Programmable DNA Nanomachine Integrated with Electrochemically Controlled Atom Transfer Radical Polymerization for Antibody Detection at Picomolar Level.

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 CuΙ-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.

Wedged DNA Walker Coupled with a Bimetallic Metal-Organic Framework Electrocatalyst for Rapid and Sensitive Monitoring of DNA Methylation.

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.

Aptamer-Functionalized and Gold Nanoparticle Array-Decorated Magnetic Graphene Nanosheets Enable Multiplexed and Sensitive Electrochemical Detection of Rare Circulating Tumor Cells in Whole Blood.

The identification and monitoring of circulating tumor cells (CTCs) in human blood plays a pivotal role in the convenient diagnosis of different cancers. However, it remains a major challenge to monitor these CTCs because of their extremely low abundance in human blood. Here, we describe the synthesis of a new aptamer-functionalized and gold nanoparticle (AuNP) array-decorated magnetic graphene nanosheet recognition probe to capture and isolate rare CTCs from human whole blood. In addition, by employing the aptamer/electroactive species-loaded AuNP signal amplification probes, multiplexed electrochemical detection of these low levels of CTCs can be realized. The incubation of the probes with the sample solutions containing the target CTCs can lead to the efficient separation of the CTCs and result in the generation of two distinct voltammetric peaks on a screen-printed carbon electrode, whose potentials and current intensities, respectively, reflect the identity and number of CTCs for the multiplexed detection of the Ramos and CCRF-CEM cells with detection limits down to 4 and 3 cells mL-1. With the successful demonstration of the concept, further extension of the developed sensing strategy for the determination of various CTCs in human whole blood for the screening of different cancers can be envisioned in the near future.

DNA-Templated In Situ Synthesis of Highly Dispersed AuNPs on Nitrogen-Doped Graphene for Real-Time Electrochemical Monitoring of Nitric Oxide Released from Live Cancer Cells.

Dispersion promotion of nanomaterials can significantly enhance their catalytic activities. With a new DNA-templated in situ synthesis approach, we report the preparation of highly dispersed AuNPs on nitrogen-doped graphene sheets (NGS) with significantly improved electrocatalytic ability for the monitoring of nitric oxide (NO) released from live cancer cells. The template DNA is adsorbed on NGS via π-π stacking, and the Au precursor chelates along the DNA lattice through dative bonding. Subsequent introduction of the reducing agent leads to in situ nucleation and growth of AuNPs, eventually resulting in highly dispersed AuNPs on NGS. Because of the synergistic enhancement of the catalytic activities of AuNPs and NGS, as well as the high dispersion of AuNPs, such a nanocomposite shows significant electro-oxidation capability toward NO, leading to a highly sensitive subnanomolar detection limit for NO in vitro. More importantly, the laminin glycoproteins can be readily adsorbed on the surface of the nanomaterials to render excellent biocompatibility for the adhesion and proliferation of live cells, enabling the biointerface for electrochemical detection of NO released from live cancer cells.

Trimetallic Hybrid Nanoflower-Decorated MoS2 Nanosheet Sensor for Direct in Situ Monitoring of H2O2 Secreted from Live Cancer Cells.

In situ monitoring of hydrogen peroxide (H2O2) secreted from live cells plays a critical role in elucidating many cellular signaling pathways, and it is a significant challenge to selectively detect these low levels of endogenous H2O2. To address this challenge, we report the establishment of a trimetallic hybrid nanoflower-decorated MoS2 nanosheet-modified sensor for in situ monitoring of H2O2 secreted from live MCF-7 cancer cells. The Au-Pd-Pt nanoflower-dispersed MoS2 nanosheets are synthesized by a simple wet-chemistry method, and the resulting nanosheet composites exhibit significantly enhanced catalytic activity toward electrochemical reduction of H2O2, due to the synergistic effect of the highly dispersed trimetallic hybrid nanoflowers and the MoS2 nanosheets, thereby resulting in ultrasensitive detection of H2O2 with a subnanomolar level detection limit in vitro. Also the immobilization of the laminin glycoproteins on the surface of the nanocomposites increases its biocompatibility for cell adhesion and growth, which enables in situ electrochemical monitoring of H2O2 directly secreted from live cells for potential application of such sensor in cellular biology, clinical diagnosis, and pathophysiology.

