Selective nucleotide recognition for biosensor evolution requires rational probe design toward the binding-pattern-susceptible readout but without serious poison in selectivity from the context sequences. In this work, we synthesized a dual-function (trihydroxyphenyl)porphyrin (POH3) to target the abasic site (AP site) in ds-DNA using the trihydroxyphenyl substituent and the tetrapyrrole macrocycle as the recognition unit (RU) and the fluorescent signal unit (SU), respectively. RU and SU are separated from each other but are prototropically allosteric. We found that an appropriate pH favors formation of the nonfluorescent quinine/pyrrole (O–NH) conformer of POH3. However, the complementary hydrogen bonding of RU in O–NH with the target cytosine opposite the AP site switches on the SU fluorescence through prototropic allostery toward the phenol/isopyrrole (OH–N) conformer, while the bases thymine, guanine, and adenine totally silence this allostery, suggesting a superb selectivity in single-nucleotide polymorphism (SNP) analysis. The role of the prototropic allostery in achieving such SNP selectivity is also evidenced using porphyrins with other hydroxyl substituent patterns. Because of the SU separation from RU, SU is not directly involved in the interaction with the AP site, and thus, the turn-on selectivity is also realized for DNA with flanking guanine, the most easily oxidized base in DNA. This tolerance to the flanking base identity has seldom been achieved in previous studies. Additionally, other DNA structures cannot bring this allostery, indicating that the combination recipe of the AP site design and the prototropically allosteric probe will find wide applications in DNA-based sensors.

The rapid identification of biomacromolecule structure that has a specific association with chiral enantiomers especially from natural sources will be helpful in developing enantioselective sensor and in speeding up drug exploitation. Herein, owing to its existence also in living cells, apurinic/apyrimidinic site (AP site) was first engineered into ds-DNA duplex to explore its competence in enantiomer selectivity. An AP site-specific fluorophore was utilized as an enantioselective discrimination probe to develop a straightforward chiral sensor using natural tetrahydropalmatine (L- and D-THP) as enantiomer representatives. We found that only L-THP can efficiently replace the prebound fluorophore to cause a significant fluorescence increase due to its specific binding with the AP site (two orders magnitude higher in affinity than binding with D-THP). The AP site binding specificity of L-THP over D-THP was assessed via intrinsic fluorescence, isothermal titration calorimetry, and DNA stability. The enantioselective performance can be easily tuned by the sequences near the AP site and the number of AP sites. A single AP site provides a perfect binding pocket to differentiate the chiral atom-induced structure discrepancy. We expect that our work will inspire interest in engineering local structures into a ds-DNA duplex for developing novel enantioselective sensors.

•A novel signal enhancement strategy was designed for electrochemical immunoassay.•PAMAM-Au-MWCNT nanocomposites serve as an ideal sensing platform.•Core-shell SiO2@Au serves as tracing tags to immobilize HRP-Ab2 as nanobioprobes.•The immunosensor was successfully applied to evaluate SirT1 expression in cells samples.An ultra-highly sensitive electrochemical immunosensor for SirT1 (a key protein in age-related diseases) evaluation has been designed, employing polymeric nanocomposites as sensing platform and core-shell SiO2@Au to immobilize HRP-Ab2 as nanobioprobes. The approach includes chemical synthesis of PAMAM-Au-MWCNT nanocomposites (PNCs) with abundant PAMAM-Au nanoparticles immobilized on the MWCNT matrix, and biochemically synthesis of nanobioprobes with highly dispersed SiO2@Au tracing tags for successive efficient load functionalized enzyme-antibodies (HRP-Ab2). The PNCs nanocomposites could improve the efficiency of immune response via abundant capture antibodies (Ab1#1) on the electrode surface, and accelerate electron transfer through MWCNT and Au nanoparticles. Besides, the SiO2@Au was employed as tracing tags to label numerous HRP-Ab2 to further enhance signal readout during HRP−thionine−H2O2 system. Under optimal conditions, the signal intensity was linearly related to the concentration of SirT1 in the range of 20 pg mL-1 to 500 ng ml-1, and the limit of detection was 12.5 pg mL-1. It is noteworthy that the proposed immunoassay protocol has been successfully applied to evaluate SirT1 expression in cells by different treatment with high sensitivity and accuracy.Download high-res image (309KB)Download full-size image

Multi-doped carbon dots (C-dots) were synthesized using a facile one-pot solvothermal method, and the C-dots can be used as a ratiometric pH probe directly without integrating with other dyes, which was demonstrated by monitoring the proton-producing enzyme catalytic reactions.

