HRP-Mimicking DNAzyme-Catalyzed in Situ Generation of Polyaniline To Assist Signal Amplification for Ultrasensitive Surface Plasmon Resonance Biosensing

It is well-known that the horseradish peroxidase- (HRP-) mimicking DNAzyme, namely, hemin/G-quadruplex, can effectively catalyze the polymerization of aniline to form DNA-guided polyaniline. Meanwhile, polyaniline exhibits extraordinary electrical, electrochemical, and redox properties, as well as excellent SPR signal-enhancing ability. Herein, we report a novel ultrasensitive surface plasmon resonance (SPR) biosensor based on HRP-mimicking DNAzyme-catalyzed in situ formation of polyaniline for signal amplification, using bleomycin (BLM) as the proof-of-concept analyte. The recognition and the subsequent cleavage of DNA probe P1 by BLM switches off the hybridization between P1 and the G-rich DNA probe P2, resulting in less hemin/G-quadruplex complexes and reduced DNA-guided polyaniline deposition on the SPR Au disk surface. As compared to the case when BLM is absent, a significant shift in SPR angle is observed, which is dependent on the BLM concentration. Therefore, ultrasensitive SPR detection of the target BLM is realized, with a detection limit down to 0.35 pM, much lower than those reported in the literature. Moreover, the proposed SPR biosensor has been successfully applied for the detection of BLM spiked in human serum samples. The HRP-mimicking DNAzyme-catalyzed in situ polyaniline deposition and polyaniline-assisted signal amplification not only significantly improves the specificity and the sensitivity of the BLM assay but also allows the ultrasensitive detection of other biomolecules by simply changing the specific target recognition DNA sequences, thus providing a versatile SPR biosensing platform for the ultrasensitive detection of a variety of analytes and showing great potential for application in the fields of bioanalysis and clinical biomedicine

Integration of Biofuel Cell-Based Self-Powered Biosensing and Homogeneous Electrochemical Strategy for Ultrasensitive and Easy-To-Use Bioassays of MicroRNA

Biofuel cell (BFC)-based self-powered biosensors have attracted substantial attentions because of their unique merits such as having no need for power sources (only two electrodes are needed). More importantly, in case it can also work in a homogeneous system, more efficient and easy-to-use bioassays could come true. Thus, herein, we proposed a novel homogeneous self-powered biosensing strategy via the integration of BFCs and a homogeneous electrochemical method, which was further utilized for ultrasensitive microRNA (miRNA) detection. To construct such an assay protocol, the cathodic electron acceptor [Fe­(CN)₆]^3– was entrapped in the pores of positively charged mesoporous silica nanoparticles and capped by the biogate DNAs. Once the target miRNA existed, it would trigger the controlled release of [Fe­(CN)₆]^3–, leading to the dramatic increase of the open circuit voltage. Consequently, the “signal-on” homogeneous self-powered biosensor for the ultrasensitive miRNA assay was realized. Encouragingly, the limit of detection for the miRNA-21 assay was down to 2.7 aM (S/N = 3), obviously superior to those of other analogous reported approaches. This work not only provides an ingenious idea to construct the ultrasensitive and easy-to-use bioassays of miRNA but also exhibits a successful prototype of a portable and on-site biomedical sensor

Label-Free Homogeneous Electroanalytical Platform for Pesticide Detection Based on Acetylcholinesterase-Mediated DNA Conformational Switch Integrated with Rolling Circle Amplification

This study addresses the need for sensitive pesticide assay by reporting a new label-free and immobilization-free homogeneous electroanalytical strategy, which combines acetylcholinesterase (AChE)-catalyzed hydrolysis product-mediated DNA conformational switch and rolling circle amplification (RCA) to detect organophosphorous and carbamate pesticides in a “signal-on” mode. When target pesticides were present, AChE activity was inhibited and could not trigger the following DNA conformational change and the RCA reaction, which results in numerous methylene blue (MB) molecules in a free state, generating a strong electrochemical response. This proposed strategy was highly sensitive for omethoate detection with a detection limit as low as 2.1 μg/L and a linear range from 10 to 10 000 μg/L. Furthermore, this strategy was demonstrated to be applicable for pesticide detection in real samples. Thus, this novel label-free homogeneous electroanalytical strategy holds great promise for pesticide detection and can be further exploited for sensing applications in the environment and the food safety field

