•A motor-based microprobe powered by bio-assembled catalase is designed.•The microprobe is fabricated based on PEDOT/Au microtube along with DNA assembling.•Motion signal of microprobe can be regulated by the target DNA directly.•The microprobe can detect DNA without any washing and separation step.•The motion-based design can be extended to detect other analyte.A motor-based microprobe is proposed using a tubular microengine powered by bio-assembled enzyme as catalyst and exploited for washing-free detection of DNA through motion readout. The microprobe is fabricated by assembling a catalase layer on the inner surface of poly(3,4-ethylenedioxythiophene)/Au (PEDOT/Au) microtube through DNA conjugate, which is responsible for the biocatalytic bubble propulsion. The sensing concept of the microprobe relies on the target-induced release of catalase through the DNA strand-replacement hybridization, which decreases the amount of enzyme assembled on microtube to slow down the movement of the microprobe. Therefore, the motion speed is negatively correlated with the target concentration. At the optimal conditions, the microprobe can conveniently distinguish the concentration of specific DNA in a range of 0.5–10 µM without any washing and separation step. This microprobe can be prepared in batch with good reproducibility and stability, and its motion speed can be conveniently visualized by optical microscope. The proposed motor-based microprobe and its dynamic sensing method provide a novel platform for the development of intelligent microprobe and clinical diagnostic strategy.

An efficient enzyme-powered micromotor device was fabricated by assembling multiple layers of catalase on the inner surface of a poly(3,4-ethylenedioxythiophene and sodium 4-styrenesulfonate)/Au microtube (PEDOT-PSS/Au). The catalase assembly was achieved by programmed DNA hybridization, which was performed by immobilizing a designed sandwich DNA structure as the sensing unit on the PEDOT-PSS/Au, and then alternately hybridizing with two assisting DNA to bind the enzyme for efficient motor motion. The micromotor device showed unique features of good reproducibility, stability and motion performance. Under optimal conditions, it showed a speed of 420 μm s−1 in 2% H2O2 and even 51 μm s−1 in 0.25% H2O2. In the presence of target DNA, the sensing unit hybridized with target DNA to release the multi-layer DNA as well as the multi-catalase, resulting in a decrease of the motion speed. By using the speed as a signal, the micromotor device could detect DNA from 10 nM to 1 μM. The proposed micromotor device along with the cyclic alternate DNA hybridization assembly technique provided a new path to fabricate efficient and versatile micromotors, which would be an exceptional tool for rapid and simple detection of biomolecules.

A target-induced cyclic strategy for DNAzyme formation was proposed to achieve simple, sensitive and universal detection of protein biomarkers with convenient colorimetric or chemiluminescence imaging readout. In the assay, the target protein was recognized by a pair of DNA-labeled antibodies (Ab1-DNA1 and Ab2-DNA2) to form a proximate complex, which could hybridize with the conjugate DNA3/DNA4 to release the guanine-rich DNA4 and thus formed G-quadruplex/hemin horseradish peroxidase-mimicking DNAzyme. The process could be further recycled with Exonuclease III by cleaving DNA3 to free the proximate complex, resulting in the cyclic formation of DNAzyme. The G-quadruplex/hemin DNAzyme could catalyze the H2O2-mediated oxidation of 3,3,5,5-tetramethylbenzidine to produce the color change from colorless to blue or enhance the chemiluminescence of a luminol–H2O2 system. Thus the signal could be read out with the naked eye, and by colorimetry and chemiluminescence imaging. Using a carcinoembryonic antigen as a model target, the proposed assay showed a detection range of 4 orders of magnitude along with detection limits of 170 and 16 pg mL−1 for colorimetric and chemiluminescence imaging assays respectively. This assay had the advantages of easy operation, sensitive detection, target flexibility and diversified signal readout, providing a great opportunity for commercial application.

