Enzyme-free Amplification Strategy Research Articles

A enzyme-free fluorescence quenching sensor for amplified detection of kanamycin in milk based on competitive triggering strategies.

In this work, we constructed a FAM fluorescence quenching biosensor based on an aptamer competition recognition and enzyme-free amplification strategy. We design a competing unit consisting of an aptamer chain and a complementary chain, and a catalytic hairpin self-assembly (CHA) unit consisting of two hairpins in which the complementary chain can trigger the catalytic hairpin self-assembly. In the initial state, the aptamer chain is combined with the complementary chain, the catalytic hairpin self-assembly unit is inhibited, the FAM fluorescence group was far away from the BHQ1 quenching group, and the fluorescence is turn-on. In the presence of kanamycin, the aptamer chain recognizes kanamycin and doesn't form double chains, resulting in the free complementary chain triggering hairpin 1 (H1), and then H1 triggering hairpin 2 (H2), FAM fluorophore is close to the BHQ1 quenching group, and the fluorescence is off-on. When H1 and H2 form a cyclic reaction, enzyme-free amplification is achieved and there is significant output of the fluorescence signal. Therefore, the biosensor has good performance in detecting kanamycin, the detection line is 54 nM, the linear range is 54 nM-0.9 μM, and it can achieve highly selective detection of kanamycin. Kanamycin residue may cause serious harm to human health. The high sensitivity detection of kanamycin is urgent, so this project has a great application potential for food detection.

UIO66 low background signal and fluorescence synergism strategy for highly sensitive detection of Salmonella typhimurium

Successful construction of a detection method for Salmonella typhimurium (S. typhimurium) based on the synergy of hybridization chain reaction (HCR) and fluorescence was realized in this paper. First, the aptamer modified with the quenching group Black Hole Quencher-1 acid (BHQ1) was immobilized on the magnetic beads in combination with the complementary chain of the aptamer modified with 6-carboxyfluorescein (6-FAM). Second, S. typhimurium and cDNA-6-FAM immobilized on magnetic beads competitively bound to the aptamer. Finally, the cDNA-6-FAM was released after magnetic separation acted as a promoter to trigger HCR amplification when the target presented. The fluorescence signal could be significantly improved by the combination of green SYBR Green I (SGI) and HCR long double-stranded DNA and the fluorescent synergy of 6-FAM and SGI. Because of the separation of target and its aptamer, the trigger strand was abstracted by magnetic separation. There was no HCR to generate long double-stranded DNA, and the fluorescence of excess hairpin/SGI could be adsorbed through UIO66 so that only a very low background signal was detected. This fluorescent sensor was capable of monitoring S. typhimurium in the range of 10–3.2 × 107 CFU mL−1 with a limit of detection as low as 1.5 CFU mL−1. Because of the excellent properties of the aptasensor and the validity of SGI fluorescence synergy, this HCR enzyme-free amplification strategy could be generalized to other areas.

Designing a Hybrid Chain Reaction Probe for Multiplex Transcripts Assay with High-Level Imaging.

The hybrid chain reaction (HCR), an isothermal and enzyme-free amplification strategy, has found extensive use in fluorescent in situ hybridization (FISH) assays. However, the existing HCRs are limited, being time-consuming processes and low-efficiency imaging due to weak signal, significantly restricting their application in transcriptomic assays. To address the limitations, we developed nine orthogonal HCR hairpin-pair (hp) probes in this study to enable efficient signal amplification for multiplex assays. To enhance the efficiency and imaging quality of multiplex assays using these HCR probes, we employed two strategies. First, we coupled fluorescent molecules to HCR hairpins via disulfide bonds, facilitating easy removal through chemical cleavage. As a result, the workflow was greatly simplified. Second, we combined HCR with in situ rolling circle amplification (ISRCA), creating ISRCA-HCR, which achieved a 17-fold signal amplification. ISRCA-HCR demonstrated a high-level imaging capability for spatial cell type assays. This study shows the application for cell typing based on the developed HCR probes, enabling accurate and high-level signal amplification for multiplex FISH imaging. This provides an effective research tool for transcriptome and spatial cell type analysis.

