Exponential Signal Amplification Research Articles

Advanced design of target-driven self-powered sensor assisted by cascade catalytic strategy

In this work, a self-powered microsensor platform based on enzyme biofuel cells (EBFCs) was developed for intelligent monitoring of disease markers miRNA-451. The cascade catalysis system constructed by using the strategy of enzyme-like ZIF-8 nanocapsule incorporation with biological enzymes, which could simultaneously take into account the specificity of biological enzymes and the high activity of nano-enzymes, significantly promoted the electron transfer between glucose and the bio-anode surface, and improved the sensitivity and stability of the sensing system. Meanwhile, the target-triggered hybridization chain reaction (HCR) amplification strategy to achieve exponential signal amplification based on accurate recognition, and jointly improve the detection sensitivity. As expected, the micro-sensor platform has a wide linear range of 0.5–1.0 fmol/L with a low limit of detection (LOD) of 0.13 fmol/L (S/N = 3) and exhibits excellent selectivity, reproducibility and stability in interference assays under optimal detection conditions. The designed self-powered system is simple to construct, easy to transport and the data transmission mode is intelligent and controllable, which is expected to be used in basic biochemical research, clinical diagnosis and environmental monitoring.

Dual-Function DNA Nanowires with Self-Feedback Amplification and Efficient Signal Transduction for Intracellular Imaging of MicroRNA-155.

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

High‐Performance Cooperative DNA Nanodevice Enables Sensitive Circular RNA Imaging and Precise Tumor Growth Suppression

The utility of circular RNAs (circRNAs) as emerging biomarkers and regulatory factors in medical diagnostics and therapeutics is hampered by the challenges associated with their sensitive detection and precise modulation. Herein, a high‐performance cooperative DNA nanodevice (HCDN) based on DNA tetrahedron‐confined catalytic DNA assembly reaction (DT‐CDA) that enables both imaging and regulation of circRNAs is developed. Activation of the DT‐CDA is contingent upon the presence of the target circRNA, which, together with a replicative fuel probe, catalyzes the sequential opening of additional DT‐CDAs. This cooperative exponential signal amplification with negligible background interference allows HCDN to effectively detect minute quantities of circRNAs. Employing circSATB2 as a model, the HCDN demonstrates substantial downregulation of Cyclin D1 (CCND1) mRNA and protein levels in cellular and in vivo models, thereby inhibiting tumor growth. The innovative design of HCDN sets the stage for a powerful methodology conducive to enhanced clinical diagnostics and biomolecule manipulation, thereby advancing the capabilities and applications of DNA nanotechnology.

Hook-Like DNAzyme-Activated Autocatalytic Biosensor for the Universal Detection of Pathogenic Bacteria.

DNAzyme-based assays have found extensive utility in pathogenic bacteria detection but often suffer from limited sensitivity and specificity. The integration of a signal amplification strategy could address this challenge, while the existing combination methods require extensive modification to accommodate various DNAzymes, limiting the wide-spectrum bacteria detection. We introduced a novel hook-like DNAzyme-activated autocatalytic nucleic acid circuit for universal pathogenic bacteria detection. The hook-like connector DNA was employed to seamlessly integrate the recognition element DNAzyme with the isothermal enzyme-free autocatalytic hybridization chain reaction and catalytic hairpin assembly for robust exponential signal amplification. This innovative autocatalytic circuit substantially amplifies the output signals from the DNAzyme recognition module, effectively overcoming DNAzyme's inherent sensitivity constraints in pathogen identification. The biosensor exhibits a strong linear response within a range of 1.5 × 103 to 3.7 × 107 CFU/mL, achieving a detection limit of 1.3 × 103 CFU/mL. Noted that the sensor's adaptability as a universal detection platform is established by simply modifying the hook-like connector module, enabling the detection of various pathogenic bacteria of considerable public health importance reported by the World Health Organization, including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella typhimurium. Additionally, the specificity of DNAzyme in bacterial detection is markedly improved due to the signal amplification process of the autocatalytic circuit. This hook-like DNAzyme-activated autocatalytic platform presents a versatile, sensitive, and specific approach for pathogenic bacteria detection, promising to significantly expand the applications of DNAzyme in bacteria detection.

Isothermal Reciprocal Catalytic DNA Circuit for Sensitive Analysis of Kanamycin.

