Film-forming, stable, conductive composites of polyhistidine/graphene oxide for electrochemical quantification of trace Pb2.

Nanomaterials with unique properties, such as good film-formation and plentiful active atoms, play a vital role in the construction of electrochemical sensors. In this work, an in situ electrochemical synthesis of conductive polyhistidine (PHIS)/graphene oxide (GO) composite film (PHIS/GO) was designed to construct an electrochemical sensor for the sensitive detection of Pb2+. Herein, GO as an active material can directly form homogeneous and stable thin films on the electrode surface because of its excellent film-forming property. Then GO film was further functionalized by in situ electrochemical polymerization of histidine to obtain plentiful active atoms (N). Due to strong van der Waals forces between GO and PHIS, PHIS/GO film exhibited high stability. Furthermore, the electrical conductivity of PHIS/GO films was greatly improved by in situ electrochemical reduction technology and the plentiful active atoms (N) in PHIS are profitable for adsorbing Pb2+ from solution, tremendously enhancing the assay sensitivity. With the above unique property, the proposed electrochemical sensor showed high stability, a low detection limit (0.045 µg L-1) and a wide linear range (0.1-300 µg L-1) for the quantification of Pb2+. The method can also be extended to the synthesis of other film-forming nanomaterials to functionalize themselves and widen their potential applications, avoiding the addition of non-conductive film-forming substances.

Programmable High-Speed and Hyper-Efficiency DNA Signal Magnifier.

Herein, a programmable dual-catalyst hairpin assembly (DCHA) for realizing the synchronous recycle of two catalysts is developed, displaying high reaction rate and outstanding conversion efficiency beyond traditional nucleic acid signal amplifications (NASA). Once catalyst I interacts with the catalyst II, the DCHA can be triggered to realize the simultaneous recycle of catalysts I and II to keep the highly concentrated intermediate product duplex I-II instead of the steadily decreased one in typical NASA, which can accomplish in about only 16 min and achieves the outstanding conversion efficiency up to 4.54 × 108 , easily conquering the main predicaments of NASA: time-consuming and low-efficiency. As a proof of the concept, the proposed DCHA as a high-speed and hyper-efficiency DNA signal magnifier is successfully applied in the rapid and ultrasensitive detection of miRNA-21 in cancer cell lysates, which exploits the new generation of universal strategy for the applications in biosensing assay, clinic diagnose, and DNA nanobiotechnology.

Programmable mismatch-fueled high-efficiency DNA signal converter.

Herein, by directly introducing mismatched reactant DNA, high-reactivity and high-threshold enzyme-free target recycling amplification (EFTRA) is explored. The developed high-efficiency EFTRA (HEEFTRA) was applied as a programmable DNA signal converter, possessing higher conversion efficiency than the traditional one with perfect complement owing to the more negative reaction standard free energy (ΔG). Once traces of input target miRNA interact with the mismatched reactant DNA, amounts of ferrocene (Fc)-labeled output DNA could be converted via the EFTRA. Impressively, the Fc-labeled output DNA could be easily captured by the DNA tetrahedron nanoprobes (DTNPs) on the electrode surface to form triplex-forming oligonucleotide (TFO) at pH = 7.0 for sensitive electrochemical signal generation and the DTNPs could be regenerated at pH = 10.0, from which the conversion efficiency (N) will be accurately obtained, benefiting the selection of suitable mismatched bases to obtain high-efficiency EFTRA (HEEFTRA). As a proof of concept, the HEEFTRA as an evolved DNA signal converter is successfully applied for the ultrasensitive detection of miRNA-21, which gives impetus to the design of other signal converters with excellent efficiency for ultimate applications in sensing analysis, clinical diagnosis, and other areas.

Highly sensitive electrochemical nuclear factor kappa B aptasensor based on target-induced dual-signal ratiometric and polymerase-assisted protein recycling amplification strategy.