Recent Advances in the Preparation and Application of DNA-Encoded Metal Nanoclusters

DNA as an intriguing organic ligand has been widely employed for synthesizing metal nanoclusters and engineering their properties. This review aims to present recent progress on DNA-encoded metal (Ag, Cu, Au, Ag/Pt, Cu/Ag, etc.) nanoclusters (DNA-MNCs), focusing on their applications in the fields of analysis, logic operation, and therapy based on properties including fluorescence, electrochemiluminescence (ECL), and antibacterial and catalytic activity, and summarizes the attractive features of the latest research. The key points are briefly described as follows. (1) Analytical systems have been constructed based on fluorescence regulation, and nuclease-assisted and enzyme-free amplification strategies have been extensively adopted with fluorescent DNA-MNCs for amplified analysis. (2) DNA-MNCs may play more than one role (emitter, quencher, or catalyst) in ECL-based analytical systems. (3) Apart from antibacterial activity, DNA-MNCs also possess apparent catalytic capability, such as enzyme-like activity (i.e., nanozymes), which has been applied in colorimetric systems. (4) Reversibly regulating the catalytic activity of DNA-MNCs has been attained with DNA systems. It is believed that through in-depth investigation of the relationship between atomic structure and property, more novel DNA-MNCs will be explored and applied in the future.

An amplifiable ratiometric fluorescent aptasensor for aflatoxin B1 detection based on dendrimer-like DNA nanostructures coupled with catalytic hairpin self-assembly

Herein, an amplifiable ratiometric fluorescent aptasensor was constructed based on dendrimer-like DNA nanostructures coupled with catalytic hairpin self-assembly for the specific detection of aflatoxin B1 (AFB1) in food. Compared with the single-signal mode, the ratio-signal mode based on FCy3/FCy5 has a wider linear range and a higher R2 for the detection of AFB1. Moreover, for the spiking experiment of peanut, soybean and wine samples, taking the average of the recovery obtained by three standard curves of FCy3, FCy5, FCy3/FCy5 as the reference value, the ratio-signal-based aptasensor showed higher accuracy than the single-signal-based aptasensor. In addition, the presence of a dendrimer-like scaffold can not only improve the stability of the sensor but also enhance FRET efficiency. In the whole detection process, the specific signal difference with dendrimer-like DNA nanostructures was 5.7 times of that without dendrimer-like DNA nanostructures. Besides, catalytic hairpin self-assembly, an enzyme-free amplification strategy, was introduced to achieve signal amplification by the complementary chain of the aptamer (c-DNA) cycle. Furthermore, the sensor can detect various mycotoxins in food by different aptamer recognition.

Structure-switching aptamer triggering signal amplification strategy for tobramycin detection based on hybridization chain reaction and fluorescence synergism.

Based on hybridization chain reaction (HCR) and fluorescence synergism, a novel aptasensor for tobramycin was successfully constructed. Tobramycin competed with cDNA-FAM to bind aptamers immobilized on magnetic beads. After magnetic separation, the released cDNA-FAM acted as initiator to trigger HCR amplification, thus the fluorescence was significantly enhanced due to binding of SYBR Green Ⅰ (SGI) to the formed long double-stranded DNA and the synergistic fluorescence of FAM. In the absence of tobramycin, the initiator was magnetically separated and no HCR occurred, more importantly, graphene oxide can quench the fluorescence of excessive hairpins/SGI and cDNA-FAM, so almost no background signal was detected. This aptasensor can monitor tobramycin in the range of 0.3-50μM with low detection limit of 17.37nM. Due to the potential generality of structure-switching aptamers and effectiveness of fluorescence synergism, this enzyme-free amplification strategy can be extended to other applications by rational design of nucleic acid sequences.

