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In this work, a lateral moving multi-pedal DNA tetrahedron machine (MTM) is designed and coupled with dual-signal output system to construct a biomimetic electrochemical ratiometric strategy for ultrasensitive target DNA analysis. The tetrahedral structure provided rigid support for the pedal, ensuring efficient replacement of the rail chain modified with ferrocene. By conjugating cholesterol molecules to one vertex of MTM, it is decorated on a lipid bilayer. This molecular architecture confers lateral movement of MTM on an electrode surface while prevents its detachment from the system. The methylene blue tagged hairpin probe provides constant power to support MTM swim on lipid bilayer. Compared with the conventional motion mode, the lateral moving mechanism has the fastest reaction rate and the highest signal-to-noise ratio. Additionally, the dual-signal reporting system further improves the accuracy of target detection on the basis of ensuring motion efficiency. The work improved movement efficiency and shortened time fragment. A linear relationship between the ratio value of two reporters and target DNA concentration was observed from 0.5 fM to 50 pM with a detection limit of 28 aM. The lateral motion mode of DNA machine coalescing with ratiometric system made this sensing platform ultrasensitive and accurate, which holds new avenue of early diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A novel electrochemiluminescence (ECL) and electrochemical dual-mode sensor was developed for detecting the activity of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and screening its inhibitor. Specifically, the adamantane (ADA)-functionalized peptide (P1), a designed substrate peptide for BACE1, was immobilized on the electrode surface via host-guest interaction between β-cyclodextrin (β-CD) and ADA. The aggregation of the peptide (P2) and luminol co-functionalized silver nanoparticles could be induced by cucurbit [8]uril (CB[8] due to the ability of CB[8] to accommodate two aromatic residues simultaneously. The obtained (CB[8]-P2-AgNPs-luminol) n aggregates with both ECL and electrochemical activity, used as the dual-mode signal probe, could be captured to the N-terminal of P1 through CB[8]. Once the substrate P1 was cleaved by BACE1, the probe-binding polypeptide fragment detached from the electrode surface, resulting in a remarkable decrease in the ECL and electrochemical signals. Taking advantage of the signal amplification function of the signal probe, the sensitive dual-mode assay for BACE1 activity can be achieved with the low detection limits of 33.11 pM for ECL and 53.19 pM for electrochemical mode. The superior analytical performance of this novel dual-mode sensor toward BACE1 activity suggested the promising application in early diagnosis of Alzheimer's disease (AD)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023 Elsevier B.V. All rights reserved.)

Rapid, simple, accurate and highly sensitive detection of enzymes is essential for early screening and clinical diagnosis of many diseases. In this study, we report the fabrication of a turn-on ratiometric electrochemical sensor for the in situ determination of β-Galactosidase (β-Gal) based on surface engineering and the design of a molecular probe (Pygal) specific for β-Gal recognition. First, Pygal probe was synthesized and characterized, and then co-assembled with the methylene blue (MB) internal reference probe on the surface of single-wall carbon nanotubes (SWCNT)-modified carbon fiber microelectrode (CFME). The resulting CFME/SWCNT/MB + Pygal sensor is activated in the presence of β-Gal giving one peak at 0.33 V originating from the oxidation of the product of Pygal enzymatic hydrolysis (PyOH). Another oxidation peak attributed to MB appears simultaneously at -0.28 V allowing the construction of a ratiometric electrochemical sensor for β-Gal detection with improved sensitivity and accuracy. The sensor showed a linear response to β-Gal in a wide concentration range from 1.5 to 30 U L -1 and a low detection limit of 0.1 U L -1 . Moreover, the sensor demonstrated excellent selectivity against several biologically relevant hydrolases and redox-active molecules. Finally, the combination of excellent electrochemical performance and favorable physicochemical properties of CFME allowed the determination of β-Gal in the whole blood of Parkinson's Disease (PD) model mice. The workflow reported in this study provides a strategy for the design and development of sensors for the in vivo monitoring of other enzymes important for the early diagnosis of different health issues., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Interferon-gamma (IFN-γ) is one kind of crucial inflammatory cytokines, and its expression level is closely associated with various disease progressions. This work addresses the development of a sensitive and specific electrochemical assay for detection of IFN-γ by combing the recognition unit of aptamer with the signal reporter of target-induced silver nanoclusters (AgNCs). For biosensor preparation, the gold nanoparticles (AuNPs) immobilized on the amine-terminated electrode surface provided electrochemical interfaces for the self-assembly of C-rich modified aptamers. Then, the aptamer recognized IFN-γ and the free aptamer hybridized with conjugated DNA sequences. After the nuclease-catalyzed cleavage of DNA duplex, in situ-generated AgNCs in the C-rich template was utilized as the electrochemical indicator for IFN-γ detection. The present method demonstrated a good performance for