In this work, an amplified electrochemical ratiometric aptasensor for nuclear factor kappa B (NF-κB) assay based on target binding-triggered ratiometric signal readout and polymerase-assisted protein recycling amplification strategy is described. To demonstrate the effect of "signal-off" and "signal-on" change for the dual-signal electrochemical ratiometric readout, the thiol-hairpin DNA (SH-HD) hybridizes with methylene blue (MB)-modified protection DNA (MB-PD) to form capture probes, which is rationally introduced for the construction of the assay platform. On the interface, the probes can specifically bind to target NF-κB and expose a toehold region which subsequently hybridizes with the ferrocene (Fc)-modified DNA strand to take the Fc group to the electrode surface, accompanied by displacing MB-PD to release the MB group from the electrode surface, leading to the both "signal-on" of Fc (IFc) and "signal-off" of MB (IMB). In order to improve the sensitivity of the electrochemical aptasensor, phi29-assisted target protein recycling amplification strategy was designed to achieve an amplified ratiometric signal. With the above advantages, the prepared aptasensor exhibits a wide linear range of 0.1pgmL-1 to 15ngmL-1 with a low detection limit of 0.03pgmL-1. This strategy provides a simple and ingenious approach to construct ratiometric electrochemical aptasensor and shows promising potential applications in multiple disease marker detection by changing the recognition probe.

Cu/Mn Double-Doped CeO2 Nanocomposites as Signal Tags and Signal Amplifiers for Sensitive Electrochemical Detection of Procalcitonin.

Nanomaterials themselves as redox probes and nanocatalysts have many advantages for electrochemical biosensors. However, most nanomaterials with excellent catalytic activity cannot be directly used as redox probe to construct electrochemical biosensor because the redox signal of these nanomaterials can only be obtained in strong acid or alkali solution at high positive or negative potential, which greatly limits their applications in biologic assay. In this study, Cu/Mn double-doped CeO2 nanocomposite (CuMn-CeO2) was synthesized to use as signal tags and signal amplifiers for the construction of electrochemical immunosensor for sensitive assay of procalcitonin (PCT). Herein, CuMn-CeO2 not only possesses excellent catalytic activity toward H2O2 for signal amplification, but also can be directly used as redox probe for electrochemical signal readout achieved in neutral mild buffer solution at low positive potential. Importantly, since doping Cu, Mn into CeO2 lattice structure can generate extra oxygen vacancies, the redox and catalytic performance of obtained CuMn-CeO2 was much better than that of pure CeO2, which improves the performance of proposed immunosensor. Furthermore, CuMn-CeO2 can be implemented as a matrix for immobilizing amounts of secondary antibody anti-PCT by forming ester-like bridging between carboxylic groups of Ab2 and CeO2 without extra chemical modifications, which greatly simplifies the preparative steps. The prepared immunosensor exhibited a wide linear range of 0.1 pg mL-1 to 36.0 ng mL-1 with a low detection limit of 0.03 pg mL-1. This study implements nanomaterial themselves as redox probes and signal amplifiers and paves a new way for constructing electrochemical immunosensor.

Amplified impedimetric aptasensor combining target-induced DNA hydrogel formation with pH-stimulated signal amplification for the heparanase assay.

Herein, a novel electrochemical impedimetric biosensor for the heparanase (HPA) assay was developed based on target protein-induced DNA hydrogel formation, followed by pH-stimulation of the hydrogel density to increase the signal amplification. The method involved the synthesis of two different copolymer chains, consisting of two cooperatively functioning cross-linking elements, where one element was associated with the HPA-response and the other one with the pH-response. Initially, single-strand DNA as a capture probe was modified on the electrode surface. In the presence of HPA, the HPA-responsive element binding to HPA-induced DNA hybridization between the two copolymer chains and captured DNA, giving rise to the formation of a low-density polymer hydrogel film on the electrode surface and it obtaining an obvious impedimetric response. A significant signal enhancement was observed when changing the pH of the hydrogel film to 5.0, which could be ascribed to the fact that the pH-responsive element can fold into four-stranded i-motif structures at pH 5.0, leading to the increase in density of the hydrogel film. By implementing the DNA hydrogel to induce an impedimetric response change, this impedimetric biosensor exhibited an excellent analytical performance towards the HPA quantitative assay, with a low detection limit of 0.003 pg mL-1. This new method provides a versatile signal amplification method and paves a new way to construct impedimetric sensors for bioassays.

Highly Effective Protein Converting Strategy for Ultrasensitive Electrochemical Assay of Cystatin C.