An enzyme-free amplification strategy for sensitive assay of circulating tumor DNA based on wheel-like catalytic hairpin assembly and frame hybridization chain reaction

As an ideal liquid biopsy biomarker candidate, circulating tumor DNA (ctDNA) plays an important role in the non-invasive diagnosis of cancer. Sensitive and precise analysis of ctDNA plays a vital role in promoting clinical applications. Herein, we have developed a novel electrochemical biosensor for ctDNA assay coupling wheel-like catalytic hairpin assembly (WCHA) and frame hybridization chain reaction (FHCR). The ctDNA bound to the DNA transducer by the toehold strand displacement and released the initiator to promote the recycle amplification of WCHA, which lead to the formation of a great number of wheel-like DNA products hybridized with the capture probes immobilized on the gold electrode surface. In addition, the metastable dumbbell probes (DSH1 and DSH2) are designed for hybridization chain reaction. Compared with the traditional HCR, this novel form of HCR (FHCR) can fold up more compact and dense DNA nanostructures, which greatly reduce the space potential resistance and enhance the electronic transmission ability. The combination of dual signal amplification strategies (WCHA/FHCR) significantly improve the sensitivity of ctDNA detection, creating a limit of detection (LOD) as low as 8.3 fM. More importantly, the proposed method is successfully applied in the analysis of clinical samples, and has an effective classifier with an AUC of 0.909, showing a sensitivity of 86.7 % and a specificity of 80.0 % for the diagnosis of breast tumors. Therefore, this platform has great potential in bioanalysis and clinical diagnostics.

A homogeneous DNA nanosphere for fluorescence detection of microRNAs with high-ordered aggregation enhanced emission and enzyme-free cascade amplification

Detection of microRNAs (miRNAs) by using precise and convenient biosensing methods is desired in biochemical study and early clinical diagnosis. We constructed an innovative programmed all-DNA biosensing platform by using homogeneous 3D DNA nanospheres as signaling reporter and entropy-driven enzyme-free amplification strategy to detect miRNA-141. The self-assembly 3D DNA nanosphere was obtained by the highly ordered aggregation of two kinds of single strand of DNA (ssDNA) labeled with 6-carboxyfluorescein (FAM) and two kinds of linker DNA strands in terms of Watson-Crick base pairing rule. The highly ordered aggregation of the ssDNA resulted in an enhancement of fluorescence signal by 69 % compared to that of monodisperse one, which significantly improved the sensitivity of the biosensor. Target microRNA triggered the release of bulk BHQ1-DNA from the quencher complex, leading to the fluorescence quenching of the DNA nanosphere. The present biosensing platform achieves a detection limit of 0.2 nM with a linear range from 0.4 nM to 4.0 nM. The results based on human serum samples indicate that the developed DNA biosensing platform could be potentially applied to early clinical diagnosis.

Real-time and rapid quantification of microRNAs in cells and tissues using target-recycled enzyme-free amplification strategy

In this study, a real-time target-recycled enzyme-free amplification strategy-based test (Trefas test) was developed for rapid, simple, isothermal, and highly sensitive microRNA (miRNA) detection. The Trefas relies on rationally designed sequence-specific hairpins (HPs, HP1 and HP2) and the strand displacement process completely free of environment-susceptible enzymes, enhancing the stability and reproducibility of the test. In the absence of target miRNA, the HP2, modified with a fluorophore and a quencher, maintains stem-loop structure so that the fluorescent signal is quenched. However, in the presence of target miRNA, the target miRNA is repeatedly used to trigger continuous HP1-HP2 hybridizations, restoring fluorescence due to the opening of HP2. The developed miR-21 real-time Trefas test exhibited a broad linear dynamic range of 1 pM to 1 μM and a detection limit of 0.58 pM for miR-21 detection in vitro. In particular, the high specificity of the developed miR-21 real-time Trefas test was prominently exhibited by discriminating single base differences in miRNA sequences. Finally, the expression level of miR-21 in the cell lines and clinical tissues was evaluated by the developed miR-21 real-time Trefas test, and the detection results were highly consistent with the results obtained by stem-loop RT-PCR. In summary, our developed test exhibited great potential for further application in biomedical research and early clinical diagnosis.