detection of IFN-γ with a low detection limit of 1.7 pg mL -1 . This aptasensor was verified to be applied for the evaluation of IFN-γ secreted by cell., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Small-molecule photosensitizers have great application prospects in photoelectrochemical (PEC) sensing due to their defined composition, diversified structure, and adjustable photophysical properties. Herein, we propose a new strategy for PEC analysis based on the target-induced in situ formation of the organic photosensitizer. Taking thiophenol (PhSH) as a model analyte, we designed and synthesized a 2,4-dinitrophenyl (DNP)-caged coumarin precursor (Dye-PhSH), which was then covalently coupled onto the TiO 2 nanoarray substrate to obtain the working photoanode. Due to the intramolecular photoinduced electron transfer process, Dye-PhSH has only a very weak photoelectric response. Upon reacting with the target, Dye-PhSH undergoes a tandem reaction of the detachment of the DNP moiety and the intramolecular cyclization process, which leads to a coumarin dye with a pronounced photoelectric effect, thus achieving a highly selective turn-on PEC response to PhSH. For the first time, this study was to construct a PEC sensor by exploiting specific organic reactions for the in situ generation of small molecule-based photoactive material. It can be anticipated that the proposed strategy will expand the paradigm of PEC sensing and holds great potential for detecting various other analytes.

Insulin, a peptide hormone secreted by pancreatic β cells, affects the development of diabetes and associated complications. Herein, we propose an electrochemical aptasensor for sensitive and selective detection of insulin using laser-scribed graphene electrodes (LSGEs). Before using disposable LSGEs, the development and proof-of-concept sensing experiments were firstly carried out on research-grade glassy carbon electrode (GCE). The aptasensor is based on using Exonuclease I (Exo I) that catalyses the hydrolysis of single-stranded aptamers attached to the electrode surface; however, the hydrolysis does not occur if the insulin is bound to the aptamer. Therefore, the unbound aptamers are cleaved by Exo I while insulin-bound aptamers remain on the electrode surface. In the next step, the gold nanoparticle - aptamer (AuNPs-Apt) probes are introduced to the electrode surface to form a 'sandwich' structure with the insulin on the surface-attached aptamer. The redox probe, methylene blue (MB), intercalates into the aptamers' guanine bases and the sandwich structure of AuNPs-Apt/insulin/surface-bound aptamer amplifies electrochemical signal from MBs. The signal can be well-correlated to the concentrations of insulin. A limit of detection of 22.7 fM was found for the LSGE-based sensors and 9.8 fM for GCE-based sensors used for comparison and initial sensor development. The results demonstrate successful fabrication of the single-use and sensitive LSGEs-based sensors for insulin detection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Oxidative stress plays an important role in the pathogenesis of many diseases, while the exact mechanism that hydrogen peroxide (H 2 O 2 ) as one of the most abundant reactive oxygen species (ROS) exerts its influence on oxidative stress remains unclear. We developed a novel turn-on ratiometric electrochemical sensor for the detection of H 2 O 2 in blood samples. The electrochemical probe 5-(1,2-dithiolan-3-yl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pent-anamide (BA) was designed and synthesized for the selective detection of H 2 O 2 via a one-step amide reaction. Meanwhile, Nile Blue A (NB) was optimized as an internal reference molecule, thus enabling accurate quantification of H 2 O 2 in a complex environment. BA and NB were then co-assembled onto a carbon fiber microelectrode (CFME) coated with Au cones. The oxidation peak current ratio between BA and NB demonstrated good linearity with the logarithm of the H 2 O 2 concentration values ranging from 0.5 μM to 400 μM with a low detection limit of 0.02 μM. The developed sensor showed remarkable selectivity against potential interferences in whole blood samples, especially for ascorbic acid, uric acid, and dopamine. In combination with the unique characteristics of CFME, such as a small size and good biocompatibility, the microsensor was used for rapid analysis of one drop of whole blood sample. This sensor not only creates a new platform for the detection of H 2 O 2 in whole blood samples, but also provides a new design strategy of other ROS analysis for early diagnosis of ROS-related diseases, drug discovery processes, and pathological mechanisms., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this paper, a Merocyanine-based turn-on probe (McyA) has been developed for colorimetric and fluorescent dual-mode detection of N 2 H 4 via an intra-molecular charge transfer (ICT) mechanism. In the presence of N 2 H 4 , the probe shows an obvious chromogenic response and fluorescent enhancement. Based on this feature, the synthesized McyA can be applied to quantify N 2 H 4 concentration from 0.12 to 5.0 μM and 0.5-10 μM with a detection limit of 0.042 μM (1.3 ppb, S/N = 3) and 0.140 μM (4.5 ppb, S/N = 3), respectively. Moreover, McyA has also be successfully employed for imaging analysis of N 2 H 4 distribution in different organs from the N 2 H 4 ingested mice model, revealing the potential application of McyA as a powerful fluorescent sensor for tracking detection and risk assessment of hydrazine in vivo., (Copyright © 2019 Elsevier B.V. All rights reserved.)

