Twice-walk strategy based on three-dimensional DNA walking machine driven by duplex-specific nuclease.

A twice-walk strategy based on a three-dimensional (3D) cleat-equipped DNA walking machine with a high signal amplification efficiency was investigated for ultrasensitive detection of miRNA. Impressively, addition of duplex-specific nuclease (DSN) just once drove the twice-walk strategy, making the strategy simpler. With the advantages of being simple, rapid and ultrasensitive, the biosensor offers potential for use in early clinical diagnosis.

A rapid one-step electrochemical method based on cleat-equipped molecular walking machine.

Various nucleic acid molecular machines have emerged in recent years. However, when the nucleic acid tracks are fully depleted, these walkers are highly susceptible to premature release or stalling in regions where the tracks are locally exhausted. In this work, a molecular walking machine with a cleat domain preventing dissociation from the track was explored for ultrasensitive detection of miRNA. It has been verified that the cleat design can enhance the signal amplification efficiency of molecular walking machines for electrochemical miRNA-141 detection. Notably, the single-step electrochemical biosensing platform utilizing the cleat-equipped molecular walking machine (CMWM) is exceptionally straightforward and rapid, concluding the reaction within 90 min and achieving a remarkable low detection limit of 0.26 fM. The proposed molecular walking machine with this specific cleat structure was utilized for the identification of miRNA-141 in cellular lysates, exhibiting remarkable selectivity and consistent reproducibility, showcasing its effective utility in bioanalysis. Therefore, the cleat walker developed in this study introduces an innovative method for constructing a miRNA electrochemical biosensing platform, offering new perspectives for its application in biomolecule detection and clinical disease diagnosis.

An ultrasensitive label-free photoelectrochemical aptasensor based on terminal deoxynucleotidyl transferase amplification and catalytic reaction of G-quadruplex/hemin.

An ultrasensitive photoelectrochemical (PEC) analysis method based on terminal deoxynucleotidyl transferase (TdT) amplification effect and G-quadruplex/hemin (G4/hemin) catalytic reaction assisted signal amplification was prepared for the determination of ampicillin. A novel Au NPs@SnIn4S8-graphene sensitized structure was used as photoactive material to attain an intense basic photocurrent. In the presence of the target, P1 obtained by strand displacement reaction was introduced into the electrode surface, and abundant G4/hemin was formed in the presence of TdT and hemin. Subsequently, G4/hemin with horseradish peroxidase-mimicking activity catalyzed the oxidation of 4-chloro-1-naphthol by H2O2 to form benzo-4-chlorohexadienone precipitation on the modified electrode surface, which severely hindered the electron transfer and effectively inhibited the photocurrent output. The detection range of the PEC aptasensor for ampicillin was 10 fM-30 nM, and the limit of detection was 4.97 fM. The aptasensor had good stability and high sensitivity, and possessed a promising application in biological analysis and environmental monitoring.

An ultrasensitive carcinoembryonic antigen electrochemical aptasensor based on 3D DNA nanoprobe and Exo III.

In this study, a highly ordered three dimensional (3D) DNA nanostructure was self-assembled by label-free DNA nanotweezers, which was used as recognized probe to interact with target. Once the target was recognized by the 3D DNA nanoprobe (3D DNT), DNA nanotweezers opened to release target analog (T1). This recognition process was proceeded in homogeneous solution, which can avoid complex electrode modification and improve reaction efficiency. Then these target analogs were captured by the signal DNA probes (E1) modified on the electrode. In the assistance of Exo III, E1 was digested and the T1 was released to participate in the next cycle to realize signal amplification. Finally, an ultrasensitive carcinoembryonic antigen (CEA) electrochemical biosensing with a detection limit of 4.88 fg mL-1 was developed.

Ratiometric fluorescence detection of ciprofloxacin using the terbium-based coordination polymers.