Inappropriate use of veterinary drugs can result in the presence of antibiotic residues in animal-derived foods, which is a threat to human health. A simple yet efficient antibiotic-sensing method is highly desirable. Programmable DNA amplification circuits have supplemented robust toolkits for food contaminants monitoring. However, they currently face limitations in terms of their intricate design and low signal gain. Herein, we have engineered a robust reciprocal catalytic DNA (RCD) circuit for highly efficient bioanalysis. The trigger initiates the cascade hybridization reaction (CHR) to yield plenty of repeated initiators for activating the rolling circle amplification (RCA) circuit. Then the RCA-generated numerous reconstituted triggers can reversely stimulate the CHR circuit. This results in a self-sufficient supply of numerous initiators and triggers for the successive cross-invasion of CHR and RCA amplifiers, thus leading to exponential signal amplification for the highly efficient detection of analytes. With its flexible programmability and modular features, the RCD amplifier can serve as a universal toolbox for the high-performance and accurate sensing of kanamycin in buffer and food samples including milk, honey, and fish, highlighting its enormous promise for low-abundance contaminant analysis in foodstuffs.

Simultaneous detection of breast cancer biomarkers HER2 and miRNA-21 based on duplex-specific nuclease signal amplification.

The detection of a single biomarker is prone to false negative or false positive results. Simultaneous analysis of two biomarkers can greatly improve the accuracy of diagnosis. In this work, we designed a new method for coinstantaneous detection of two breast cancer biomarkers miRNA-21 and HER2 using the properties of duplex-specific nuclease (DSN). Cy5-labeled DNA1 and FAM-labeled DNA2 are used as signal probes to distinguish the two signals. When the sample contains the targets HER2 and miRNA-21, HER2 binds to the HER2 aptamer on the double-stranded DNA2, while miRNA-21 binds to the complementary DNA1. Then, DSN enzyme is added to cut the DNA probes adsorbed on the HER2 aptamer and miRNA-21, releasing the fluorescent groups, which can be readsorbed to the empty sites, thus repeating the cutting of the probes and producing an exponential signal amplification with two distinct fluorescent signals. The detection limits of miRNA-21 and HER2 are 1.12 pM and 0.36 ng mL-1, respectively, with linear ranges of 5 pM to 50 pM and 1 ng mL-1 to 15 ng mL-1. The method was validated in real biological samples, providing a new approach for synchronous analysis of important markers in breast cancer.

Label-free and sensitive detection of N6-methyladenosine demethylase activity in crude cell extracts and clinical cancer tissues based on demethylation-triggered exponential signal amplification

The m6A demethylase catalyzes the removal of m6A modification to establish proper RNA methylation patterns, and it has emerged as a promising disease biomarker and a therapeutic target. The reported m6A demethylase assays often suffer from tedious producers, expensive reagents, radioactive risk, limited sensitivity, and poor specificity. Herein, we develop a simple, selective, label-free, and highly sensitive fluorescent biosensor for m6A demethylase assay based on demethylation-triggered exponential signal amplification. In this biosensor, m6A demethylase-catalyzed demethylation can protect the circular DNA from the digestion by DpnI, subsequently triggering hyperbranched rolling circle amplification to achieve exponential signal amplification for producing abundant ssDNA and dsDNA products. The amplified DNA signal can be sensitively and simply detected by SYBR Gold in a label-free manner. This biosensor avoids any antibodies, washing/separation procedures, and fluorophore-/quencher-labeled probes, great simplifying the assay procedures and reducing the assay cost. Moreover, this biosensor achieves good specificity and excellent sensitivity with a detection limit of 1.2 fg/μL, which is superior to conventional ELISA (36.3 pg/μL). Especially, this biosensor enables direct monitoring of m6A demethylase activity in crude cell extracts with high accuracy, and it can be further applied for the screening of m6A demethylase inhibitor, measurement of m6A demethylase activity in different cell lines, and discrimination of m6A demethylase level in clinical cancer and healthy tissues, providing a facile and robust platform for RNA methylation-related biomedical research, disease diagnosis, and drug discovery.

HCR/DNAzyme-triggered cascaded feedback cycle amplification for self-powered dual-photoelectrode detection of femtomolar HPV16

For high-performance dual-photoelectrode assay, developing a pair of photoactive materials with well-matched band structure and the design of a powerful sensing strategy are highly desirable. Herein, the Zn-TBAPy pyrene-based MOF and BiVO4/Ti3C2 Schottky junction were employed as photocathode and photoanode to form an efficient dual-photoelectrode system. The integration of the cascaded hybridization chain reaction (HCR)/DNAzyme-assisted feedback amplification with DNA walker-mediated cycle amplification strategy realizes femtomolar HPV16 dual-photoelectrode bioassay. Through the activation of the HCR cascaded with the DNAzyme system in the presence of HPV16, plentiful HPV16 analogs are generated that leads to exponential positive feedback signal amplification. Meanwhile on the Zn-TBAPy photocathode, the NDNA hybridizes with the bipedal DNA walker followed by circular cleavage by Nb.BbvCI NEase, producing a dramatically enhanced PEC readout. The achieved ultralow detection limit of 0.57 fM and a wide linear range of 10−6 nM–103 nM showcase the excellent performance of the developed dual-photoelectrode system.

