The accurate and timely determination of dipicolinic acid (DPA) is of great concern to prevent an anthrax epidemic. However, conventional methods suffer from poor detection efficiency and limited signal amplification. Herein, we report a highly efficient electrochemical biosensor for DPA analysis, which is based on two core conceptual and mechanistic breakthroughs: (i) a confined intramolecular DNA machine amplification paradigm that replaces the traditional intermolecular diffusion-driven design and (ii) the synergistic coupling of target-triggered intramolecular DNA machine activation with CeO2 catalytic redox-recycling amplification. The sensor designs a sandwich-structured complex (SSC) as the recognition component, and DNA circuit probes are assembled in the X-shaped probe (XSP) structure. The disassembly of the SSC triggered by the strong chelation function between DPA and Zr4+ results in the release of the trigger probe and the initiation of the intramolecular DNA machine on the XSP nanostructure. Nanoceria (CeO2) nanoparticles move close to the sensing electrode, catalyzing the conversion of p-aminophenylphosphate to p-aminophenol to amplify current responses during the potential sweep in the presence of the co-reactant nicotinamide adenine dinucleotide. The reported sensing strategy allows for effective determination of DPA with a limit of detection down to 7.4 pM within a reaction time of just 40 min. Significantly, the sensitivity of the intramolecular DNA machine surpassed that of the intermolecular DNA machine sensor by up to 3 orders of magnitude. Furthermore, the sensor has proven to effectively and consistently monitor bacterial spore samples, which demonstrates a wide applicability in hazardous pathogens on site analysis and biomedical research.

17β-Estradiol (E2) is crucial for the development of the genital system and sexual maturation in females, but it is still challenging to monitor E2 because of its low concentrations in bodily fluids. This study demonstrates an efficient electrochemical sensor based on a DNA-fueled molecule machine-mediated atom transfer radical polymerization amplification for the sensitive detection of E2 in clinical samples. The involved toehold-mediated strand displacement leads to the attachment of azido group-modified probes on the sensing electrode, with Cu(I)-catalyzed azide-alkyne cycloaddition to facilitate the introduction of electrochemically mediated atom transfer radical polymerization (eATRP) initiators onto the electrode. The following eATRP reaction further creates lengthy electroactive polymers (poly-FcMMA) to generate a significantly amplified current response for the quantitative determination of E2. By harnessing the considerably increased current, the sensor exhibits outstanding specificity over other interferents, a small detection limit of 35 fM, and a wide linear range from 100 fM to 80 nM. The effectiveness of the reported sensor to analyze complex samples is demonstrated to accurately detect E2 in clinical serum samples from different menstrual cycle phases and weeks of pregnancy, providing an effective prediction of pregnancy outcomes and the endocrine system.

It is crucial to monitor the level of uric acid (UA) in body fluids for the clinical diagnosis and treatment of related diseases. However, the complex composition of human body fluids often means that UA is present alongside substances that have similar chemical structures and redox potentials, making direct determination of UA in biological samples difficult. This study addresses this challenge by modifying the sensing electrode with poly(3-methylthiophene) (P3MT) and assembling gold nanocages (GNCs) to develop an efficient electrochemical sensor for the determination of UA. The inherent catalytic and conductive properties of P3MT provide a suitable substrate and microenvironment, enhancing the assembly efficiency of GNCs. The synergistic catalysis of P3MT and GNCs contributes to the efficient distinguishing of the electrochemical signals of UA against interfering signals. The electrooxidation reaction of UA is found to involve equal proton and electron transfer, and the UA concentration and current display an excellent linear relationship, ranging from 1 to 26 mu M under the coexistence of 20 mu M ascorbic acid. A detection limit of 0.3 mu M is achieved, enabling the sensitive detection of UA in serum samples from both healthy volunteers and gout patients. With its high accuracy, good selectivity, and sensitivity, the sensor is a valuable asset in clinical diagnosis and drug research related to purine metabolism disorders.