Ultrasensitive Self-Powered Aptasensor Based on Enzyme Biofuel Cell and DNA Bioconjugate: A Facile and Powerful Tool for Antibiotic Residue Detection

Herein, we reported a novel ultrasensitive one-compartment enzyme biofuel cells (EBFCs)-based self-powered aptasensing platform for antibiotic residue detection. By taking full advantage of the unique features of both EBFCs-based self-powered sensors and aptamers, the as-proposed aptasensing platform has the merits of simple instrumentation, anti-interference ability, high selectivity, and low cost. In this study, DNA bioconjugate, i.e., SiO₂@gold nanoparticles–complementary strand of aptamer (SiO₂@AuNPs–csDNA), was elaborately designed and played a key role in blocking the mass transport of glucose to the bioanode. While in the presence of the target antibiotic, SiO₂@AuNPs–csDNA bioconjugate broke away from the bioanode due to the aptamer recognition of the target. Without the blocking of glucose by the DNA bioconjugate, a significantly elevated open circuit voltage of the EBFCs-based aptasensor was obtained, whose amplitude was dependent on the antibiotic concentration. In addition, this proposed aptasensor was the first reported self-powered aptasensing platform for antibiotic determination and featured high sensitivity owing to the elaborate design of the DNA bioconjugate modified bioanode of EBFC, which was superior to those previously reported in the literature. Furthermore, due to the anti-interference ability and the excellent selectivity of the aptasensor, no special sample pretreatment was needed for the detection of antibiotics in milk samples. Therefore, the proposed EBFCs-based self-powered aptasensor has a great promise to be applied as a powerful tool for on-site assay in the field of food safety

Amphiphile-Mediated Ultrasmall Aggregation Induced Emission Dots for Ultrasensitive Fluorescence Biosensing

The development of ultrasensitive and highly selective fluorescence biosensors for diverse analytes is highly desirable but remains a challenge. It is attributable to the scarcity of fluorogens with promising brightness, stability, and nontoxicity, which primarily determine the performance of fluorescence biosensors. Herein, we report the design and preparation of aggregation induced emission (AIE) dots with high brightness, exceptional colloidal stability, ultrasmall size, and functional groups for developing ultrasensitive biosensor through the electrostatic conjugation to biological molecules, and use blemycin (BLM) as the proof-of-concept analyte. The recognition and the subsequent cleavage of the quencher-labeled DNA (Q-DNA) by BLM result in the formation of three-mer quencher-linked oligonucleotide fragments (Q-DNA-1), which significantly decreases the amount of quenchers anchored on AIE dot surfaces and subsequently reduces the fluorescence resonance energy transfer (FRET) effect. As compared to the case in which BLM is absent, remarkable fluorescence enhancement is observed, and is dependent on BLM concentration. Thus, ultrasensitive fluorescence detection of target BLM is realized, with a detection limit down to 3.4 fM, the lowest value reported so far. Moreover, the proposed fluorescence biosensor has also been successfully utilized for detection of BLM spiked in human serum samples. The as-proposed strategy not only significantly improves the selectivity and sensitivity of BLM assay, but also allows the ultrasensitive detection of a variety of bioactive molecules by simply changing the specific target recognition substances, thus providing a versatile fluorescence platform, and showing great potential to be applied in chemo-/bioanalysis and clinical biomedicine

Label-Free and Enzyme-Free Homogeneous Electrochemical Biosensing Strategy Based on Hybridization Chain Reaction: A Facile, Sensitive, and Highly Specific MicroRNA Assay