•A photoelectrochemical AChE biosensor based on semiconductor nanocomposite was developed.•The combination of ZnO and TiO2 enhanced the photoelectrochemical activity.•Cd2 + showed dose-dependent effects on AChE activity.Acetylcholinesterase (AChE) is a well-known serine hydrolase and its dysfunction could disturb cholinergic neurotransmission which is related to the pathogenesis of neurodegenerative disorders. In this report, a photoelectrochemical biosensor based on metal oxide semiconductor nanocomposite was fabricated to investigate the effect of cadmium ion (Cd2 +) on the activity of AChE. The photoelectrochemical nanocomposite was prepared by anodic oxidation of titanium (Ti) foil to form titanium dioxide (TiO2) nanotubes (TNs) array followed by cathodic deposition of zinc oxide nanorods (ZnONRs) onto the TNs. AChE was immobilized on the obtained nanocomposites and the biosensor showed enhanced photoelectrochemical response under visible light irradiation. The experimental results showed that Cd2 + exhibited interesting dose-dependent and time-dependent effects on AChE activity. Specifically, high concentration of Cd2 + inhibited while low level of Cd2 + could stimulate the activity of AChE. These findings are of great significance for the study of enzyme activity influenced by metal ions and related pathogenesis investigation of neurodegenerative disorders.A photoelectrochemical biosensor based on metal oxide semiconductor nanocomposite was developed to investigate the effect of Cd2 + on AChE activity.

A label-free molecular beacon (MB) system integrated with enzyme-free amplification strategy was developed for simple and highly selective assay of Aβ oligomers.Highly selective response for Aβ oligomers.The method could monitor the Aβ aggregation process.Without the organic fluorophore labeling for signaling.Without the use of enzyme for amplification assay.Amyloid-beta (Aβ) oligomers are highly toxic species in the process of Aβ aggregation and are regarded as potent therapeutic targets and diagnostic markers for Alzheimer's disease (AD). Herein, a label-free molecular beacon (MB) system integrated with enzyme-free amplification strategy was developed for simple and highly selective assay of Aβ oligomers. The MB system was constructed with abasic site (AP site)-containing stem-loop DNA and a fluorescent ligand 2-amino-5,6,7-trimethyl-1,8-naphyridine (ATMND), of which the fluorescence was quenched upon binding to the AP site in DNA stem. Enzyme-free amplification was realized by target-triggered continuous opening of two delicately designed MBs (MB1 and MB2). Target DNA hybridization with MB1 and then MB2 resulted in the release of two ATMND molecules in one binding event. Subsequent target recycling could greatly amplify the detection sensitivity due to the greatly enhanced turn-on emission of ATMND fluorescence. Combining with Aβ oligomers aptamers, the strategy was applied to analyze Aβ oligomers and the results showed that it could quantify Aβ oligomers with high selectivity and monitor the Aβ aggregation process. This novel method may be conducive to improve the diagnosis and pathogenic study of Alzheimer's disease.

Based on melamine binding-triggered triplex formation and subsequent activation of Mg2+-dependent DNAzymes, a novel strategy for DNAzyme regulation was proposed and developed for melamine recognition.

It is widely acknowledged that sialic acid (SA) overexpression on cell surfaces has been thought to be a characteristic feature associated with many malignant diseases. In this paper, we report a new strategy for SA expression evaluation and electrochemical cytosensing of living cells. High selectivity and sensitivity were achieved by combining 3-aminophenylboronic acid (APBA) modified carbon nanospheres (CNS-APBA) for SA recognition and horseradish peroxidases (HRP) decorated on gold nanoparticles (Au-HRP NPs) for signal amplification. The HRP labels can effectively catalyze the oxidation of aniline in the presence of H2O2 to form polyaniline (PAn). The relatively positive detection potential range for PAn completely excluded the conventional interference from dissolved oxygen. Based on the dual signal amplification by functionalized carbon nanospheres and enzymatically produced polyaniline, the designed cytosensor can be used for SA evaluation on cancer cell surfaces and highly sensitive cell detection with a wide calibration range (from 5.0 × 102 to 1.0 × 106 cells per mL for MCF-7 cells and from 1.0 × 103 to 1.0 × 106 cells per mL for BGC-823 cells) and a low detection limit (25 cells per mL for MCF-7 cells and 800 cells per mL for BGC-823 cells). This strategy provides a valuable tool for the evaluation of sialic acid on cancer cells and great potency for cancer study.