The human milk protein beta-2-microglobulin (B2M) plays a vital role as a diagnostic biomarker for infants, with conventional detection methods frequently relying on intricate instruments that have constraints in sensitivity and selectivity. Here, an innovative heteroaptamer recognition-mediated strand displacement amplification for electrochemical analysis of B2M in breast milk is described. Two heteroaptamers associating with distinct sites on B2M kickstart the toehold-mediated strand displacement on the magnetic bead, facilitating the reuse of heteroaptamer/B2M complexes. The molecular machine's operation induced the formation of numerous G-quadruplex structures on the sensing interface, which could bind with hemin and produce a significantly increased current for the detection of B2M, with a detection limit of 0.2 fg/mL. With its unique capabilities, the sensor can differentiate the target B2M from other non-target proteins, guaranteeing the specific detection of B2M in milk samples. This system could be modified to detect different targets by using the correct aptamer/ligand combinations.

Metastasis leads to poor breast cancer prognosis, urging early detection of metastatic biomarkers. We developed an innovative electrochemical biosensor that integrates a polygonal nucleic acid frame-accelerated DNA nanomachine and substrate-free electrocatalytic AuPd@CoMOF nanocomposites. The multi-site recognition probe (msRP) serves as the core dual-target recognition element, enabling the sequence-specific binding of miRNA-21 and miRNA-105. It is co-localized with other reaction probes on a triangular DNA nanostructure (TDN) scaffold, which induces a robust spatial confinement effect to accelerate DNA walker reaction kinetics and construct a localized, accelerated, higher-order signal amplification system. AuPd@CoMOF enhances electrode electron transfer efficiency and catalyzes thionine without exogenous substrates, eliminating substrate-induced analytical errors. Under optimal conditions, the sensor ultra-sensitively detected miRNA-21 (LOD 4.6 aM) and miRNA-105 (LOD 5.4 aM) in the range of 20 aM–500 pM, with excellent selectivity against non-target miRNAs. It distinguished breast cancer cells from other cancer cells and metastatic from non-metastatic breast cancer cells, and its clinical serum detection results were highly consistent with qRT-PCR, providing a novel strategy for high-efficiency DNA nanomachine design and a reliable tool for the early clinical diagnosis of breast cancer metastasis.

Reactive oxygen species (ROS) play a critical role in regulating various physiological processes. To gain a comprehensive understanding of their distinct functions in different physiological events, it is imperative to detect binary ROS simultaneously. However, the development of the sensing method capable of binary ROS detection remains a significant challenge. In this study, we address this challenge by integrating chemically modified DNAzyme probes with a functionalized metal-organic framework (MOF) to create an efficient electrochemical sensing platform for the binary detection of ROS. ROS targets would activate the DNAzyme cleavage activity by removing the phenylboronate (BO) and phosphorothioate (PS) modifications, leading to the controlled release of doxorubicin (DOX) and methylene blue (MB) enclosed within MOF nanocomposites. This process generates two distinct voltammetric current peaks, with their potentials and intensities reflecting the identity and concentration of the ROS targets. The sensor demonstrates simultaneous detection of multiple ROS (H2O2 and HClO) produced by cancer cells with high sensitivity across a broad linear range of 1 to 200 nM and a low detection limit in the sub-nanomolar range. The design strategies behind the developed ROS sensing system could also be exploited to create other biosensors with highly sensitive and binary detection to promote clinical research and revolutionize disease diagnostics.

Doxorubicin (DOX) is a frequently prescribed chemotherapy medication for a range of cancers. It is essential to monitor the levels of doxorubicin in human biological fluids to ensure effective treatment. An efficient electrochemical sensor for amplified detection of DOX is developed in this work. The strand displacement reaction is initiated when the blocking probe, which attaches to the terminal toehold sequence of the immobilization probe, is released due to the selective recognition of DOX with the aptamer probe. The secondary toehold region is then available for hybridization with the methylene blue (MB)-labeled signal probe, causing the blocking probe to be displaced and activating the molecular machine. Consequently, the blocking probe is reused in a cyclical manner, with numerous signal probes being attached to the sensor surface, resulting in a significant increase in current for quantitative analysis. This technique successfully detected DOX in human serum samples with high selectivity and sensitivity, reaching a detection limit of 670 fM. The proposed research offers a novel approach to identifying various biomarkers, with the potential to significantly advance clinical diagnosis and drug research.