Aptamer/Protein Proximity Binding-Triggered Molecular Machine for Amplified Electrochemical Sensing of Thrombin.

The development of convenient and sensitive methods without involving any enzymes or complex nanomaterials for the monitoring of proteins is of great significance in disease diagnostics. In this work, we describe the validation of a new aptamer/protein proximity binding-triggered molecular machinery amplification strategy for sensitive electrochemical assay of thrombin in complex serum samples. The sensing interface is prepared by self-assembly of three-stranded DNA complexes on the gold electrode. The association of two distinct functional aptamers with different sites of thrombin triggers proximity binding-induced displacement of one of the short single-stranded DNAs (ssDNAs) from the surface-immobilized three-stranded DNA complexes, exposing a prelocked toehold domain to hybridize with a methylene blue (MB)-tagged fuel ssDNA strand (MB-DNA). Subsequent toehold-mediated strand displacement by the MB-DNA leads to the release and recycling of the aptamer/protein complexes and the function of the molecular machine. Eventually, a large number of MB-DNA strands are captured by the sensor surface, generating drastically amplified electrochemical responses from the MB tags for sensitive detection of thrombin. Our signal amplified sensor is completely enzyme-free and shows a dynamic range from 5 pM to 1 nM with a detection limit of 1.7 pM. Such sensor also has a high specificity for thrombin assay in serum samples. By changing the affinity probe pairs, the developed sensor can be readily expanded as a more general platform for sensitive detection of different types of proteins.

Proximity Binding and Metal Ion-Dependent DNAzyme Cyclic Amplification-Integrated Aptasensor for Label-Free and Sensitive Electrochemical Detection of Thrombin.

Thrombin plays important roles for the diagnosis of neurodegenerative and cardiovascular diseases. By integrating proximity binding-induced strand displacement and metal ion-dependent DNAzyme recycling amplification, we demonstrate here the development of a simple and sensitive strategy for the detection of thrombin in human serums. The binding of the two distinct aptamers to the thrombin targets increases the local concentration of the aptamers and facilitates the release of the enzymatic sequences through proximity binding-induced strand displacement. The liberated enzymatic sequences further hybridize with the G-quadruplex containing and hairpin-structured substrate sequences on the sensor electrode to form the metal-ion dependent DNAzymes. Subsequently, the metal ions catalyze the cleavage of the substrate sequences to unlock the G-quadruplex forming sequences and to release the enzymatic sequences to trigger another cleavage cycle. Such metal ion-dependent DNAzyme recycling amplification leads to the formation of many active G-quadruplex forming sequences, which associate with hemin to form G-quadruplex/hemin complexes on the electrode surface. Direct electron transfer of hemin to the electrode during the potential scan can thus generate significantly amplified current for sensitive detection of thrombin at the low picomolar level. The work demonstrated here can thus offer new opportunities for the development of convenient signal amplification strategies for detecting various protein targets.

DNA-fueled molecular machine enables enzyme-free target recycling amplification for electronic detection of microRNA from cancer cells with highly minimized background noise.

The variations in microRNA (miRNA) expression levels can be useful biomarkers for the diagnosis of different cancers. In this work, on the basis of a new miRNA-triggered molecular machine for enzyme-free target recycling signal amplification, the development of a simple electronic sensor for highly sensitive detection of miRNA-21 from human breast cancer cells is described. The three-stand DNA duplex probes are self-assembled on the gold electrode surface to fabricate the sensor. The miRNA-21 target binds to the terminal toehold region of the probes, displaces one of the short strands through toehold-mediated strand displacement reactions, and exposes the secondary toehold region for subsequent hybridization with the methylene blue (MB)-modified DNA fuel strand, which further displaces both the miRNA-21 target and the other short strand to activate the operation of the molecular machine. As a result, the miRNA-21 target is cyclically reused, and many MB-DNA fuel strands are attached to the sensor surface, leading to a significantly amplified current response for sensitive detection of miRNA-21 down to 1.4 fM. The developed sensor also shows high sequence discrimination capability and can be used to monitor miRNA-21 expression levels in cancer cells. Moreover, this sensor avoids the involvement of any enzymes for target recycling amplification and features with highly minimized background noise for miRNA detection, which makes this method hold great potential for convenient monitoring of different miRNA biomarkers for early diagnosis of various cancers.