Human sirtuin1 (SirT1), which is a member of the sirtuin family, plays an important role in a wide range of cellular processes. Here we demonstrate a new strategy for the photoelectrochemical assay of SirT1 in different cell lines based on a semiconductor–polymer hybrid system consisting of Au–polymer and TiO2-Au nanocomposites. Au–polymer (GC-HBAP) hybrids were synthesized from crosslinked hyperbranched azo-polymer and gold colloids and then used as an immobilization platform for SirT1 antibody. Gold-doped TiO2 (TiO2-Au) nanocomposites were prepared as the photoelectrochemical labels for signal readout in the sandwiched immunoassay. The integration of GC-HBAP with TiO2-Au facilitated the electron transfer and the photoelectrocatalytic reaction, resulting in good analytical performance with high sensitivity, selectivity and rapid response for the analysis of SirT1 levels in different cell lines. This proposed semiconductor–polymer system might open a new perspective for the development of a highly sensitive photoelectrochemical immunosensor, and have potentially promising applications in assays of other proteins.

•A highly sensitive immunosensor adopting a dual-layered enzyme strategy was proposed.•This immunosensor exhibited satisfactory analytical performances for CaM detection.•It was successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF-7) with high sensitivity, which has shown great potency for cancer study.Calmodulin (CaM) is a ubiquitous protein in eukaryotic cells, and it plays an important role in cancer progression. In this paper, a highly sensitive immunosensor adopting a dual-layered enzyme strategy was proposed for electrochemical detection of CaM. This immunosensor was constructed by introducing honeycomb-like mesoporous carbon (HMPC) as a sensor platform to sequentially immobilize antibody (Ab1), CaM and a multi-functionalized label. The label (HRP-PAupc-Ab1) was synthesized by covalently binding Ab1 and horseradish peroxidase (HRP) to poly(acrylic acid)-functionalized Au popcorn (PAupc) nanoparticles. A novel dual-layered enzyme strategy was employed by incubating HRP-secondary antibody (HRP-Ab2) onto the label surface and the enhanced biocatalyzed precipitation was therefore induced. This immunosensor exhibited satisfactory analytical performances for CaM detection with a linear response ranging from 5.0 pg mL−1 to 100 ng mL−1 and a detection limit of 1.5 pg mL−1. The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF-7) with high sensitivity, which has shown great potency for cancer study.

Here we report a label-free colorimetric method for protein assay based on the intrinsic peroxidase-like catalytic activity of DNA-hemin-graphene (DNA-GH) composite. By using aptamers as protein recognition elements, protein-mediated aggregation of the DNA-GH composite leads to the decrease or increase of the colorimetric signal depending on the sandwich or competitive design strategy. Thrombin and PDGF-BB were chosen as model analytes and the detection limits (LOD) by this method were estimated to be 0.5 nM and 5 nM, respectively. Compared to traditional ELISA method for protein detection, this method possesses the advantages of high sensitivity, simplicity, and low cost. In addition, by designing different DNA-modified hemin-graphene (GH) constructs, using proteins as inputs, the “OR” and “INHIBIT” logic gates were built. This procedure does not require chemical modification on the aptamer probes or analytes and circumvents the limitation associated with the number of target binding sites. Given the attractive analytical characteristics and distinct advantages of DNA-GH composite, the universal approach can be widely applied for the detection of diverse proteins and for the design of versatile logic gates.

Cell surface glycans are a class of sophisticated biomolecules related to cancer development and progression, and their analysis is of great significance for early cancer diagnosis and treatment. In this paper, we proposed a fluorescence assay to evaluate glycan expression on living cancer cells based on a competitive strategy coupled with dual-functionalized nanobiocomposites. The competitive assay was conducted between living cancer cells and thiomannosyl derivatives using concanavalin A (Con A)-modified electrode as the interaction platform. To impart fluorescence signaling ability to competitive derivatives, quantum dots (QDs) were anchored on BSA-protected Au nanoparticles, and thiomannosyl derivatives were further immobilized on the nanoparticle surface through Au–S binding. Due to the spacing between QDs and Au nanoparticles by BSA, the {QDs–Au–BSA–mannose} nanobiocomposites maintained the fluorescence of QDs and showed binding ability with the Con A-modified electrode. Au nanorods (AuNRs)-modified electrode was used as an effective substrate to immobilize Con A. This assay was successfully applied to the analysis of two cancer cells lines (A549 and QGY-7701). The method is simple and shows promise for the study of glycan expression on living cancer cells.