Homogenous electrochemical biosensing strategies have attracted substantial attention, because of their advantages of being immobilization-free and having rapid response and improved recognition efficiency, compared to heterogeneous biosensors; however, the high cost of labeling and the strict reaction conditions of tool enzymes associated with current homogeneous electrochemical methods limit their potential applications. To address these issues, herein we reported, for the first time, a simple label-free and enzyme-free homogeneous electrochemical strategy based on hybridization chain reaction (HCR) for sensitive and highly specific detection of microRNA (miRNA). The target miRNA triggers the HCR of two species of metastable DNA hairpin probes, resulting in the formation of multiple G-quadruplex-incorporated long duplex DNA chains. Thus, with the electrochemical indicator Methylene Blue (MB) selectively intercalated into the duplex DNA chain and the multiple G-quadruplexes, a significant electrochemical signal drop is observed, which is dependent on the concentration of the target miRNA. Thus, using this “signal-off” mode, a simple, label-free and enzyme-free homogeneous electrochemical strategy for sensitive miRNA assay is readily realized. This strategy also exhibits excellent selectivity to distinguish even single-base mismatched miRNA. Furthermore, this method also exhibits additional advantages of simplicity and low cost, since both expensive labeling and sophisticated probe immobilization processes are avoided. Therefore, the as-proposed label-free and enzyme-free homogeneous electrochemical strategy may become an alternative method for simple, sensitive, and selective miRNA detection, and it has great potential to be applied in miRNA-related clinical diagnostics and biochemical research

Versatile and Programmable DNA Logic Gates on Universal and Label-Free Homogeneous Electrochemical Platform

Herein, a novel universal and label-free homogeneous electrochemical platform is demonstrated, on which a complete set of DNA-based two-input Boolean logic gates (OR, NAND, AND, NOR, INHIBIT, IMPLICATION, XOR, and XNOR) is constructed by simply and rationally deploying the designed DNA polymerization/nicking machines without complicated sequence modulation. Single-stranded DNA is employed as the proof-of-concept target/input to initiate or prevent the DNA polymerization/nicking cyclic reactions on these DNA machines to synthesize numerous intact G-quadruplex sequences or binary G-quadruplex subunits as the output. The generated output strands then self-assemble into G-quadruplexes that render remarkable decrease to the diffusion current response of methylene blue and, thus, provide the amplified homogeneous electrochemical readout signal not only for the logic gate operations but also for the ultrasensitive detection of the target/input. This system represents the first example of homogeneous electrochemical logic operation. Importantly, the proposed homogeneous electrochemical logic gates possess the input/output homogeneity and share a constant output threshold value. Moreover, the modular design of DNA polymerization/nicking machines enables the adaptation of these homogeneous electrochemical logic gates to various input and output sequences. The results of this study demonstrate the versatility and universality of the label-free homogeneous electrochemical platform in the design of biomolecular logic gates and provide a potential platform for the further development of large-scale DNA-based biocomputing circuits and advanced biosensors for multiple molecular targets

Affinity-Mediated Homogeneous Electrochemical Aptasensor on a Graphene Platform for Ultrasensitive Biomolecule Detection via Exonuclease-Assisted Target-Analog Recycling Amplification