•A wash-free and homogeneous strategy is proposed to detect carcino-embryonic antigen.•Proximity hybridization is introduced to regulate chemiluminescence resonance energy transfer.•Graphene oxide is used as energy acceptor to construct a universal method.•The assay takes place in homogeneous solution.•The sensing approach shows low detection limit, wide linear range and excellent selectivity.Chemiluminescence resonance energy transfer (CRET) and the proximity ligation assay have been widely used in design of sensors for the bioanalysis. Here, a wash-free and homogeneous strategy was proposed to detect carcino-embryonic antigen (CEA) based on proximity hybridization-regulated CRET. The Cy5 demonstrated strong chemiluminescence (CL) via the oxidation of TCPO in the presence of H2O2 and energy transfer between excited TCPO and Cy5. Graphene oxide (GO) as an excellent quencher was used to produce the “Signal off” mode that little CL emission was observed through CRET between GO and the Cy5-labelled DNA3. Once CEA was introduced, the target-induced proximity hybridization occurred to form a proximate complex, which inhibited the CRET by preventing GO from absorbing Cy5-labelled DNA3. Furthermore, taking advantage of nicking endonuclease Nt.BbvCI for in situ recycling, the signal could be further amplified for highly sensitive CL detection. Our results showed that this strategy enabled a specific response to CEA with a detection range of 5 orders of magnitude, along with a detection limit of 3.2 pg mL−1. Apart from its easy operation, high sensitivity and acceptable accuracy, the proposed method needed only 0.3 μL of sample, indicating its great opportunity for commercial application.A wash-free and homogeneous strategy is designed to detect carcino-embryonic antigen based on proximity hybridization-regulated chemiluminescence resonance energy transfer. The assay shows advantages of easy operation, wide detection range, low sample consumption, high sensitivity and acceptable accuracy, indicating great opportunity for commercial application.

A highly efficient polymeric tubular micromotor doped with Pt nanoparticle@carbon nanotubes is fabricated by template-assisted electrochemical growth. The micromotors preserve good navigation in multi-media and surface modification, along with simple synthesis, easy functionalization and good biocompatibility, displaying great promise in biological applications.

A motor-based autonomous microsensor is proposed for in situ visualization immunoassay of cancer biomarkers through motion readout or tag counting. The microsensor is prepared by functionalizing a newly designed gold-nanoparticle-modified self-propelled polyaniline/Pt (AuNP/PANI/Pt) micromotor with capture antibody. The autonomous movement of the microsensor in the fuel-enhanced sample mixture results in the fast and selective recognition of the protein target and subsequent loading of the secondary-antibody-modified glycidyl methacrylate microspheres (GMA), which slows down the movement of the sensing microengine. The velocity of the microsensor and the number of GMA conjugated on the microsensor can be conveniently visualized using optical microscopy. They are negatively and positively correlated with the target concentration, respectively. Therefore, the microsensor can conveniently distinguish the concentration of carcinoembryonic antigen in a range of 1–1000 ng/mL. The motor-based microsensor can be easily prepared in batch using AuNP/PANI/Pt. The whole detection procedure for protein target can be completed in 5 min without any washing and separation step. This method shows considerable promise for diverse clinical and diagnostic applications.

•An ultrasensitive enzyme-free electrochemical immunosensor is developed.•A double strand DNA@Au nanoparticle tag is employed for signal amplification.•The tag is able to load a high amount of RuHex via electrostatic interaction.•The immunosensor is of high sensitivity and selectivity for CEA detection.An ultrasensitive enzyme-free electrochemical immunoassay was developed for detection of the fg/mL level carcinoembryonic antigen (CEA) by using a double strand DNA@Au nanoparticle (dsDNA@AuNP) tag and hexaammineruthenium(III) chloride (RuHex) as the electroactive indicator. The dsDNA@AuNP was synthesized by one-pot hybrid polymerization of dsDNA on initiator DNA modified AuNPs via hybridization chain reaction. The immunosensor was prepared by covalently cross-linking capture antibody on chitosan/AuNP nanocomposite modified glass carbon electrode. The AuNPs accelerated the electron transfer and led to high detection sensitivity. With a sandwich-type immunoreaction and a biotin–streptavidin affinity reaction, the dsDNA@AuNP tag was conjugated on the immunocomplex to bring a high amount of RuHex to the electrode surface via electrostatic interaction, resulting in an amplified electrochemical signal. Under optimal conditions, the proposed sensing platform showed a wide linear detection range from 10 fg/mL to 10 ng/mL along with a detection limit of 3.2 fg/mL for CEA. The immunosensor exhibited high sensitivity and good stability, showing a promising application in early cancer diagnosis and could be extended to sensitive electrochemical biosensing of other analytes.An ultrasensitive enzyme-free electrochemical immunoassay was developed for detection of fg/mL level carcinoembryonic antigen by using a double strand DNA@Au nanoparticle (dsDNA@AuNP) tag and hexaammineruthenium(III) chloride (RuHex) as the electroactive indicator.