In this report, a novel visible-light-activated photoelectrochemical biosensor was fabricated to study the inhibition of acetylcholinesterase (AChE) activity induced by two endogenous neurotoxins, 1(R)-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [(R)-Sal] and 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetra-hydroisoquinoline [(R)-NMSal], which have drawn much attention in the study of the pathogenesis of neurodegenerative diseases such as Parkinson's disease. The photoelectrode was prepared by three steps, as follows. At first, nitrogen and fluorine co-doped TiO2 nanotubes (TNs) were obtained by anodic oxidation of a Ti sheet. Secondly, silver nanoparticles (AgNPs) were deposited onto the TNs through a microwave-assisted heating polyol (MAHP) process. At last, AChE was immobilized on the obtained photoelectrode and the biosensor was marked as AChE/Ag/NFTNs. Due to the nitrogen and fluorine co-doping, the photoelectrochemical biosensors can produce high photocurrent under visible light irradiation. Moreover, the presence of AgNPs greatly increased the photocurrent response of the biosensor. AChE/Ag/NFTNs hybrid system was used to study AChE inhibition induced by (R)-Sal and (R)-NMSal. The result proved that both (R)-Sal and (R)-NMSal exhibited mixed and reversible inhibition against AChE. This strategy is of great significance for the development of novel photoelectrochemical biosensors in the future.Highlights► A novel visible-light-activated photoelectrochemical biosensor was fabricated. ► The inhibition of acetylcholinesterase activity induced by two endogenous neurotoxins was studied. ► Both (R)-Sal and (R)-NMSal exhibited mixed and reversible inhibition against AChE.

•A carbon-dots-based fluorescent polymeric nanoprobe for Cu2+ was prepared.•The probe can detect Cu2+ ratiometrically with single-wavelength excitation.•The probe was used to image Cu2+ in liver cells.Copper is closely related to liver damage, therefore, it is essential to develop a simple and sensitive strategy to detect copper ions (Cu2+) in liver cells. A hydrophobic carbon dots (HCDs)-based dual-emission fluorescent probe for Cu2+ was prepared by encapsulating HCDs in micelles formed by self-assembly of amphiphilic polymer DSPE-PEG and tetrakis (4-carboxyphenyl) porphyrin (TCPP)-modified DSPE-PEG. The obtained probe showed characteristic fluorescence emissions of HCDs and TCPP with large emission shift of 170 nm with single-wavelength excitation. In the presence of Cu2+, the fluorescence of TCPP was quenched and that of HCDs remained unchanged, displaying ratiometric fluorescence response to Cu2+. The developed probe exhibited high sensitivity (detection limit down to 36 nM) and selectivity to Cu2+ over other substances, and the probe was used to image the changes of Cu2+ level in liver cells successfully.

Multi-doped carbon dots (C-dots) were synthesized using a facile one-pot solvothermal method, and the C-dots can be used as a ratiometric pH probe directly without integrating with other dyes, which was demonstrated by monitoring the proton-producing enzyme catalytic reactions.

Human sirtuin1 (SirT1), which is a member of the sirtuin family, plays an important role in a wide range of cellular processes. Here we demonstrate a new strategy for the photoelectrochemical assay of SirT1 in different cell lines based on a semiconductor–polymer hybrid system consisting of Au–polymer and TiO2-Au nanocomposites. Au–polymer (GC-HBAP) hybrids were synthesized from crosslinked hyperbranched azo-polymer and gold colloids and then used as an immobilization platform for SirT1 antibody. Gold-doped TiO2 (TiO2-Au) nanocomposites were prepared as the photoelectrochemical labels for signal readout in the sandwiched immunoassay. The integration of GC-HBAP with TiO2-Au facilitated the electron transfer and the photoelectrocatalytic reaction, resulting in good analytical performance with high sensitivity, selectivity and rapid response for the analysis of SirT1 levels in different cell lines. This proposed semiconductor–polymer system might open a new perspective for the development of a highly sensitive photoelectrochemical immunosensor, and have potentially promising applications in assays of other proteins.