Herein, a facile self-assembly through adenosine monophosphate (AMP) and luminol with Tb3+ was employed to construct a dual-ligand coordinated AMP-Tb-luminol coordination polymers (CPs), which emitted the typical fluorescence of luminol. Based on the sensitization effect of ciprofloxacin (CIP) on the luminescence of Tb3+, a ratiometric sensor was fabricated using the fluorescence of luminol as an inert reference. The fluorescent intensity ratios of Tb3+ to that of luminol enhanced linearly with the CIP concentration in the range from 5 nM to 2.5 µM with a lower limit of detection of 2 nM. In addition, the proposed ratiometric fluorescent sensor exhibited high selectivity for CIP, which could also be used to detect CIP in human blood serum (HBS) with satisfactory results. To our knowledge, this is the first demonstration of using dual-ligand coordination lanthanide (Ln)-based CPs for ratio-metric CIP assay, and this straightforward strategy may open up a new platform for designing the ratio-metric sensors based on the Ln CPs.

Sensitive electrochemical detection of oxytetracycline based on target triggered CHA and poly adenine assisted probe immobilization.

Here, we developed a homogeneous electrochemical biosensor for the sensitive determination of antibiotic by the CHA reaction and the consecutive adenine mediated probe fixation. The binding of target to the target recognition sequences in the triple-helix DNA can release the trigger. It can initiate the catalytic hairpin assembly (CHA) to generate lots of mimic targets, which were labeled with electroactive substance ferrocene (Fc). Because the generated mimic target has consecutive sequence of adenines (PolyA), they can be self-assembled on the AuNPs modified electrode and finally realize electrochemical detection. Under optimal conditions, this developed biosensor achieved a satisfactory limit of detection of 0.089 nM (S/N = 3) and a linear range from 0.1 nM to 100 nM for sensitive detection of oxytetracycline with good specificity. The whole process is carried out in homogeneous solution, not only realizes signal amplification, but also avoids the complex modification process of electrode surface. Compared with some reported electrochemical sensors, the method is easier to operate and has good precision.

Signal-off photoelectrochemical aptasensor for kanamycin: Strand displacement reaction combines p-n competition.

A"signal-off" photoelectrochemical aptasensor based on p-n type semiconductor competitive quenching effect and strand displacement reaction was constructed for the determination of kanamycin. Au NPs@MgIn2S4-graphene composite was used as n-type photoactive semiconductor material. In the presence of the kanamycin, strand displacement reaction was triggered and the p-type CuInS2 quantum dots labeled aptamer was introduced on the Au NPs@MgIn2S4-graphene surface. The CuInS2 quantum dots can competitive consume the electron donors (AA) and light energy of the PEC system, thus quenched the anodic photocurrent of Au NPs@MgIn2S4-graphene. The photocurrent decreased with the increase of kanamycin concentration. The linear range of kanamycin was 1.0 pM-10 µM, and the detection limit was 1.7 pM. In addition, the method can be used for the determination of kanamycin in milk and honey.

Novel preparation method of bipedal DNA walker based on hybridization chain reaction for ultrasensitive DNA biosensing.

In this work, a novel strategy for preparation of bipedal DNA walker (BDW) based on hybridization chain reaction (HCR) with the assistance of Exonuclease III (Exo III) was proposed. Based on this strategy, an electrochemical biosensor was constructed to achieve sensitive detection of CYFRA 21-1 DNA. Firstly, target recognition and circulation were achieved through a one-step catalytic hairpin assembly (CHA) reaction. For further amplification, hybridization chain reaction (HCR) was employed to form duplex-stranded DNA (dsDNA) nanostructure in homogeneous solution. In particular, the elongated single strand of the hairpin DNA for HCR was designed as the Mg2+ DNAzyme sequence. With the assistance of Exo III, dsDNA nanostructure can be digested and transformed into large amounts of BDW. These BDW can cleave the signal probe driven by Mg2+, which was modified on the electrode surface and thus achieved "signal-off" detection of target. This BDW preparation method based on HCR with the digestion of Exo III converted one target input into large amount of BDW. Coupled with the walking cleavage of BDW, a series of cascade amplification endowed high sensitivity with this biosensor and realized ultrasensitive detection of target DNA with the detection limit as low as 3.01 aM.

Enhanced oxidase-mimicking activity of Ce4+ by complexing with nucleotides and its tunable activity for colorimetric detection of Fe2.