In this work, a highly effective protein converting strategy based on immunoreaction-induced DNA strand displacement and T7 Exonuclease (T7 Exo)-assisted protein cyclic enzymatic amplification for ultrasensitive detection of cystatin C was described. Herein, Au@Fe3O4 as magnetic separator was labeled by antibody 1-conjungated DNA (DNA1) and the DNA substrate of T7 Exo (DNA3) which initially hybridized with output DNA (S1) to form a stable S1/DNA1 duplex (S1/DNA3). Antibody 2 was labeled by competing DNA (DNA 2). In the presence of cystatin C, sandwich immunoreaction would induce proximity hybridization between DNA2 and DNA3 and thus displace S1 from the S1/DNA3 duplex with formation of a stable DNA2/DNA3 duplex, realizing the conversion of input target cystatin C into output S1. To enhance the conversion ratio, the DNA2/DNA3 duplex was then digested by T7 Exo with release of DNA2 which could act as competing DNA again to displace S1 from the S1/DNA3 duplex in adjacent locations and initiate another cleavage reaction. Through such a cyclic process, each input cystatin C could induce more than one output S1, enhancing detection sensitivity. A hairpin DNA modified electrode was used to capture the output S1, and then, a hybridization chain reaction is triggered on the biosensor surface. Then, thionine as electron mediator was embedded into the dsDNA polymers to produce a detection signal. The electrochemical biosensor exhibited a much wider linear range of 0.01 pg mL(-1) to 30 ng mL(-1) with low detection limit of 3 fg mL(-1). Moreover, this method introduced protein unrelated to nucleic acids into the realm of potential inputs for translation, which might create a new immunoassay method for sensitive detection of protein.

A nanohybrid of platinum nanoparticles-porous ZnO-hemin with electrocatalytic activity to construct an amplified immunosensor for detection of influenza.

In this work, a nanohybrid of platinum nanoparticles-porous ZnO spheres-hemin (Pt-pZnO-hemin) was synthesized for construction of alkaline phosphatase-based immunosensor for detection of influenza. Briefly, porous ZnO spheres (pZnO) were prepared using soluble starches as the capping agent, followed by surface functionalization of platinum nanoparticles via a hydrothermal method (Pt-pZnO). Then, hemin with carboxylic functionality was spontaneously adsorbed onto Pt-pZnO by ester-like binding between carboxylic group of hemin and ZnO. Compared with platinum nanoparticles and hemin, the resulting Pt-pZnO-hemin nanohybrid showed more excellent electrocatalysis activity toward 1-naphthol (1-NP). Taking advantage of the Pt-pZnO-hemin, we have developed an amplified electrochemical immunosensor based on in situ generation of redox probe by alkaline phosphatase (ALP) and Pt-pZnO-hemin as signal enhancer. Herein, electrochemically active 1-NP was generated by enzymatic hydrolysis of inactive 1-naphthyl phosphate by ALP, then Pt-pZnO-hemin was used as catalyst to catalytically oxidize 1-NP, resulting in electrochemical signal amplification. Furthermore, in comparison with other nanomaterials including Au-pZnO, Pt-pZnO and Au-pZnO-hemin, the excellent catalytical property of Pt-pZnO-hemin make it a promising nanohybrid material for ALP-based immunosensor for signal amplification.

Amplified thrombin aptasensor based on alkaline phosphatase and hemin/G-quadruplex-catalyzed oxidation of 1-naphthol.

An alkaline phosphatase (ALP)-based biosensor can in situ generate an electroactive product by enzymatic hydrolysis of inactive substrates. To obtain a higher signal-to-background ratio, a chemical redox cycling signal-amplified strategy based on the addition of a strong reducing agent has often be applied in the construction of ALP-based biosensors. However, the strong reducing agent not only affects the activity of ALP but also readily reacts with dissolved oxygen, leading to inaccurate results. In this work, a new signal-amplified strategy for a thrombin (TB) aptasensor based on the catalytic oxidation of ALP-generated products, 1-naphthol (NP), using hemin/G-quadruplex DNAzymes was reported. We implemented gold-nanoparticle-decorated zinc oxide nanoflowers (Au-ZnO) as the matrix for immobilizing ALP and TB aptamer (TBA) and then labeled it with hemin to form hemin/G-quadruplex/ALP/Au-ZnO bioconjugates (TBA II bioconjugates). Through a "sandwich" reaction, TBA II bioconjugates were captured on the electrode surface. The amplified signal was carried out in two steps: (i) an ALP-catalyzed inactive substrate, 1-naphthyl phosphate (NPP), in situ produces NP on the surface of the electrode; (ii) on the one hand, NP as a new reactant could be directly electrooxidized and generated an electrochemical signal, but, on the other hand, NP could be oxidized by hemin/G-quadruplex in the presence of H2O2, resulting in amplification of the electrochemical signal. The proposed TB aptasensor achieved a linear range of 1 pM to 30 nM with a detection limit of 0.37 pM (defined as S/N = 3).