An enzyme-free amplification strategy based on two-photon fluorescent carbon dots for monitoring miR-9 in live neurons and brain tissues of Alzheimer's disease mice.

An enzyme-free amplification strategy based on two-photon fluorescent carbon dots for monitoring miR-9 in live neurons and brain tissues of Alzheimer's disease (AD) mice. Notably, using our developed probe, miR-9 was found to be up-regulated in early onset AD, while it was found to be down regulated to lower than the normal level in late onset AD.

3D DNA nanosphere-based photoelectrochemical biosensor combined with multiple enzyme-free amplification for ultrasensitive detection of cancer biomarkers

In this work, a new 3D DNA nanosphere was ingeniously designed and fabricated, which was used to combine with multiple enzyme-free amplification strategy to develop a photoelectrochemical (PEC) biosensing platform for ultrasensitive detection of carcinoembryonic antigen (CEA). The 3D DNA nanostructure was self-assembled by base complementary pairing in a few minutes and rolling circle amplification (RCA) reaction. The intense photocurrent derived from Au NPs/ZnSe QDs can be effectively decreased by 3D DNA nanospheres assembled on the electrode, making photoelectric signal present “off” state. The specific binding of target CEA with its hairpin (HP1) aptamer opens HP1 structure, which initiated multiple enzyme-free strand displacement amplification (SDA) reaction and generated a large number of single strands DNA S1. Then S1 competitively binds to capture DNA on the electrode to release 3D DNA nanospheres, thus the photocurrent signal became “on” state for achieving amplified assay of target CEA. The proposed PEC biosensor exhibits excellent performance with a wide linear range of 1.0 fg/mL to 10 ng/mL and a low detection limit of 0.12 fg/mL for CEA, which was successfully applied for the assay of real serum samples with good precision. The reported strategy opens a new simple way for PEC biosensor using DNA nanostructure, showing huge potential in clinical application research.

Integrating PDA microtube waveguide system with heterogeneous CHA amplification strategy towards superior sensitive detection of miRNA

Catalytic hairpin assembly (CHA) is a typical enzyme-free amplification strategy, in which the target can catalyze two hairpin probes to form a duplex and yield multiple outputs signal. However, the non-specific hybridization of two hairpin probes in CHA circuit usually occurred even in the absence of target, causing significant background leakage and impeding its practical applications in trace miRNA analysis. Herein, we proposed a novel heterogeneous CHA (hetero-CHA) design integrating with PDA microtube waveguide system, offering the advantages to enhance the target signal, but suppress the background leakage simultaneously. In hetero-CHA strategy, single-stranded targets are enriched nearby the surface of PDA microtube, facilitating the target-triggered CHA amplification and strand displacement reactions. In contrast, double-stranded DNA complexes formed by uncatalyzed hybridizations are isolated from PDA microtube, impeding the leakage signal. By combination with condensing enrichment effect, the proposed hetero-CHA probe exhibited high selectivity and sensitivity to miRNA target, giving a detection limit as low as 3.3 fM. More importantly, the proposed hetero-CHA probe can be applied directly to distinguish the expression of miRNA-21 in clinical serum of cancer patients (including lung, breast and pancreatic) from those of healthy human beings, favoring the cancer diagnosis and therapeutic evaluation.