Complexing with adenosine-5'-monophosphate (AMP) was proven to be a facile way to enhance the oxidase-mimicking activity of Ce4+, and enabled nanoenzyme recovery and reuse. Additionally, the oxidase-mimicking activity of AMP-Ce4+ infinite coordination polymers (ICPs) could be specifically inhibited by Fe2+. Based on this finding, we developed a simple and highly selective colorimetric assay to detect Fe2+.

Novel electrochemical biosensor based on Exo III-assisted digestion of dsDNA polymer from hybridization chain reaction in homogeneous solution for CYFRA 21-1 DNA assay.

A novel electrochemical biosensing strategy was proposed to detect cytokeratin fragment antigen 21-1 (CYFRA 21-1) DNA based on Exo III-assisted digestion of dsDNA polymer (EADDP) from hybridization chain reaction (HCR). Primarily, the presence of target can drive a catalytic hairpin assembly (CHA) reaction, which was aimed to achieve target recognition and circulation. Then the HCR can be triggered for further signal amplification and generate long dsDNA polymer with signal tags. Subsequently, the introduction of Exo III can digest the long dsDNA polymer to produce large amounts of double signal fragments (DSFs). The above experiments were all carried out in homogeneous solution. Finally, the released DSF can be captured onto the electrode directly by capture probe (CP) and a highly amplified electrochemical signal can be detected. The EADDP in homogeneous solution circumvented complex solid-liquid interface reaction and tedious operation steps on electrode. Besides, one target can be converted into abundant DSFs, which greatly improved the sensitivity. This biosensor exhibited a low detection limit (0.0348 fM) and wide linear range (5 fM âˆ¼ 50 nM) for CYFRA 21-1 DNA biosensing with reliable specificity and stability.

Raloxifene, identified as a novel LSD1 inhibitor, suppresses the migration of renal cell carcinoma.

Aim: As an important epigenetic modulator, histone lysine-specific demethylase 1 (LSD1) has been proved to be associated with the progression of renal cell carcinoma (RCC). Discovering novel LSD1 inhibitors offers therapeutic potential for RCC treatment. Methods & Results: We identified raloxifene as a novel LSD1 inhibitor (IC50 = 2.08 µM) through small compound library screening. Molecular docking indicated raloxifene might bind LSD1 in the flavin adenine dinucleotide (FAD) binding cavity in a reversible manner. Cell viability and migration assays showed raloxifene could suppress the proliferation and migration of RCC cells bearing overexpressed LSD1. Conclusion: Our findings indicated that LSD1 might be a promising therapeutic target for RCC and that raloxifene could serve as a lead compound for further anti-RCC metastasis drug discovery.

Ratiometric fluorescence sensing of glutathione by using the oxidase-mimicking activity of MnO2 nanosheet.

In this work, a facile ratiometric fluorescence sensor for GSH measurement was designed based on MnO2 nanosheet (NS), carbon dots (CDs), as well as a simple substrate o-phenylenediamine (OPD). Herein, MnO2 NS played triple essential roles in the sensing system. First, it could be reduced by GSH through a special reaction, and therefore served as GSH recognizer. Second, it played as a fluorescence nanoquencher to strongly quench the fluorescence of CDs. Third, it could directly oxidize OPD to yield a luminescent product 2, 3-diaminophenazine (DAP) via the intrinsic oxidase-like activity. It revealed that MnO2 NS could be reduced to Mn2+ in the presence of GSH. Thus its oxidase-like activity and fluorescence quenching abilities were inhibited, which then restricted the generation of DAP and recovered the fluorescence of CDs. Based on this phenomenon, a novel ratiometric fluorescence sensor for GSH determination was fabricated by measuring the ratio of fluorescent intensity of DAP to that of CDs. Besides, the constructed ratiometric fluorescent sensor, which could be facilely operated with single-wavelength excitation, exhibited high sensitivity and selectivity with a wider linear range and a lower detection limit.

Dual-Response Ratiometric Electrochemical Microsensor for Effective Simultaneous Monitoring of Hypochlorous Acid and Ascorbic Acid in Human Body Fluids.