Target-induced reconfiguration of DNA probes for recycling amplification and signal-on electrochemical detection of hereditary tyrosinemia type I gene.

By coupling target DNA-induced reconfiguration of the dsDNA probes with enzyme-assisted target recycling amplification, we describe the development of a signal-on electrochemical sensing approach for sensitive detection of hereditary tyrosinemia type I gene. The dsDNA probes are self-assembled on the sensing electrode, and the addition of the target DNA reconfigures and switches the dsDNA probes into active substrates for exonuclease III, which catalytically digests the probes and leads to cyclic reuse of the target DNA. The target DNA recycling and the removal of one of the ssDNA from the dsDNA probes by exonuclease III result in the formation of many hairpin structures on the sensor surface, which brings the electroactive methylene blue labels into proximity with the electrode and produces a significantly amplified current response for sensitive detection of the target gene down to 0.24 pM. This method is also selective to discriminate single-base mismatch and can be employed to detect the target gene in human serum samples. With the demonstration for the detection of the target gene, we expect the developed method to be a universal sensitive sensing platform for the detection of different nucleic acid sequences.

Multiplexed and amplified electronic sensor for the detection of microRNAs from cancer cells.

The detection of microRNA expression profiles plays an important role in early diagnosis of different cancers. On the basis of the employment of redox labels with distinct potential positions and duplex specific nuclease (DSN)-assisted target recycling signal amplifications, we have developed a multiplexed and convenient electronic sensor for highly sensitive detection of microRNA (miRNA)-141 and miRNA-21. The sensor is constructed by self-assembly of thiol-modified, redox species-labeled hairpin probes on the gold sensing electrode. The hybridizations between the target miRNAs and the surface-immobilized probes lead to the formation of RNA/DNA duplexes, and DSN subsequently cleaves the redox-labeled hairpin probes of the RNA/DNA duplexes to recycle the target miRNAs and to generate significantly amplified current suppression at different potentials for multiplexed detection of miRNA-141 and miRNA-21 down to 4.2 and 3.0 fM, respectively. The sensor is also highly selective toward the target miRNAs and can be employed to monitor miRNAs from human prostate carcinoma (22Rv1) and breast cancer (MCF-7) cell lysates simultaneously. The sensor reported here thus holds great potential for the development of multiplexed, sensitive, selective, and simple sensing platforms for simultaneous detection of a variety of miRNA biomarkers for clinic applications with careful selection of the labels.

Two-step resonance-energy-transfer-based ratiometric biosensor for sensing and annihilation of Staphylococcus aureus.

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.

An amplified logic gate driven by in situ synthesis of silver nanoclusters for identification of biomarkers.

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.

Cross-triggered and cascaded recycling amplification system for electrochemical detection of circulating microRNA in human serum.

A cross-triggered and cascaded recycling amplification system was developed for electrochemical sensing of microRNA 122 based on the DNAzyme/multicomponent nucleic acid enzyme cleavage technique and a dumbbell-shaped probe. The linear range and detection limit were obtained to be 1 fM-100 pM and 0.34 fM, respectively. Compared with some reported studies, the proposed system can achieve the selective detection of endogenous miRNA in liver injury patients and healthy human serums with the advantages of high sensitivity, low cost, and easy manipulation, which are significant for disease diagnosis as well as the fundamental research of molecular biology.