Circulating tumor DNA (ctDNA) assay in blood is important for the diagnosis of cancer-related diseases. Because of its low abundance and high background of wild-type DNA, the development of reliable and sensitive strategies for ctDNA assay still faces enormous challenges. Herein, an innovative dual-mode ratiometric biosensor was constructed for ctDNA assay based on DNA triangle-corbelled target recycling coupled with resonance energy transfer (RET). DNA triangle as anchoring substrate enabled target recycling with high reaction efficiency, bringing in the sensitive and reliable detection of PIK3CA E545K ctDNA, a key biomarker of human breast carcinoma. Fluorescence (FL) reagent and electrochemiluminescence (ECL) energy acceptor labeled two probes competitively hybridized with capture DNA, resulting in the reverse change tendencies of FL and ECL signals. By measuring the ratio of FL/ECL, the analysis of ctDNA was readily acquired with a detection limit of 20 aM. Compared with some reported works, the designed sensor could realize the reliable detection of ctDNA in serum samples from breast cancer patients and healthy volunteers with advantages of high sensitivity, specificity, stability, and low cost. Therefore, this work offered an efficient dual-mode sensing strategy, which is promising for clinical and diagnostic application scenarios.

A conceptual innovative electrochemical sensing platform was constructed for microRNA (miRNA) detection based on terminal deoxynucleotidyl transferase (TdT)-mediated synthesis of copper nanoclusters. In principle, the substrate strand immobilized on working electrode acted as the template for the TdT-mediated DNA extension reaction, while the DNAzyme probe would be activated in the absence of miRNA 21, which led to the cleavage of the substrate strand to produce the 5’ phosphate terminus and inhibited the TdT-mediated DNA extension reaction. On the other hand, the presence of miRNA 21 initiated the toehold-mediated strand displacement reaction, causing the disassembly of the DNAzyme structure and preventing the substrate strand from being cleaved. This enabled the recycle amplification of target miRNA 21, and TdT catalyzed the DNA extension reaction on the 3’ hydroxyl terminus of the substrate strand, producing a large number of poly thymine (polyT) sequences. These sequences bound with copper ions for in-situ synthesis of copper nanoclusters on the sensing electrode, and the increased electrochemical signal of copper was recorded by differential pulse stripping voltammetry (DPSV), which paved the way for sensitive determination of miRNA 21 extracted from cancer cells with a detection limit of 36 aM. Due to its high sensitivity, user-friendly operation, and cost-effectiveness, this method is helpful for the fundamental research of sensing mechanism as well as the diagnosis of related diseases.

Conventional solid/liquid electrochemical interfaces typically encounter challenges with impeded mass transport for poor electrochemical quantification due to the intricate pathways of reactants from the bulk solution. To address this issue, this work reports an innovative approach integrating a target-activated DNA framework nanomachine with electrochemically driven metal-organic framework (MOF) conversion for self-sacrificial biosensing. The presence of the target biomarker serotonin initiates the DNA framework nanomachine by an entropy-driven circuit to form a cross-linked nanostructure and subsequently release the Fe-MOF probe. Acting as a natural metal precursor and a nanoconfined source of reactant, the Fe-MOF probe is converted into electroactive Prussian Blue during electrochemical processes. Taking advantage of the confinement effect, our proposed biosensor exhibits the excellent capability to detect serotonin in a linear range from 1 pM to 5 μM with a remarkable detection limit of 0.4 pM and exceptional specificity against other interferents. The proof-of-concept demonstration of serotonin detection in clinical serum samples from patients with carcinoid tumors highlights the utility of a complex sample analysis. The design could be applied for other biomarker detection with a high potential to inspire innovative sensing approaches, holding promise for applications in biomedical research and disease diagnosis.

A growing number of studies have shown the crucial role of microRNA (miRNA) sensing in cancer clinical diagnosis and prognosis research. In this study, a label-free and enzyme-free electrochemical sensor was presented to achieve sensitive determination of miRNA 122, which was constructed based on in situ synthesis of silver nanoclusters (AgNCs) mediated by cascaded recycling amplification. The efficient and cascaded recycling amplification was initiated by the target miRNA 122, resulting in the release of signal probe with rich cytosine bases, which would be loaded on the sensing interface for in situ synthesis of silver nanoclusters, leading to dramatically increased current for quantitative analysis. This method achieved selective and robust miRNA 122 sensing in human serum samples with a detection limit down to 27 aM. The proposed work would provide an innovative method for determining different biomarkers, holding great potential for boosting the development of clinical diagnosis and drug investigation.