Enzyme-free isothermal target-recycled amplification combined with PAGE for direct detection of microRNA-21

MicroRNA-21 (miR-21) has been regarded as a kind of potential biomarker in several types of cancers. Herein, in this study, a simple, sensitive and cost-effective miR-21 approach was developed utilizing the isothermal target-recycled enzyme-free amplification strategy and polyacrylamide gel electrophoresis (PAGE). In the target-recycled enzyme-free amplification strategy, two rational designed hairpin probes (HPs, HP1 and HP2) can form into HP1-HP2 duplex in the presence of miR-21 under the isothermal condition, producing target signal in PAGE. The sensitivity and the linear range of miR-21 approach were demonstrated by the in vitro detection of miR-21, with the detection limit of 10 pM and the linear range of 50 pM to 8 nM. In particular, the contents of miR-21 in cell extractions of different cell lines were successfully detected through miR-21 approach and the relative expression was highly coincident with the results of stem-loop RT-PCR approach. In summary, the developed approach can detect miR-21 sensitively and easily.

Dual enzyme-free amplification strategy for ultra-sensitive fluorescent detection of bisphenol A in water

An ultra-sensitive strategy for bisphenol A (BPA) detection based on dual enzyme-free strategies: hybridization chain reaction (HCR) and enzyme-strand recycling reaction has been developed. The BPA aptamer can form hairpins structure by the partly self-complementary sequence. In the presence of BPA, the released BPA aptamer sequence can trigger the HCR between two hairpins to from a long nicked double-helix DNA. The tails of hairpins on the duplex DNA were closely enough to hybridize with molecular beacon (MB) on the gold nanoparticles (AuNPs) to circularly cleave the loop of MB, leading to a “turn-on” fluorescent signal. This method exhibited high sensitivity for BPA detection in a linear rang from 0.2 to 1000 pM with 0.05 pM of limit of detection. Moreover, it was successfully used for BPA detection in real water samples. Importantly, this method was simple without complex enzymatic procedure and high cost, showing a promising future for on-site detection of BPA in practical application.

Enzyme-Free Amplification Strategy for Biosensing Using Fe3+-Poly(glutamic acid) Coordination Chemistry.

In this work, we outline a signal amplification strategy using the coordination chemistry between Fe3+ and poly(glutamic acid) (PGA) for biosensing applications. The theoretical calculation based on density functional theory shows that PGA has a much higher binding affinity with Fe3+ than the other metal ions. Guided by this rationale, we prepare a PGA-mediated signal probe through conjugating PGA onto polystyrene (PS) nanoparticles to form a brushlike nanostructure for Fe3+ coordination. This PGA-PS brush (PPB) has a large loading capacity of Fe3+ with a number of 1.92 × 108 Fe atoms per nanoparticle that greatly amplifies the signals for assays in an enzyme-free way. Combined with ferrozine coloration-based readout, this PPB-mediated amplification is further applied for the enzyme-free immunoassay that shows an ultrahigh sensitivity for detection of microcystins-LR (12 pg/mL), a 5-fold enhancement compared with that of traditional enzyme-linked immunosorbent assay (ELISA) (60 pg/mL). In addition, the good stability, rapid response, and long shelf life make this enzyme-free amplification strategy a promising platform for point-of-care biosensing applications.

Ultra-sensitive fluorescent and colorimetric detection of UO22+ based on dual enzyme-free amplification strategies

A novel fluorescent and colorimetric strategy with enzyme-free dual amplification was developed for ultra-sensitively detection of uranyl in river waters samples. The enzyme-strand recycling and hairpin catalytic assembly (HCA) reaction were used as dual enzyme-free amplification strategies. Three hairpins labeled with fluorophore can absorb on the surface of gold nanoparticles (AuNPs) to prevent them from aggregation in salt solution and quench the fluorescent signal simultaneously. In the presence of UO22+, the HCA reaction between hairpins was induced to form rigid DNA triangles, resulting in the aggregation of AuNPs with a red to blue color change and a “turn-on” fluorescent signal. The limit of detection was as low as 0.1pM. Importantly, this strategy provided colorimetric and fluorescent dual signals for lower false-positives and higher anti-interference ability. It was successfully used for detection of uranyl in real waters samples.