Detection of small molecules by extending the terminal protection to the polymerase

Since Terminal Protection of Small Molecule-Linked DNA (abbreviated as TPSMLD) was first found, TPSMLD-based assays have speedily prospered in small molecule-protein interaction study and sensing. However, most of the TPSMLD-based sensing strategies have adopted nuclease-assisted signal amplification, mainly by exonucleases, which adversely affect triggering the downstream reactions. Here we describe a simple, sensitive, and selective strategy for small molecule measurement taking biotin as a model. Due to the extension of the terminal protection mechanism to the polymerase and the introduction of the competition assay, a turn-on and exponential amplification signal was conveniently achieved. Benefiting from the high selectivity of TPSMLD and the exponential signal amplification assisted by terminal deoxynucleotidyl transferase (TdT)-endonuclease IV (Endo IV), biotin could be sensitively detected with a detection limit of 0.21 nM. Additionally, the recovery test was also executed in 1% spiked human serum with a satisfying recovery in the range of 95.8–98.0%, which demonstrated the robust anti-interference ability and potential clinical application of our proposed strategy. Predictably, by changing the small molecule-protein pair, this work could serve as a universal small molecule detection platform.

Closed Cyclic DNA Machine for Sensitive Logic Operation and APE1 Detection.

DNA self-assembly has been developed as a kind of robust signal amplification strategy, but most of reported assembly pathways are programmed to amplify signal in one direction. Herein, based on mutual-activated cascade cycle of hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA), a closed cycle circuit (CCC) based DNA machine is developed for sensitive logic operation and molecular recognition. Benefiting from the synergistically accelerated signal amplification, the closed cyclic DNA machine enabled the logic computing with strong and significant output signals even at weak input signals. The typical logic operations such as OR, YES, AND, INHIBIT, NOR, and NAND gate, are conveniently and clearly executed with this DNA machine through rational design of the input and computing elements. Moreover, by integrating the target recognition module with the CCC module, the proposed DNA machine is further employed in the homogeneous detection of apurinic/apyrimidinic endonuclease 1 (APE1). The precise recognition and exponential signal amplification facilitated the highly selective and sensitive detection of APE1 with limit of detection (LOD) of 7.8×10-5 U mL-1 . Besides, the normal cells and tumor cells are distinguished unambiguously by this method according to the detected concentration difference of cellular APE1, which indicates the robustness and practicability of this method.

Iodine-mediated photoinduced autoinductive tandem chromogenic system for visual colorimetric detection of triacetone triperoxide explosive.

A long-standing challenge in colorimetric detection of triacetone triperoxide (TATP) explosive is low sensitivity. We herein developed an iodine-mediated photoinduced auto-inductive tandem chromogenic system to achieve exponential signal amplification. The strategy employs the KI-TATP reaction and photo-induced autocatalytical oxidation of o-phenylenediamine (OPD) that work in tandem. The resulting I3- from the KI-TATP reaction oxidizes OPD to yellow 2,3-diaminophenazine (DAP) that is further excited by blue light illumination to produce reactive oxygen species (ROS). The obtained ROS, in turn, promotes the oxidation of OPD to gain more DAP, causing the auto-inductive chromogenic reaction processes. This tandem chromogenic system is applied for visual colorimetric detection of TATP, allowing the selective and sensitive detection of TATP down to 42.8μM. Moreover, analyses of TATP in real samples are performed, and the satisfactory recovery results are achieved. Furthermore, a field detection kit is also developed.