The quantitative detection of antibodies is crucial for the diagnosis of infectious and autoimmune diseases, while the traditional methods experience high background signal noise and restricted signal gain. In this work, we have developed a highly efficient electrochemical biosensor by constructing a programmable DNA nanomachine integrated with electrochemically controlled atom transfer radical polymerization (eATRP). The sensor works by binding the target antidigoxin antibody (anti-Dig) to the epitope of the recognization probe, which then initiates the cascaded strand displacement reaction on a magnetic bead, leading to the capture of cupric oxide (CuO) nanoparticles through magnetic separation. After CuO was dissolved, the eATRP initiators were attached to the electrode based on the Cu-Iota-catalyzed azide-alkyne cycloaddition. The subsequent eATRP reaction results in the formation of long electroactive polymers (poly-FcMMA), producing an amplified current response for sensitive detection of anti-Dig. This method achieved a detection limit at clinically relevant picomolar concentration in human serum, offering a sensitive, convenient, and cost-effective tool for detecting various biomarkers in a wide range of applications.

Herein, a disposable electrochemiluminescence (ECL) biosensor sensitized with target-triggered multi-DNA release and multipedal DNA walker was designed for in situ quenching detection of methylated target DNA. In this work, zirconium(IV)-based metal organic frameworks encapsulated with Ru(bpy)32+ emitter (UiO-66-NH2@Ru) were attached onto indium tin oxide (ITO) electrode as sensing substrate. With the assistance of magnetic bead separation, one methylated DNA released three kinds of DNAs, achieving a one-to-multiple signal transduction and amplification. Then, these multi-DNA strands hybridized with swing arm immobilized on ITO surface as DNA walking strands. The further hybridization between multi-DNA strands and DNA tracks resulted in the formation of cleavage sites, which were digested by a restriction endonuclease, causing the cleavage of DNA tracks into two short fragments. Subsequently, the dissociated DNA walking strands explored adjacent DNA tracks, and the multipedal walking procedure was conducted. Relying on the in situ quenching of H2O2 for ECL emission of Ru(bpy)32+ system, this method achieved the quantification of methylated target DNA. Moreover, taking the advantages of inexpensiveness, high walking kinetics, great amplification efficiency, good reproducibility and practicability, this disposable biosensor held great potential for analyzing DNA methylation levels in resource-limited environments.

The dissociation of the walking strand from the track gives rise to decreased efficiency and long reaction time of DNA walkers. In this work, we constructed a DNA walker combining the introduction of a wedge segment with a bimetallic metal-organic framework (MOF) electrocatalyst to solve this problem. The target methylated DNA acted as a single-legged walker, and the immobilization probe assembled on the track contained a wedge segment that was complementary to the target methylated DNA persistently, inhibiting its dissociation from the track. The fuel strand modified with a bimetallic MOF would drive the target strand to conduct branch migration and move processively along the track. The stepwise movement of the target strand resulted in the loading of numerous bimetallic MOF catalysts to reduce H2O2 at the electrode interface, thereby a significantly increased current response would be obtained for the detection of methylated DNA. This DNA walker achieved a detection limit of 200 aM within 20 min and effectively distinguished DNA with different methylation statuses, which would pave a way for rapid and sensitive monitoring of DNA methylation.

An amplified DNA logic sensor was constructed for the identification of multiple biomarkers, in which the inputs of targets triggered the disassembly of a V-shaped probe (VSP) structure by a strand displacement reaction, leading to the synthesis of silver nanoclusters (AgNCs) for electrocatalytic reduction of H2O2. The sensing platform achieved sensitive detection of methylated DNA and microRNA 122 with detection limits down to 3.4 and 4.1 fM, respectively, and can be used for the assay of clinical serum samples from healthy volunteers and liver injury patients with satisfactory results. The DNA logic sensor exhibited the advantages of convenience, low cost, and versatility without the involvement of electroactive label modification, which is helpful for disease diagnosis as well as the fundamental investigation of interfacial electrochemistry and molecular biology.

A two-step resonance energy transfer (RET)-based fluorescence/electrochemiluminescence (FL/ECL) biosensor was developed for ratiometric measurement and annihilation of Staphylococcus aureus (S. aureus). Using coupled dual-recognition-triggered target conversion with the catalytic hairpin assembly (CHA) technique, the monitoring of S. aureus was obtained at the single-cell level.