DNA 3'-phosphatase is an essential enzyme, which plays a pivotal role in repairing DNA damage. The peculiar activity of DNA 3'-phosphatase has been proved to associate with a variety of human pathologies. Therefore, sensitive determination of DNA 3'-phosphatase is necessary for clinical diagnosis and therapy. Here, we develop a simple, sensitive, and specific fluorescent biosensor including three DNA chains of hairpin DNA1, hairpin DNA2 and fluorescence probe DNA (FP) for detecting the activity of DNA 3'-phosphatase. First, biotin-modified hairpin DNA1 binds with streptavidin-modified magnetic beads (MB) to get MB-DNA1. DNA 3'-phosphatase can hydrolyze phosphate groups on MB-DNA1 to form hydroxyl groups, which leads to the polymerization extension and nicking endonuclease cleavage reaction to obtain the trigger DNA1 fragment (tDNA1). Next, two cyclic amplification reactions are designed. In cycle I, the tDNA1 hybridizes with the hairpin DNA2, which leads the hairpin structure of DNA2 opened and the fluorescence signal of 6-carboxy-fluorescein (FAM) labeled on hairpin DNA2 turned on. This cyclic reaction is amplified by exonuclease III (Exo III). At the same time, the trigger DNA2 fragment (tDNA2) is obtained. In cycle II, similarly, the tDNA2 hybridizes with FP. Thus, the fluorescence signal of FAM labeled on FP released, which multiplies with the fluorescence signal from cycle I. Finally, this strategy is applied to determine two typical DNA 3'-phosphatases including T4 polynucleotide kinase (T4 PNK) and alkaline phosphatase (ALP) with the detection limit (LOD) of 0.0033 and 0.00037 U/mL, respectively. The method provides a promising platform to evaluate the DNA 3'-phosphatase activity in the complicated biological samples and can be potentially applied in the relevant fields such as biomedical research, drug discovery and clinical diagnosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Split aptamer strategy was often used to improve the sensitivity of aptasensor. However, traditional split aptamer strategy can not be directly used to improve the label-free aptamer based Thioflavin T (ThT) displacement assay for ATP because the split ATP aptamer display much lower enhancement effects on the fluorescence of ThT than intact aptamer. In order to address this issue, this is the first report using G-rich DNA sequence to enhance the affinity of the two split ATP aptamer halves to ThT and offer lower limit of detection (LOD), wider linear range and higher selectivity through the enhanced molecular recognition. Compared to the intact aptamer/ThT complex, the ensemble of two G-rich split ATP aptamer fragments/ThT are higher fluorescent. Consequently, G-rich sequences would improve the fluorescent signal and thus the sensing performance of the proposed assay. In the optimized conditions, the LOD of the proposed fluorescent ATP aptasensor is 2 nM, which is lower than the reported ThT/ATP aptamer based methods. Additionally, our aptasensor has a wider dynamic linear range (0.1 μM - 120 μM) and higher selectivity. The proposed aptasensor has been successfully applied to detect ATP in 15% human serum. More importantly, the current study not only provides a novel method for ATP assay but also presents a way to construct a label-free split aptamer based fluorescent sensor for other species where aptamer can be generated., (Copyright © 2019 Elsevier B.V. All rights reserved.)