Manganese porphyrin (MnTMPyP)–dsDNA complex was reported as an excellent mimicking enzyme of peroxidase. It possessed high catalytic activity and much quicker catalytic kinetics and better stability with exposure to light irradiation and high temperature than both horseradish peroxidase and hemin/G-quadruplex DNAzyme. The groove binding of MnTMPyP to the dsDNA scaffold efficiently maintained the catalytic activity of the MnTMPyP center and improved its stability. By combining with an isothermal hybridization chain reaction (HCR) and in situ formation of MnTMPyP-dsDNA, a highly efficient chemiluminescent (CL) immunosensing method was proposed. After a sandwich immunoreaction, a biotinylated DNA strand, which was bound to biotinylated signal antibody by streptavidin, triggered the HCR and growth of MnTMPyP-dsDNA on the immunocomplex. The in situ, HCR-assisted enzyme formation brought numerous enzymatic catalytic centers, MnTMPyP, on the immunocomplex, resulting in significant CL signal amplification and highly sensitive CL detection. Using carcinoembryonic antigen as the model target, the proposed CL immunoassay method showed a wide linear range from 10 pg/mL to 100 ng/mL with a detection limit of 6.8 pg/mL. The new MnTMPyP-dsDNA complex could be conveniently synthesized, functionalized, and combined with DNA amplification strategies, showing a promising potential in bioanalysis and other relative fields.

A DNA nanopolylinker was designed as a three dimensional nanoprobe with high loading of signal molecules for amplifying the biosensing signal. The nanoprobe was prepared by hybridization chain reaction engineering dsDNA polymerization on initiator DNA modified Au nanoparticle with two kinds of small molecule, for example, FITC-labeled DNA hairpins. The core–shell conjugate that was formed contained approximately 320 FITC molecules for further binding of signal molecules. With a sandwich-type immunoreaction and a biotin-streptavidin affinity reaction, the biotinylated core–shell nanoprobe was immobilized on the immunosensor surface, and the FITC molecules then bound enzyme labeled anti-FITC antibody to catalyze a silver deposition process, leading to a novel cascade signal amplification strategy. By combining the proposed strategy with stripping analysis of the deposited silver, an ultrasensitive immunoassay method for biomarker detection was developed. Under optimal conditions, this method showed a linear detection range over 5 orders of magnitude for carcinoembryonic antigen with a detection limit of 1.2 fg mL−1 (about 18 molecules in 5.0 μL sample). The preparation of DNA nanopolylinker was simple and economic, and it could be used as a universal and multifarious probe for different bioanalytical techniques and showed the promising potential of the signal amplification strategy in the future design of biosensing methodology.

A triple signal amplification strategy was designed for ultrasensitive immunosensing of cancer biomarker. This strategy was achieved using graphene to modify immunosensor surface for accelerating electron transfer, poly(styrene-co-acrylic acid) microbead (PSA) carried gold nanoparticles (AuNPs) as tracing tag to label signal antibody (Ab2) and AuNPs induced silver deposition for anodic stripping analysis. The immunosensor was constructed by covalently immobilizing capture antibody on chitosan/electrochemically reduced graphene oxide film modified glass carbon electrode. The in situ synthesis of AuNPs led to the loading of numerous AuNPs on PSA surface and convenient labeling of the tag to Ab2. With a sandwich-type immunoreaction, the AuNPs/PSA labeled Ab2 was captured on the surface of an immunosensor to further induce a silver deposition process. The electrochemical stripping signal of the deposited silver nanoparticles in KCl was used to monitor the immunoreaction. The triple signal amplification greatly enhanced the sensitivity for biomarker detection. The proposed method could detect carcinoembryonic antigen with a linear range of 0.5 pg mL–1 to 0.5 ng mL–1 and a detection limit down to 0.12 pg mL–1. The immunosensor exhibited good stability and acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics.

An electrochemical genosensor in which signal amplification is achieved using p-aminophenol (p-AP) redox cycling by nicotinamide adenine dinucleotide (NADH) is presented. An immobilized thiolated capture probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the detection probe, and streptavidin-alkaline phosphatase as reporter enzyme. The phosphatase liberates the electrochemical mediator p-AP from its electrically inactive phosphate derivative. This generated p-AP is electrooxidized at an Au electrode modified self-assembled monolayer to p-quinone imine (p-QI). In the presence of NADH, p-QI is reduced back to p-AP, which can be re-oxidized on the electrode and produce amplified signal. A detection limit of 1 pM DNA target is offered by this simple one-electrode, one-enzyme format redox cycling strategy. The redox cycling design is applied successfully to the monitoring of the 16S rRNA of E. coli pathogenic bacteria, and provides a detection limit of 250 CFU μL−1.

A highly efficient polymeric tubular micromotor doped with Pt nanoparticle@carbon nanotubes is fabricated by template-assisted electrochemical growth. The micromotors preserve good navigation in multi-media and surface modification, along with simple synthesis, easy functionalization and good biocompatibility, displaying great promise in biological applications.