An amplified electrochemical immunosensor based on in situ-produced 1-naphthol as electroactive substance and graphene oxide and Pt nanoparticles functionalized CeO2 nanocomposites as signal enhancer.

In this work, an amplified electrochemical immunosensor based on 1-naphthol as electroactive substance and Pt/CeO2/GO composites as catalytic amplifier was constructed for sensitive detection influenza. Through "sandwich" reaction, the Pt/CeO2/GO functionalized bioconjugates were captured on electrode surface and the electrochemical signal directly originated from 1-naphthol, which was in situ produced with high local concentration though the hydrolysis of 1-naphthyl phosphate catalyzed by ALP. Then, 1-naphthol as new reactant was oxidized by Pt/CeO2/GO composites with outstanding catalytic performance, resulting in detection signal amplification. In addition, as compared to label electroactive substance to antibodies, a simplified preparative step of immunosensor could be achieved because the signal probe get rid of introducation other electroactive substances. The proposed immunosensor achieved a linear range of 1.0×10(-3)-1.0ngmL(-1) and 5.0 to 1.0×10(2)ngmL(-1) with a detection limit of 0.43pgmL(-1) (defined as S/N=3).

Enzyme-free electrochemical immunosensor based on host-guest nanonets catalyzing amplification for procalcitonin detection.

An enzyme-free electrochemical immunosensor based on the host-guest nanonets of N,N-bis(ferrocenoyl)-diaminoethane/ß-cyclodextrins/poly(amidoamine) dendrimer-encapsulated Au nanoparticles (Fc-Fc/ß-CD/PAMAM-Au) for procalcitonin (PCT) detection has been developed in this study. The signal probe was constructed as follows: amine-terminated ß-CD was adsorbed to PAMAM-Au first, and then the prepared Fc-Fc was recognized by the ß-CD to form stable host-guest nanonets. Next, secondary antibodies (Ab2) were attached into the formed netlike nanostructure of Fc-Fc/ß-CD/PAMAM-Au by chemical absorption between PAMAM-Au and -NH2 of ß-CD. Herein, the PAMAM-Au act not only as nanocarriers for anchoring large amounts of the ß-CD and Ab2 but also as nanocatalysts to catalyze the oxidation of ascorbic acid (AA) for signal amplification. Moreover, the Fc-Fc could be stably immobilized by the hydrophobic inner cavity of ß-CD as well as improving solubility by the hydrophilic exterior of ß-CD. With the unique structure of two ferrocene units, Fc-Fc not only affords more electroactive groups to make the electrochemical response more sensitive but also plays a role of combining dispersive ß-CD-functionalized PAMAM-Au to form the netlike nanostructure. Furthermore, Fc-Fc exhibits good catalytic activity for AA oxidation. When the detection solution contained AA, the synergetic catalysis of PAMAM-Au and Fc-Fc to AA oxidation could be obtained, realizing enzyme-free signal amplification. The proposed immunosensor provided a linear range from 1.80 pg/mL to 500 ng/mL for PCT detection and a detection limit of 0.36 pg/mL under optimal experimental conditions. Moreover, the immunosensor has shown potential application in clinical detection of PCT.

High throughput immunosenor based on multi-label strategy and a novel array electrode.

Accurate prediction of a particular cancer can be achieved by measuring multiplex biomarkers. Traditional methods for multi-biomarkers detection are either multi-spots assay with chip or multi-label assay with one detection spot. However, the detection throughput of these two approaches is limited by the substrate area and the numbers of available label respectively. To solve this problem, in the present study, an immunoassay was firstly prepared by combining multi-label strategy and multi-spot assay with a novel array electrode for simultaneous detection of six biomarkers for hepatocellular carcinoma (HCC). The detection throughput of the proposed method was doubled in comparison with traditional multi-spots assay (one target protein was detected on each analytic spot), which could greatly enhance the sensitivity and specificity of HCC diagnosis. This detection model may serve as the starting point for high throughput of multianalyte assay.