Redox homeostasis between hypochlorous acid (HClO/ClO-) and ascorbic acid (AA) significantly impacts many physiological and pathological processes. Herein, we report a new electrochemical sensor for the simultaneous determination of HClO and AA in body fluids. We first coated a carbon fiber microelectrode (CFME) with a three-dimensional nanocomposite consisting of graphene oxide (GO) and carbon nanotubes (CNTs) to fabricate the CFME/GO-CNT electrode. After the electrochemical reduction of GO (ERGO), we integrated a latent 1-(3,7-bis(dimethylamino)-10H-phenothiazin-10-yl)-2-methylpropan-1-one (MBS) electrochemical molecular recognition probe to monitor HClO and employed anthraquinone (AQ) as an internal reference. The compact CFME/ERGO-CNT/AQ + MBS sensor enabled the accurate and simultaneous measurement of HClO and AA with excellent selectivity and sensitivity. Measurements were highly reproducible, and the sensor was stable and exceptionally biocompatible. We successfully detected changes in the redox cycles of HClO and AA in human body fluids. This sensor is a significant advance for the investigation of reactions involved in cellular redox regulation. More importantly, we have devised a strategy for the design and construction of ratiometric electrochemical biosensors for the simultaneous determination of various bioactive species.

Grafting homogenous electrochemical biosensing strategy based on reverse proximity ligation and Exo III assisted target circulation for multiplexed communicable disease DNA assay.

Rapid and effective diagnosis of communicable disease is one of the critical issues of the modern society, especially for detecting different targets at the same time. In this work, a grafting homogenous electrochemical biosensing strategy is proposed by integrating of reverse proximity ligation and exonuclease III (Exo III) assisted target circulation to analyze hepatitis B (HBV) and human immunodeficiency (HIV). Specially, a two-wing nanodevice (TWD) with two detection paths is elaborately designed based on analogous proximity ligation assay. The reverse proximity ligation process provides a new way of signal conversion and amplification, what accomplished by demolishing the TWD in the presence of targets. Meanwhile, a vast number of signal probes are released via Exo III assisted target circulation. Then the signal probes are grafted on the universal sensing interface, which is decorated with graftable tetrahedron DNA (GTD). These lead to a highly amplified electrochemical signal. Compared with the conventional strategies, the grafting homogenous electrochemical biosensing strategy not only achieves convenient sensitive detection of multiple communicable diseases DNA simultaneously, but also performs well in the detection of sole target. This strategy effectively decreases the background, homogenizes the distribution of probes, and avoids the complex and time-consuming modification process of the working electrode, which holds great potential application in early diagnosis for communicable disease in the future.

"Turn-on" ratiometric electrochemical detection of H2O2 in one drop of whole blood sample via a novel microelectrode sensor.

Oxidative stress plays an important role in the pathogenesis of many diseases, while the exact mechanism that hydrogen peroxide (H2O2) 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 H2O2 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 H2O2 via a one-step amide reaction. Meanwhile, Nile Blue A (NB) was optimized as an internal reference molecule, thus enabling accurate quantification of H2O2 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 H2O2 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 H2O2 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.

Photoelectrochemical aptasensor for thrombin based on Au-rGO-CuS as signal amplification elements.

A photoelectrochemical platform for thrombin determination was developed based on Au-rGO-CuS as multiple signal amplification elements. CuInS2 QDs was used to sensitize burr-shape TiO2 (b-TiO2) to obtain a strong photocurrent. Under the specific recognition between aptamer and thrombin, a sandwichlike structure was formed and the Au-rGO-CuS-labeled aptamer (S2@Au-rGO-CuS) was immobilized on the electrode surface. This induced a sharp decrease in photocurrent. The phenomenon is mainly due to the fact that CuS NPs can competitively consume the light energy and electron donor with CuInS2/b-TiO2. The rGO can increase the amount of CuS NPs and the Au NPs can accelerate charge transferring which depress the recombination of photogenerated electrons and holes in CuS to further enhance the competitive capacity of CuS. The sandwichlike structure has a steric hindrance effect. Therefore, the S2@Au-rGO-CuS has a multiple signal amplification function for thrombin determination. Under optimal conditions, the PEC aptasensor exhibited a wide linear concentration range from 0.1 pM to 10 nM with a low detection limit of 30 fM (S/N = 3) for thrombin. Besides, the designed aptasensor performed well in the assay of human serum sample, indicating good potential for the determination of thrombin in real samples. Graphical abstract.