Label-free and enzyme-free fluorescence detection of microRNA based on sulfydryl-functionalized carbon dots via target-initiated hemin/G-quadruplex-catalyzed oxidation.

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.

Construction of an Electrochemical Biosensing Platform Based on Hierarchical Mesoporous NiO@N-Doped C Microspheres Coupled with Catalytic Hairpin Assembly.

A critical challenge for improving the detection performance of sensors is building a favorable sensing interface. Herein, an innovative electrochemical biosensing system relying on hierarchical mesoporous NiO@N-doped C microspheres coupled with catalytic hairpin assembly was developed for DNA analysis. In this strategy, the utilization of NiO@N-doped C microspheres and multiwalled carbon nanotubes as electrode materials effectively enhanced the interfacial electron transfer and improved the surface active sites for subsequent reactions. By designing a target-assisted catalytic hairpin assembly, single target DNA could initiate the introduction of multiple signal probes labeled with ferrocene (Fc) onto a working electrode surface. Because the change in the Fc signal is dependent on the amount of target DNA, the resulting electrochemical sensing platform is highly sensitive. Under optimized reaction conditions, this testing platform has a wide linear range for target DNA detection from 100 aM to 100 pM with a detection limit of 45 aM. Furthermore, the platform displayed excellent selectivity, acceptable reproducibility, and long-term stability, highlighting the application potential of this sensing system.

Electrochemical screening of single nucleotide polymorphisms with significantly enhanced discrimination factor by an amplified ratiometric sensor.

The detection of single nucleotide polymorphisms (SNPs) is of great clinical significance to the diagnosis of various genetic diseases and cancers. In this work, the development of an ultrasensitive ratiometric electrochemical sensor for screening SNP with a significantly enhanced discrimination factor is reported. The ferrocene (Fc) and methylene blue (MB) dual-tagged triple helix complex (THC) probes are self-assembled on the gold electrode to construct the sensing interface. The addition of the mutant p53 gene causes the disassembly of the THC probes with the release of the Fc-tagged sequence and the folding of the MB-labeled sequence into a hairpin structure, causing the change in the current response ratio of MB to Fc for monitoring the mutant p53 gene. Such ratio is dramatically enhanced by the toehold-mediated displacement reaction-assisted target recycling amplification with the presence of an assistance hairpin sequence. With the significant signal amplification and the advantageous specificity of the THC probes, sub-femtomolar detection limit and a highly enhanced SNP discrimination factor for the mutant p53 gene can be obtained. Besides, the proof-of-demonstration application of the sensor for diluted real samples has been verified, offering such sensor new opportunities for monitoring various genetic related diseases.

Target-triggered catalytic hairpin assembly and TdT-catalyzed DNA polymerization for amplified electronic detection of thrombin in human serums.

Specific and sensitive detection of protein biomarkers is of great importance in biomedical and bioanalytical applications. In this work, a dual amplified signal enhancement approach based on the integration of catalytic hairpin assembly (CHA) and terminal deoxynucleotidyl transferase (TdT)-mediated in situ DNA polymerization has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. The presence of the target thrombin leads to the unfolding and capture of a significant number of hairpin signal probes with free 3'-OH termini on the sensor electrode. Subsequently, TdT can catalyze the elongation of the signal probes and formation of many G-quadruplex sequence replicates with the presence of dGTP and 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 completely label-free fashion. The sensor shows a linear range of 0.5pM-10.0nM and a detection limit of 0.12pM for thrombin. Moreover, the developed sensor can selectively discriminate the target thrombin against other non-target proteins and can be employed to monitor thrombin in human serum samples.

Cross-triggered and cascaded recycling amplification for ultrasensitive electrochemical sensing of the mutant human p53 gene.

Based on an endonuclease-assisted, cross-triggered and cascaded recycling amplification strategy, the construction of a simple electrochemical sensing platform for the ultrasensitive detection of the mutant p53 gene in human serum is described. Using this new signal amplification approach, the sub-femtomolar level of the mutant p53 gene can be selectively detected.

DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples.

Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells.

The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells.