides, the absorbance ratio A650/A520 has a linear correlation with the P4 concentration of 0.89–500 nM. Due to the excellent recoveries in serum and urine, this biosensor has great potential with respect to the visual and instrumental detection of P4 in biological fluids.     

Glucose biosensor based on nano-SiO2 and “unprotected” Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

Formation of electrochemically reduced graphene oxide on melamine electrografted layers and its application toward the determination of methylxanthines

The current study describes the electrografting of 2,4-diamino-1,3,5-triazine (AT) groups at the surfaces of glassy carbon electrode (GCE) and indium tin oxide (ITO) through in situ diazotization of melamine. The presence of AT groups at the surface of the electrode was confirmed by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). Furthermore, graphene oxide (GO) was self-assembled on AT grafted GCE. The oxygen functional groups present on the surface of GO were electrochemically reduced to form an electrochemically reduced graphene oxide (ERGO) on AT grafted electrode surface. Raman spectra show the characteristic D and G bands at 1340 and 1605 cm⁻¹, respectively, which confirms the successful attachment of GO on AT grafted surface, and the ratio of D and G bands was increased after the electrochemical reduction of GO. EIS shows that the electron transfer reaction of [Fe(CN)₆]^3−/4− was higher at the ERGO modified electrode than at bare, AT grafted, and GO modified GCEs. The electrocatalytic activity of ERGO was investigated toward the oxidation of methylxanthines. It shows excellent electrocatalytic activity toward these methylxanthines by not only shifting their oxidation potentials toward less positive potentials but also enhancing their oxidation currents.     

Individually addressable microelectrode arrays fabricated with gold-coated pencil graphite particles for multiplexed and high sensitive impedance immunoassays

A renewable, site-selective immobilization platform of microelectrode array (MEA) for multiplexed immunoassays has been initially developed using pencil graphite particles coated with gold layers as microelectrodes. The graphite particles available on the common pencil were utilized for directing the electro-deposition of gold layers with uniform microstructures which displayed a well-defined sigmoidal voltammetric response. In the concept-of-proof experiments, the resulting MEA platform was modified with functionalized monolayer, on which anti-human IgG antibodies could be stably immobilized in a site-selective way through binding chemistry to selectively capture human IgG antigens from the sample media. The subsequent introduction of anti-human IgG antibodies conjugated with 15 nm electro-active gold nanoparticles to recognize the captured IgG proteins resulted in a significant decrease in the interfacial electron-transfer resistance. High sensitive electrochemical quantification by gold nanoparticle-amplified impedance responses could thus be achieved. Experimental results show that the developed MEA sensor can allow for the detection of human IgG with wide linear range (0.05–100 ng ml⁻¹) and sensitivity over 10³ larger than that of the conventional, bulk gold electrode. The rapid regeneration of the used MEA platform can additionally be realized by a simple electrochemical treatment. The high selectivity of four individually addressable MEA platforms for multiple antigens in a single sample has been further demonstrated in the multiplexed immunoassay experiments. Such a site-selective immobilization strategy of MEA platform may open a new door towards the development of various simple, sensitive, cost-effective, and reusable biological sensors and biochips.     

Graphite Carbon-Supported Mo2C Nanocomposites by a Single-Step Solid State Reaction for Electrochemical Oxygen Reduction

Novel graphite-molybdenum carbide nanocomposites (G-Mo₂C) are synthesized by a typical solid state reaction with melamine and MoO₃ as precursors under inert atmosphere. The characterization results indicate that G-Mo₂C composites are composed of high crystallization and purity of Mo₂C and few layers of graphite carbon. Mo₂C nanoparticles with sizes ranging from 5 to 50 nm are uniformly supported by surrounding graphite layers. It is believed that Mo atom resulting from the reduction of MoO₃ is beneficial to the immobilization of graphite carbon. Moreover, the electrocatalytic performances of G-Mo₂C for ORR in alkaline medium are investigated by cyclic voltammetry (CV), rotating disk electrode (RDE) and chronoamperometry test with 3M methanol. The results show that G-Mo₂C has a considerable catalytic activity and superior methanol tolerance performance for the oxygen reduction reaction (ORR) benefiting from the chemical interaction between the carbide nanoparticles and graphite carbon.     

Plasmonic Coupling in Er3+:Au Tellurite Glass

In this paper, we report on luminescence and absorbance effects of Er⁺³:Au-doped tellurite glasses synthesized by a melting-quenching and heat treatment technique. After annealing times of 2.5, 5.0, 7.5, and 10.0 h, at 300 °C, the gold nanoparticles (GNP) effects on the Er⁺³ are verified from luminescence spectra and the corresponding levels lifetime. The localized surface plasmon resonance around 800 nm produced a maximum fluorescence enhancement for the band ranging from 800 to 840 nm, corresponding to the transitions ⁴H_11/2 → ⁴I_13/2 (805 nm) and ⁴S_3/2 → ⁴I_13/2 (840 nm), with annealing time till 7.5 h. The measured lifetime of the levels ⁴H_11/2 and ⁴S_3/2 confirmed the lifetime reduction due to the energy transfer from the GNP to Er⁺³, causing an enhanced photon emission rate in these levels.     

Charge separation in photosystem II core complexes induced by 690-730 nm excitation at 1.7 K

The illumination of oxygen-evolving PSII core complexes at very low temperatures in spectral regions not expected to excite P680 leads to charge separation in a majority of centers. The fraction of centers photoconverted as a function of the number of absorbed photons per PSII core is determined by quantification of electrochromic shifts on Pheo_D1. These shifts arise from the formation of metastable plastoquinone anion (Q_A⁻) configurations. Spectra of concentrated samples identify absorption in the 700-730 nm range. This is well beyond absorption attributable to CP47. Spectra in the 690-730 nm region can be described by the ‘trap’ CP47 absorption at 689 nm, with dipole strength of ∼1 chlorophyll a (chl a), partially overlapping a broader feature near 705 nm with a dipole strength of ∼0.15 chl a. This absorption strength in the 700-730 nm region falls by 40% in the photoconverted configuration. Quantum efficiencies of photoconversion following illumination in the 690-700 nm region are similar to those obtained with green illumination but fall significantly in the 700-730 nm range. Two possible assignments of the long-wavelength absorption are considered. Firstly, as a low intensity component of strongly exciton-coupled reaction center chlorin excitations and secondly as a nominally ‘dark’ charge-transfer excitation of the ‘special pair’ P_D1-P_D2. The opportunities offered by these observations towards the understanding of the nature of P680 and PSII fluorescence are discussed.     

Screening and development of DNA aptamers as capture probes for colorimetric detection of patulin

Patulin (PAT) is a kind of mycotoxin that has serious harmful impacts on both food quality and human health. A high-affinity ssDNA aptamer that specifically binds to patulin was generated using systemic evolution of ligands by exponential enrichment (SELEX) assisted by graphene oxide (GO). After 15 rounds of positive and negative selection, a highly enriched ssDNA pool was sequenced and the representative sequences were subjected to binding assays to evaluate their affinity and specificity. Of the eight aptamer candidates tested, the sequence PAT-11 bound to patulin with high affinity and excellent selectivity with a dissociation constant (K_d) of 21.83 ± 5.022 nM. The selected aptamer, PAT-11, was subsequently used as a recognition element to develop a detection method for patulin based on an enzyme-chromogenic substrate system. The colorimetric aptasensor exhibited a linear range from 50 to 2500 pg mL⁻¹, and the limit of detection was found to be 48 pg mL⁻¹. The results indicated that GO-SELEX technology was appropriate for the screening of aptamers against small-molecule toxins, offering a promising application for aptamer-based biosensors.     

Optical analysis of RE3+ (RE = Pr3+, Er3+ and Nd3+):cadmium lead boro tellurite glasses

This article reports on the optical characterization of Pr³⁺‐, Er³⁺‐ and Nd³⁺‐doped cadmium lead boro tellurite (CLBT) glasses prepared using the melt quenching method. The visible–near infrared (Vis–NIR) absorption spectra of these glasses were analyzed systematically. On measuring the NIR emission spectra of Er³⁺:CLBT glasses, a broad emission band centered at 1536 nm (⁴I_13/2 → ⁴I_15/2) was observed, as were three NIR emission bands at 900 nm (⁴F_3/2 → ⁴I_9/2), 1069 nm (⁴F_3/2 → ⁴I_11/2) and 1338 nm (⁴F_3/2 → ⁴I_13/2) from Nd³⁺:CLBT glasses and an NIR emission band at 1334 nm (¹G₄ → ³H₅) from Pr³⁺:CLBT glasses at an excitation wavelength (λ_ex) of 514.5 nm (Ar⁺ laser). Copyright © 2016 John Wiley & Sons, Ltd.     

Analysis of Cav1.2 and ryanodine receptor clusters in rat ventricular myocytes

We analyzed the distribution of ryanodine receptor (RyR) and Ca_v1.2 clusters in adult rat ventricular myocytes using three-dimensional object-based colocalization metrics. We found that ∼75% of the Ca_v1.2 clusters and 65% of the RyR clusters were within couplons, and both were roughly two and a half times larger than their extradyadic counterparts. Within a couplon, Ca_v1.2 was concentrated near the center of the underlying RyR cluster and accounted for ∼67% of its size. These data, together with previous findings from binding studies, enable us to estimate that a couplon contains 74 RyR tetramers and 10 copies of the α-subunit of Ca_v1.2. Extradyadic clusters of RyR contained ∼30 tetramers, whereas the extradyadic Ca_v1.2 clusters contained, on average, only four channels. Between 80% and 85% of both RyR and Ca_v1.2 molecules are within couplons. RyR clusters were in the closest proximity, with a median nearest-neighbor distance of 552 nm; comparable values for Ca_v1.2 clusters and couplons were 619 nm and 735 nm, respectively. Extradyadic RyR clusters were significantly closer together (624 nm) and closer to the couplons (674 nm) than the couplons were to each other. In contrast, the extradyadic clusters of Ca_v1.2 showed no preferential localization and were broadly distributed. These results provide a wealth of morphometric data that are essential for understanding intracellular Ca²⁺ regulation and modeling Ca²⁺ dynamics.     

Upconversion luminescence resonance energy transfer-based aptasensor for the sensitive detection of oxytetracycline

In this work, a biosensor based on luminescence resonance energy transfer (LRET) from NaYF₄:Yb,Tm upconversion nanoparticles (UCNPs) to SYBR Green I has been developed. The aptamers are covalently linked to UCNPs and hybridized with their complementary strands. The subsequent addition of SYBR Green allows SYBR Green I to insert into the formed double-stranded DNA (dsDNA) duplex and brings the energy donor and acceptor into close proximity, leading to the fluorescence of UCNPs transferred to SYBR Green I. When excited at 980 nm, the UCNPs emit luminescence at 477 nm, and this energy is transferred to SYBR Green I, which emits luminescence at 530 nm. In the presence of oxytetracycline (OTC), the aptamers prefer to bind to its corresponding analyte and dehybridize with the complementary DNA. This dehybridization leads to the liberation of SYBR Green I, which distances SYBR Green I from the UCNPs and recovers the UCNPs' luminescence. Under optimal conditions, a linear calibration is obtained between the ratio of I₅₃₀ to I₄₇₇ nm (I₅₃₀/I₄₇₇) and the OTC concentration, which ranges from 0.1 to 10 ng/ml with a limit of detection (LOD) of 0.054 ng/ml.     

Aptamer biosensor for label-free square-wave voltammetry detection of angiogenin

Angiogenin (Ang), one of the most potent angiogenic factor, is related with the growth and metastasis of numerous tumors. This paper presents a very simple and label-free square-wave voltammetry (SWV) aptasensor to detect angiogenin, in which an anti-angiogenin-aptamer was used as a molecular recognition element, and the couple ferro/ferricyanide as a redox probe. At the bare gold electrode, the redox couple (K₄[Fe(CN)₆]/K₃[Fe(CN)₆]) can be very easily accessed to the electrode surface to give a very strong SWV signal. At the anti-angiogenin/Au electrode surface, when angiogenin was added to the electrochemical cell, the binding of the analyte results in less availability for a redox reaction, which led to smaller SWV current. To quantify the amount of angiogenin, current suppressions of SWV peak were monitored using the redox couple of an [Fe(CN)₆]^4−/3− probe. The plot of signal suppression against the logarithm of angiogenin concentration is linear with over the range from 0.01 nM to 30 nM with a detection limit of 1 pM. The aptasensor also showed very good selectivity for angiogenin without being affected by the presence of other proteins in serum. It is the first time to use a very simple method to detect the cancer marker. Such an aptasensor opens a rapid, selective and sensitive route for angiogenin detection and provides a promising strategy for other protein detections.     

Characterization of PSI recovery after chilling-induced photoinhibition in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) leaves

By simultaneously analyzing the chlorophyll a fluorescence transient and light absorbance at 820 nm as well as chlorophyll fluorescence quenching, we investigated the effects of different photon flux densities (0, 15, 200 μmol m⁻² s⁻¹) with or without 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the repair process of cucumber (Cucumis sativus L.) leaves after treatment with low temperature (6°C) combined with moderate photon flux density (200 μmol m⁻² s⁻¹) for 6 h. Both the maximal photochemical efficiency of Photosystem II (PSII) (F _v/F _m) and the content of active P700 (ΔI/I _o) significantly decreased after chilling treatment under 200 μmol m⁻² s⁻¹ light. After the leaves were transferred to 25°C, F _v/F _m recovered quickly under both 200 and 15 μmol m⁻² s⁻¹ light. ΔI/I _o recovered quickly under 15 μmol m⁻² s⁻¹ light, but the recovery rate of ΔI/I _o was slower than that of F _v/F _m. The cyclic electron transport was inhibited by chilling-light treatment obviously. The recovery of ΔI/I _o was severely suppressed by 200 μmol m⁻² s⁻¹ light, whereas a pretreatment with DCMU effectively relieved this suppression. The cyclic electron transport around PSI recovered in a similar way as the active P700 content did, and the recovery of them was both accelerated by pretreatment with DCMU. The results indicate that limiting electron transport from PSII to PSI protected PSI from further photoinhibition, accelerating the recovery of PSI. Under a given photon flux density, faster recovery of PSII compared to PSI was detrimental to the recovery of PSI or even to the whole photosystem.     

Amplified electrochemical aptasensor for thrombin based on bio-barcode method

In the present study, an electrochemical aptasensor for highly sensitive detection of thrombin was developed based on bio-barcode amplification assay. For this proposed aptasensor, capture DNA aptamerI was immobilized on the Au electrode. The functional Au nanoparticles (DNA–AuNPs) are loaded with barcode binding DNA and aptamerII. Through the specific recognition for thrombin, a sandwich format of Au/aptamerI/thrombin/DNA–AuNPs was fabricated. After hybridization with the PbSNPs-labeled barcode DNA, the assembled sensor was obtained. The concentration of thrombin was monitored based on the concentration of lead ions dissolved through differential pulse anodic stripping voltammetric (DPASV). Under optimum conditions, a detection limit of 6.2 × 10⁻¹⁵ mol L⁻¹ (M) thrombin was achieved. In addition, the sensor exhibited excellent selectivity against other proteins.     

Graphene oxide supported rhombic dodecahedral Cu2O nanocrystals for the detection of carcinoembryonic antigen

In this work, a simple electrochemical immunosensor was developed for the detection of carcinoembryonic antigen (CEA) based on rhombic dodecahedral Cu₂O nanocrystals–graphene oxide–gold nanoparticles (rCu₂O–GO–AuNPs). GO as the template and surfactant resulting in rCu₂O exhibit improved rhombic dodecahedral structure uniformity and excellent electrochemical performance. Moreover, GO was found to be able to effectively improve the long stability of rCu₂O on the electrode response. Under optimal conditions, the immunosensor showed a low limit of detection (0.004 ng ml⁻¹) and a large linear range (0.01–120 ng ml⁻¹). This work presents a potential alternative for the diagnostic applications of GO-supported special morphology materials in biomedicine and biosensors.     