A new voltammetric sensor and its application in pharmaceutical analysis for rutin.

A new and sensitive electrochemical sensor for rutin determination was developed based on cetyltrimethylammonium chloride (CTAC) functionalized graphene (Gr) and palladium nanoparticles (Pd) (CTAC-Gr-PdNPs) composite. Rutin displayed remarkably increased electrochemical activity on the CTAC-Gr-PdNPs composite modified electrode due to the synergistic effect of the large surface area and electrocatalytic activity of both Gr and Pd nanoparticles, which offers the feasibility for highly sensitive determination of rutin via electrochemistry. Under the optimal experimental conditions, the oxidation peak current of rutin was proportional to its concentration in the range of 2.0 × 10-8-1.0 × 10-6 mol L-1, and the limit of detection (LOD) was 5 nM (S/N = 3). The developed method was successfully applied to determine rutin in pharmaceuticals with satisfactory recoveries, which shows that the fabricated sensor has potential in pharmaceutical analysis.

A highly sensitive and adjustable colorimetric assay of hydrogen sulfide by signal amplification based on G-quadruplex-Cu2+ peroxidase mimetics.

This work reports the first example of a colorimetric H2S sensor constructed through G-quadruplex-Cu2+ (G4-Cu2+) peroxidase mimetics employing Cu2+ ions and G-rich DNA with signal amplification. In the hydrogen peroxide (H2O2)-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), the catalytic capacity of Cu2+ can be greatly improved in the presence of 22AG DNA, where 22AG DNA acts as a signal amplifier. However, G4-Cu2+ peroxidase mimetics lose their catalytic abilities after reacting with H2S. This is employed to develop a colorimetric assay of H2S without complex synthesis and instrumentation, with a linear range from 0.01 µM to 150 µM and a detection limit of 7.5 nM. The sensitivity of the sensor can also be adjusted by changing the concentration of Cu2+. Moreover, the developed sensor is successfully applied for the quantitative determination of H2S in human serum samples.

Molybdenum sulfide-based electrochemical platform for high sensitive detection of taxifolin in Chinese medicine.

MoS2 and nitrogen doped active carbon composite (MoS2/ANC) is fabricated to detect taxifolin and exhibits superior redox current response and decreased redox potential difference. Further investigation reveals that the kinetic process of the redox reaction of taxifolin on MoS2/ANC electrode is controlled by both adsorption and diffusion process. Under the optimum conditions, the redox peak currents linearly relate with the concentration of taxifolin in the range of 1 × 10-9-1 × 10-6 mol L-1, accompanied by the low detection limit (3 × 10-10 mol L-1). Meanwhile, outstanding selectivity, stability and repeatability are also obtained at MoS2/ANC electrode. At last, the proposed method is applied to quantitatively detect taxifolin in fructus polygoni orientalis and satisfactory results have been achieved.

A "signal-on" electrochemical biosensor based on DNAzyme-driven bipedal DNA walkers and TdT-mediated cascade signal amplification strategy.

In this work, a dual amplified signal enhancement approach based on coupling deoxyribozyme (DNAzyme)-driven bipedal DNA walkers (BDW) and terminal deoxynucleotidyl transferase (TdT)-mediated DNA elongation signal amplifications has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. In presence of thrombin, the BDW complex, which is comprised from the target thrombin and two DNAzyme-containing probes, can exhibit autonomous cleavage behavior on the surface of the substrate DNA (SD) modified electrode, and remove the cleaved DNA fragment from the electrode surface. Subsequently, the TdT can catalyze the elongation of the SD with free 3'-OH termini and formation of many G-quadruplex sequence replicates with the presence of 2'-deoxyaguanosine-5'-triphosphate (dGTP) and adenosine 5'-triphosphate (dATP) at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a "signal-on" and completely label-free fashion. Under optimized conditions, the response peak current was linear with the concentration of thrombin in the range from 0.5 pM to 100000 pM with detection limit of 0.31 pM. This research provides us a sustainable idea for the hyphenated multiple amplification strategies and a stable and effective method for the detection of protein biomarkers.