As is well-known, graphene shows a remarkable difference in affinity toward nonstructured single-stranded (ss) DNA and double-stranded (ds) DNA. This property makes it popular to prepare DNA-based optical sensors. In this work, taking this unique property of graphene in combination with the sensitive electrochemical transducer, we report a novel affinity-mediated homogeneous electrochemical aptasensor using graphene modified glassy carbon electrode (GCE) as the sensing platform. In this approach, the specific aptamer-target recognition is converted into an ultrasensitive electrochemical signal output with the aid of a novel T7 exonuclease (T7Exo)-assisted target-analog recycling amplification strategy, in which the ingeniously designed methylene blue (MB)-labeled hairpin DNA reporters are digested in the presence of target and, then, converted to numerous MB-labeled long ssDNAs. The distinct difference in differential pulse voltammetry response between the designed hairpin reporters and the generated long ssDNAs on the graphene/GCE allows ultrasensitive detection of target biomolecules. Herein, the design and working principle of this homogeneous electrochemical aptasensor were elucidated, and the working conditions were optimized. The gel electrophoresis results further demonstrate that the designed T7Exo-assisted target-analog recycling amplification strategy can work well. This electrochemical aptasensor realizes the detection of biomolecule in a homogeneous solution without immobilization of any bioprobe on electrode surface. Moreover, this versatile homogeneous electrochemical sensing system was used for the determination of biomolecules in real serum samples with satisfying results

Truly Immobilization-Free Diffusivity-Mediated Photoelectrochemical Biosensing Strategy for Facile and Highly Sensitive MicroRNA Assay

In conventional photoelectrochemical (PEC) analysis, photoactive materials are usually immobilized on electrode surfaces, and such immobilization procedures are tedious and time-consuming, and it is also difficult to prepare electrodes with good reproducibility. To circumvent such limitations, we propose here a truly immobilization-free diffusivity-mediated PEC bionsensing strategy for microRNA assay, using methylene blue (MB) in solution as the photoactive probe, and nonmodified indium tin oxide (ITO) glass as the working electrode. The hybridization between the target microRNA and the MB-labeled single-stranded DNA probe (MB-DNA) triggers the digestion of MB-DNA by T7 exonuclease (T7 Exo), thus to generate MB-labeled mononucleotide, and then the released target microRNA initiates the subsequent cycling processes and generates a large amount of MB-labeled mononucleotides. Due to the diffusivity difference between MB-DNAs and MB-labeled mononucleotides, significantly increased photocurrent signal is observed for MB-labeled mononucleotides as compared to that of MB-DNAs. Therefore, via this “signal-on” mode and the T7 Exo facilitated signal amplification, a facile and highly sensitive immobilization-free PEC microRNA assay is readily realized, with a detection limit down to 27 aM. Moreover, this strategy exhibits excellent specificity and is successfully applied in detecting microRNA spiked in serum samples. Since all the reactions take place in homogeneous solutions and no electrode modification is needed, this PEC biosensing strategy exhibits the advantages of simplicity, rapidness, and good reproducibility. More significantly, it provides a novel concept to design truly immobilization-free PEC biosensing systems, and shows potential to be applied in bioanalysis and biochemical research

Graphene-Assisted Label-Free Homogeneous Electrochemical Biosensing Strategy based on Aptamer-Switched Bidirectional DNA Polymerization

In this contribution, taking the discrimination ability of graphene over single-stranded (ss) DNA/double-stranded (ds) DNA in combination with the electrochemical impedance transducer, we developed a novel label-free homogeneous electrochemical biosensor using graphene-modified glassy carbon electrode (GCE) as the sensing platform. To convert the specific aptamer-target recognition into ultrasensitive electrochemical signal output, a novel aptamer-switched bidirectional DNA polymerization (BDP) strategy, capable of both target recycling and exponential signal amplification, was compatibly developed in this study. In this strategy, all the designed DNA structures could be adsorbed on the graphene/GCE and, thus, serve as the electrochemical impedance signal reporter, while the target acts as a trigger of this BDP reaction, in which these designed DNA structures are bound together and, then, converted to long dsDNA duplex. The distinct difference in electrochemical impedance spectroscopy between the designed structures and generated long dsDNA duplex on the graphene/GCE allows label-free and homogeneous detection of target down to femto-gram level. The target can be displaced from aptamer through the polymerization to initiate the next recognition–polymerization cycle. Herein, the design and signaling principle of aptamer-switched BDP amplification system were elucidated, and the working conditions were optimized. This method not only provides a universal platform for electrochemical biosensing but also shows great potential in biological process researches and clinic diagnostics