Bimetallic core-shell nanostructures possess modifiable or tunable localized surface plasmon resonance (LSPR) characters through the alterations of element and configuration. Herein, we described the use of plasmon-tunable Ag@Au bimetallic core-shell nanostructures to improve the electrochemiluminescence (ECL) emission of quantum dots (QDs) for microRNA sensing. Varying the concentration of the initial Ag seed and Au source allowed the size of the Ag core and Au shell to be altered by stepwise galvanic replacement and chemical reduction. The absorption bands of the resultant Ag@Au core-shell nanostructures closely overlapped with the ECL emission spectra of QDs, resulting in an effective LSPR-enhanced ECL emission. Changing the distance between CdS QDs and Ag@Au nanostructures showed that the LSPR-enhanced ECL was distance-dependent. Furthermore, an innovative LSPR-enhanced ECL sensor incorporating a catalytic hairpin assembly-based signal amplification system enabled the sensitive analysis of microRNA-21 (miRNA-21). The linear response range and detection limit were obtained to be 50 aM-10 pM and 20 aM, respectively, and the practical bioassay applications of the sensor were confirmed using HeLa cells and human serums with acceptable results. This pioneering work provided insights into the role of plasmon-tunable Ag@Au bimetallic core-shell nanostructures in enhancing the sensitivity of ECL sensors.

Methylated DNA is a promising epigenetic biomarker for diseases diagnosis, therapy monitoring, and prognosis. Here, we developed a sensitive ratiometric electrochemical biosensor for the detection of methylated DNA based on the design of multistep DNA amplification circuits. The methylated target DNA firstly initiates the DNAzyme-assisted amplification circuit and produces a number of single-stranded triggers that catalyze the subsequent double-step catalytic hairpin assembly circuits to release numerous four-way DNA junctions. The four-way DNA junctions displace the methylene blue (MB)-modified signal probes for intercalating doxorubicin (DOX) molecules on the sensing interface, resulting in a significantly increased value of i(DOX)/i(MB) to achieve sensitive monitoring of methylated DNA. Taking advantage of the significant signal amplification of multistep DNA amplification circuits, the proposed method exhibited high sensitivity for target analysis with a detection limit of 4 aM and a dynamic linear range of 10 aM to 20 pM. Moreover, the proof-of-concept application of the sensing platform was investigated, suggesting that the system is promising for potential applications in early cancer diagnosis and the fundamental research of epigenetics.

The selective and sensitive monitoring of microRNAs (miRNAs) plays essential roles in cancer diagnosis. Herein, on the basis of DNA triple helix and toehold-mediated strand displacement reaction (TSDR), the construction of a simple electrochemical sensing system is reported for monitoring of let 7a in human serum. The triple helix complex probe (THCP) consisting of signal probe (MB-SP) labeled with methylene blue (MB) and capture probe (CP) is first assembled onto the sensing interface. The presence of target let 7a initiates the disassembly of THCP structure and conformational change of MB-SP. And the hairpin probe (HP) triggers the TSDR amplification and achieves the recycling of target let 7a, thus leading to massive MB-SP turning into molecular beacon structure, which causes the significantly amplified MB signal for monitoring of let 7a down to femtomolar level. In addition, taking advantage of TSDR signal amplification and the high selectivity of the THCP, the selective discrimination of let 7 miRNAs family members and circulating miRNAs detection in human serum are accomplished, which would supply a simple and sensitive monitoring approach for early clinical diagnostics.

Carbon dots (CDs)-based biosensors have attracted considerable interest in reliable and sensitive detection of microRNA (miRNA) because of their merits of ultra-small size, excellent biosafety and tunable emission, whereas complicated labeling procedure and expensive bioenzyme associated with current strategies significantly limit their practical application. Herein, we developed a label-free and enzyme-free fluorescence strategy based on strand displaced amplification (SDA) for highly sensitive detection of miRNA using sulfydryl-functionalized CDs (CDs-SH) as probe. CDs-SH displayed excellent response to G-quadruplex DNA against other DNAs based on based on the catalytic oxidation of -SH into -S-S- by hemin/G-quadruplex. Further, CDs-SH were employed to detect miRNA, using miRNA-21 as target model, which triggered the SDA reaction of P1 and P2 to generate hemin/G-quadruplex, subsequently making CDs-SH transform from dot to aggresome along with the quenched fluorescence. Therefore, label-free, enzyme-free, and highly sensitive analysis of miRNA-21 was readily acquired with a limit of detection at 0.03 pM. This proposed biosensor couples the advantages of CDs and label-free/ enzyme-free strategy, and thus has a significant potential to be used in early and accurate diagnosis of cancer.