Proximity aptasensor for protein detection based on an enzyme-free amplification strategy.

A novel electrochemical aptasensor for the detection of trace protein is proposed based on proximity binding-induced strand displacement and hybridization chain reaction. This method is proven to be highly selective and has potential practical utility, and offers new opportunities for the convenient detection of proteins with an enzyme-free amplification process.

Entropy-driven reactions in living cells for assay let-7a microRNA

Imaging of microRNA (miRNA) in living cells could facilitate the monitoring of the expression and distribution of miRNA and research on miRNA-related diseases. Given the low expression levels and even down-regulation of cellular miRNA that is associated with some diseases, enzyme-free amplification strategies are imperative for intracellular miRNA assay. In this work, we report an entropy-driven reaction for amplification assay miRNA with a detection limit of 0.27 pM. The resulting signal amplification provides excellent recognition and signal enhancement of specific miRNAs in living cells. This method supplies accurate information regarding cellular miRNA-related biological events and provides a new tool for highly sensitive and simultaneous imaging of multiple low-level biomarkers, thereby improving the accuracy of early disease diagnosis.

Sensitive detection of intracellular RNA of human telomerase by using graphene oxide as a carrier to deliver the assembly element of hybridization chain reaction.

Since the level of human telomerase RNA (hTR) in tumor cells is higher than that in normal somatic cells, the quantitative assay of hTR is of significant importance in tumor diagnosis. Herein, graphene oxide (GO) was simultaneously exploited as a fluorescence quencher and a carrier of nucleic acid to successfully deliver two hairpin DNA probes of hybridization chain reaction (HCR) into the cancer cell for detecting telomerase RNA based on DNA nanoassembly of HCR. The sticky end of HCR probes could tightly absorb on the surface of GO, resulting in fluorescence quenching of the dye which was tagged at the sticky end of two hairpin probes. When faced with hTR, the fluorescence of DNA probes is subsequently recovered because hTR could trigger HCR to autonomous assembly of a DNA polymer which released from the GO and led to fluorescence recovery. Taking advantage of nucleic acid nanoassembly of HCR, this intracellular HCR strategy creates enormous signal amplification, and enables ultra-sensitive fluorescence imaging of hTR expression. By monitoring fluorescence change, human telomerase RNA could be specifically studied and this method can also be used for detecting single-base mutation. The GO-aided HCR strategy allowed us to sensitively detect hTR in a living cell, which holds great potential for analyzing other low-abundance biomolecules in living cells via HCR.

Selective amyloid β oligomer assay based on abasic site-containing molecular beacon and enzyme-free amplification

Amyloid-beta (Aβ) oligomers are highly toxic species in the process of Aβ aggregation and are regarded as potent therapeutic targets and diagnostic markers for Alzheimer's disease (AD). Herein, a label-free molecular beacon (MB) system integrated with enzyme-free amplification strategy was developed for simple and highly selective assay of Aβ oligomers. The MB system was constructed with abasic site (AP site)-containing stem-loop DNA and a fluorescent ligand 2-amino-5,6,7-trimethyl-1,8-naphyridine (ATMND), of which the fluorescence was quenched upon binding to the AP site in DNA stem. Enzyme-free amplification was realized by target-triggered continuous opening of two delicately designed MBs (MB1 and MB2). Target DNA hybridization with MB1 and then MB2 resulted in the release of two ATMND molecules in one binding event. Subsequent target recycling could greatly amplify the detection sensitivity due to the greatly enhanced turn-on emission of ATMND fluorescence. Combining with Aβ oligomers aptamers, the strategy was applied to analyze Aβ oligomers and the results showed that it could quantify Aβ oligomers with high selectivity and monitor the Aβ aggregation process. This novel method may be conducive to improve the diagnosis and pathogenic study of Alzheimer's disease.