A three-dimensional DNA walker and silver nanoparticles promoting lattice-strain-driven photo-induced electron transfer for high-performance semi-homogeneous biosensing

As an important tumor marker, the ultra-sensitive detection of low-abundance microRNAs provides important data support for early warning of tumors. Herein, taking microRNA-141 as the target model, a novel enzyme-free semi-homogeneous photoelectrochemical (SHO-PEC) biosensor was designed by entropy-driven three-dimensional (3D) DNA walker with exponential signal amplification, magnetic enrichment and separation of pseudo-targets, bridged CdTe@ZnS quantum dots to promote photocathodic current enhancement, and in situ generated silver nanoparticles (NPs) to accelerate charge separation. In this protocol, ZnS shell passivates the surface defects of the soft core CdTe, introducing considerable strain that causes electrons to extend towards the shell, which is responsible for the higher photocurrent response. Also, the Ag NPs enable ultrafast photogenerated electron transfer by forming Schottky junctions. These two unique sensitized mechanisms were proposed for the first time in PEC sensing. Moreover, a one-step simple electrode modification strategy avoids tedious layer-by-layer assembly and rinsing steps. These make the "signal-on" SHO-PEC biosensor with remarkable advantages of high sensitivity, good specificity, strong reliability, fast speed, easy operation, and low cost. This research provides a new initiative for the development of PEC sensing platforms with excellent comprehensive performance.

Deamination-triggered exponential signal amplification for chemiluminescent detection of cytosine deaminase at the single-cell level.

We construct a sensitive chemiluminescent biosensor for sensitive detection of cytosine deaminase APOBEC3A based on deamination-triggered exponential signal amplification. This biosensor displays good specificity and high sensitivity, and it can screen APOBEC3A inhibitors and measure endogenous APOBEC3A at the single-cell level, with prospective applications in disease diagnostics and therapy.

A toehold mediated feedback rolling circle amplification with exponential signal amplification enables label-free nucleic acid sensing with high sensitivity and specificity

Rolling circle amplification (RCA) has been widely used for nucleic acid and other biomarkers analysis, however, the limited amplification efficiency and low specificity in traditional RCA hinder its further application. Here, a toehold mediated feedback rolling circle amplification (TMFRCA) strategy with exponential signal amplification was proposed for label-free nucleic acids sensing. The rational engineered TH and CT ensured exponential signal amplification by utilizing the feedback of amplicons to the reactant TH for circularly initiating new RCA. Introduction of toehold strand displacement and G-quadruplex (G4) dimers in RCA reaction allowed enhanced specific recognition and further improved fluorescence signal due to higher quantum yield of G4 dimers/ThT. Taking classic swine fever virus (CSFV) as model target, this TMFRCA method exhibited great sensitivity and high specificity with a limit of detection down to 0.29 fM and enabled to discriminate target with single base differences. In addition, the proposed TMFRCA could be used for detecting CSFV RNA in actual samples with great accuracy comparable to RT-qPCR, and possessed great universality for detecting nucleic acid without length limitation. We believe this method can promote the development of nucleic acid signal amplification and be an effective tool for nucleic acid sensing-based fundamental research and clinical diagnosis.

Computer-Aided Design of DNA Self-Limited Assembly for Relative Quantification of Membrane Proteins.

Immunofluorescence imaging of cells plays a vital role in biomedical research and clinical diagnosis. However, when it is applied to relative quantification of proteins, it suffers from insufficient fluorescence intensity or partial overexposure, resulting in inaccurate relative quantification. Herein, we report a computer-aided design of DNA self-limited assembly (CAD-SLA) technology and apply it for relative quantification of membrane proteins, a concept proposed for the first time. CAD-SLA can achieve exponential cascade signal amplification in one pot and terminate at any desired level. By conjugating CAD-SLA with immunofluorescence, in situ imaging of cell membrane proteins is achieved with a controllable amplification level. Besides, comprehensive fluorescence intensity information from fluorescent images can be obtained, accurately showing relative quantitative information. Slight protein expression differences previously indistinguishable by immunofluorescence or Western blotting can now be discriminated, making fluorescence imaging-based relative quantification a promising tool for membrane protein analysis. From the perspectives of both DNA self-assembly technology and immunofluorescence technology, this work has solved difficult problems and provided important reference for future development.

Colorimetric detection of thrombin based on signal amplification by transcription-reverse transcription concerted reaction using non-crosslinking aggregation of gold nanoparticles.

Gold nanoparticles (AuNPs) have been used as colorimetric biosensors by utilizing the difference in color between the dispersed (red) and aggregated (blue) states. We previously developed a biosensor that converts sandwich-type thrombin recognition to RNA amplification and color difference of AuNPs. But the sensitivity was insufficient because of the linear signal amplification mechanism. In this study, we designed an exponential signal amplification biosensor based on transcription-reverse transcription concerted (TRC) reaction.

Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease IV-assisted exponential signal amplification

Alkaline phosphatase (ALP) is widely expressed in human tissues. ALP plays an important role in the dephosphorylation of proteins and nucleic acids. Therefore, quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods. Terminal deoxynucleotidyl transferase (TdT) catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3′-OH end of single-stranded DNA in the absence of a template. In this study, we developed a highly sensitive and selective method based on TdT and endonuclease IV (Endo IV) to quantify ALP activity. After ALP hydrolyzes the 3′-PO4 end of the substrate and generates 3′-OH, TdT can effectively elongate the 3′-OH end with deoxynucleotide adenine triphosphate (dATP) and produce a poly A tail, which can be detected by the poly T probes. Endo IV digests the AP site in poly T probes to generate a fluorescent signal and a new 3′-OH end, leading to the generation of exponential fluorescence signal amplification. The substrate for TdT elongation was optimized, and a limit of detection of 4.3 × 10−3 U/L was achieved for ALP by the optimized substrate structure. This method can also detect ALP in the cell lysate of a single cell. This work has potential applications in disease diagnosis and biomedical detection.

Electrochemiluminescent determination of prostate-specific antigen using Au@(MoS2/GO/o-MWNTs) nanohybrids as co-reaction accelerator and hyperbranched hybridization chain reaction for signal amplification.

Three-dimensional flowerlike Au@(MoS2/GO/o-MWNTs) nanohybrids (abbreviated as AMGMs) were synthesized and then introduced into an electrochemiluminescence (ECL) system as a new co-reaction accelerator for the ultrasensitive prostate-specific antigen (PSA). The AMGMs not only served as a substrate with good conductivity and a large specific surface area for loading abundant primary antibodies but also acted as an effective co-reaction accelerator; the co-reaction accelerator could interact with a co-reactant rather than the luminophore to boost the generation of free radical intermediates, thereby producing abundant excited states of luminophores to amplify the ECL signal response. Additionally, an anticipated signal amplification strategy based on the hybridization chain reaction (HCR) was developed by gathering a large amount of a DNA initiator on gold nanoparticles. These gathered DNA initiators could generate multiple DNA concatemers and attach more signal molecules, which resulted in outstanding exponential signal amplification. Consequently, the ECL immunosensor demonstrated high sensitivity, with a linear range from 0.1pgmL-1 to 50ngmL-1 and a detection limit of 0.028pgmL-1. In addition, the immunosensor displayed excellent stability and selectivity. It was evaluated by analyzing human serum sample. The recovery obtained was 98.80-112.00% and the RSD was 1.73-3.12%, indicating the immunosensor could be applied to the simultaneous detection of PSA in human serum samples. Graphical abstract.

A Cascaded DNA Circuit in Bead Arrays for Quantitative Single-Cell MicroRNA Analysis.

Single-cell microRNA (miRNA) analysis helps people understand the causes of diseases and formulate new disease treatment strategies. However, miRNA from a single cell is usually very rare and requires signal amplification for accurate quantification. Here, to amplify the signal, we constructed the cascaded DNA circuits consisting of catalytic hairpin assembly and hybrid chain reaction into the bead array platform, on which the uniformly distributed beads were adopted for miRNA quantification. After exponential signal amplification, a consistent linear correlation between the percentage of fluorescent beads and the copy number of miRNA was detected. The proposed bead array can achieve ultrahigh sensitivity as low as 60 copies of miR-155 and high specificity for distinguishing single nucleotide differences. This method has been successfully applied to the quantitative detection of miRNA in a single cancer cell. The high sensitivity, programmability, and simple workflow of the bead array chip will give a huge advantage in basic and clinical research.

Restriction Endonuclease-Based Assays for DNA Detection and Isothermal Exponential Signal Amplification.

Application of restriction endonuclease (REase) enzymes for specific detection of nucleic acids provides for high assay specificity, convenience and low cost. A direct restriction assay format is based on the specific enzymatic cleavage of a target–probe hybrid that is accompanied with the release of a molecular marker into the solution, enabling target quantification. This format has the detection limit in nanomolar range. The assay sensitivity is improved drastically to the attomolar level by implementation of exponential signal amplification that is based on a cascade of self-perpetuating restriction endonuclease reactions. The cascade is started by action of an amplification “trigger”. The trigger is immobilized through a target-specific probe. Upon the target probe hybridization followed with specific cleavage, the trigger is released into the reaction solution. The solution is then added to the assay amplification stage, and the free trigger induces cleavage of amplification probes, thus starting the self-perpetuating cascade of REase-catalyzed events. Continuous cleavage of new amplification probes leads to the exponential release of new triggers and rapid exponential signal amplification. The proposed formats exemplify a valid isothermal alternative to qPCR with similar sensitivity achieved at a fraction of the associated costs, time and labor. Advantages and challenges of the approach are discussed.