A multi-amplification aptasensor for highly sensitive detection of thrombin based on high-quality hollow CoPt nanoparticles decorated graphene

In this work, we have successfully demonstrated a facile strategy to incorporate high-quality hollow CoPt bimetal alloy nanoparticles (HCoPt) onto reduced graphene oxide sheet (HCoPt-RGs). An advanced sandwich-type electrochemical aptasensor for thrombin was proposed by using the HCoPt-RGs conjugates as secondary label. The formed conjugates provided large surface area for loading plentiful redox probe thionine (Thi), horseradish peroxidase (HRP) and secondary aptamer (Apt II) with good stability and friendly biocompatibility, indicating their superior properties in electroactive mediator enrichment and biomolecule immobilization. Furthermore, activated by glutaraldehyde (GA), the chitosan-hollow CoPt alloy nanoparticle (CS-HCoPt) film can greatly facilitate the capture of primary aptamer (Apt I) and dramatically reduce the nonspecific binding. Excellent sensitivity was obtained by detecting the conspicuously enhanced electrochemical signal of Thi, which was amplified by HCoPt alloy nanoparticles and HRP toward the catalytic reduction of H₂O₂. The aptasensor displayed excellent performance for thrombin with a wide linearity in the range from 1.0 × 10⁻¹² to 5.0 × 10⁻⁸ M and a relatively low detection limit of 3.4 × 10⁻¹³ M. Moreover, the resulted aptasensor also exhibited good specificity, acceptable reproducibility and stability, indicating that the present strategy could pave a promising way for the wide application of graphene in clinical research.     

Mutational Studies Uncover Non-Native Structure in the Dimeric Kinetic Intermediate of the H2A-H2B Heterodimer

The folding pathway of the histone H2A-H2B heterodimer minimally includes an on-pathway, dimeric, burst-phase intermediate, I₂. The partially folded H2A and H2B monomers populated at equilibrium were characterized as potential monomeric kinetic intermediates. Folding kinetics were compared for initiation from isolated, folded monomers and the heterodimer unfolded in 4 M urea. The observed rates were virtually identical above 0.4 M urea, exhibiting a log-linear relationship on the final denaturant concentration. Below ∼ 0.4 M urea (concentrations inaccessible from the  4-M urea unfolded state), a rollover in the rates was observed; this suggests that a component of the I₂ ensemble contains non-native structure that rearranges/isomerizes to a more native-like species. The contribution of helix propensity to the stability of the I₂ ensemble was assessed with a set of H2A-H2B mutants containing Ala and Gly replacements at nine sites, focusing mainly on the long, central α2 helix. Equilibrium and kinetic folding/unfolding data were collected to determine the effects of the mutations on the stability of I₂ and the transition state between I₂ and N₂. This limited mutational study indicated that residues in the α2 helices of H2A and H2B as well as α1 of H2B and both the C-terminus of α3 and the short αC helix of H2A contribute to the stability of the I₂ burst-phase species. Interestingly, at least eight of the nine targeted residues stabilize I₂ by interactions that are non-native to some extent. Given that destabilizing I₂ and these non-native interactions does not accelerate folding, it is concluded that the native and non-native structures present in the I₂ ensemble enable efficient folding of H2A-H2B.     

Biocatalysts for fuel cells: efficient hydrogenase orientation for H<Subscript>2</Subscript> oxidation at electrodes modified with carbon nanotubes

We report the modification of gold and graphite electrodes with commercially available carbon nanotubes for immobilization of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption. Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs), and amine-modified and carboxyl-functionalized SWCNs were used and compared throughout. Two separate methods were performed: covalent attachment of oriented hydrogenase by controlled architecture of carbon nanotubes at gold electrodes, and adsorption of hydrogenase at carbon-nanotube-coated pyrolytic graphite electrodes. In the case of self-assembled carbon nanotubes at gold electrodes, hydrogenase orientation based on electrostatic interaction with the electrode surface was found to control the electrocatalytic process for H₂ oxidation. In the case of carbon nanotube coatings on pyrolytic graphite electrodes, catalysis was controlled more by the geometry of the nanotubes than by the orientation of the enzyme. Noticeably, shortened SWCNs were demonstrated to allow direct electron transfer and generate high and quite stable current densities for H₂ oxidation via adsorbed hydrogenase, despite having many carboxylic surface functions that could yield unfavorable hydrogenase orientation for direct electron transfer. This result is attributable to the high degree of oxygenated surface functions in addition to the length of shortened SWCNs that yields highly divided materials. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.