A novel fluorescent aptasensor for sensitive and selective detection of environmental toxins fumonisin B1 based on enzyme-assisted dual recycling amplification and 2D δ-FeOOH-NH2 nanosheets.

Fumonisin (FB) is a pervasive hazardous substance in the environment, presenting significant threats to human health and ecological systems. Thus, the selective and sensitive detection of fumonisin B1 (FB1) is crucial due to its high toxicity and wide distribution in corn, oats, and related products. In this work, we developed a novel and versatile fluorescent aptasensor by combining enzyme-assisted dual recycling amplification with 2D δ-FeOOH-NH2 nanosheets for the determination of FB1. The established CRISPR/Cas12a system was activated by using activator DNA (aDNA), which was released via a T7 exonuclease-assisted recycling reaction. Additionally, the activated Cas12a protein was utilized for non-specifically cleavage of the FAM-labeled single-stranded DNA (ssDNA-FAM) anchored on δ-FeOOH-NH2 nanosheets. The pre-quenched fluorescence signal was restored due to the desorption of the cleaved ssDNA-FAM. Due to the utilization of this T7 exonuclease-Cas12a-δ-FeOOH-NH2 aptasensor for signal amplification, the detection range of FB1 was expanded from 1 pg/mL to 100 ng/mL, with a limit of detection (LOD) as low as 0.45 pg/mL. This study not only provides novel insights into the development of fluorescence biosensors based on 2D nanomaterials combined with CRISPR/Cas12a, but also exhibits remarkable applicability in detecting other significant targets.

In situ enzymatic template replication on DNA microarrays.

DNA microarrays are very useful tools to study the realm of nucleic acids interactions at high throughput. The conventional approach to microarray synthesis employs phosphoramidite chemistry and yields unmodified DNA generally attached to a surface at the 3' terminus. Having a freely accessible 3'-OH instead of 5'-OH is desirable too, and being able to introduce nucleoside analogs in a combinatorial manner is highly relevant in the context of nucleic acid therapeutics and in aptamer research. Here, we describe an enzymatic approach to the synthesis of high-density DNA microarrays that can also contain chemical modifications. The method uses a standard DNA microarray, to which a DNA primer is covalently bound through photocrosslinking. The extension of the primer with a DNA polymerase yields double-stranded DNA but is also amenable to the incorporation of modified dNTPs. Further processing with T7 exonuclease, which catalyzes the degradation of DNA in a specific (5'→3') direction, results in template strand removal. Overall, the method produces surface-bound natural and non-natural DNA oligonucleotides, is applicable to commercial microarrays and paves the way for the preparation of combinatorial, chemically modified aptamer libraries.

A turn-on fluorescent strategy for alkaline phosphatase detection based on enzyme-assisted signal amplification.

As a representative biochemical indicator, alkaline phosphatase (ALP) is of great importance in indicating and diagnosing clinical diseases. Herein, we developed a signal-on fluorescence sensing method for sensitive ALP activity detection based on the enzyme-assisted target recycling (EATR) technique. In this method, a two-step signal amplification process is designed. In the presence of ALP, the 3' phosphate group of an ss-DNA is removed explicitly by ALP, thus releasing free 3'-OH. Terminal deoxynucleotidyl transferase (TdT) can subsequently extend this substrate to generate poly(A) tails, converting the trace-level ALP information into multiple sequences and achieving the first-time amplification. A poly(T) Taqman probe labeled with FAM and BHQ1 provides the second one under the assistance of T7 exonuclease (T7 Exo) through alternate hybridization and degradation of ds-DNA regions. The previously quenched fluorescence is recovered due to the departure of FAM/BHQ1 during the cleavage of T7 Exo. Thus, taking advantage of template-free TdT-mediated polymerization and T7 Exo-based EATR, this strategy shows a sensitive LOD at 0.0074 U/L (S/N = 3) and a linear range of 0.01-8 U/L between ALP concentration and fluorescence intensity. To further verify the specificity and accuracy in practical application, we challenged it in a set of co-existing interference and biological environments and have gained satisfying results. The proposed method successfully quantified the ALP levels in clinical human serum samples, suggesting its applicability in practical application. Moreover, we have used this method to investigate the inhibition effects of Na3VO4. Above all, the proposed assay is sensitive, facile, and cost-effective for ALP determining, holding a promising perspective and excellent potential in clinical diagnosis and drug screening.

Photoelectrochemical monitoring of miRNA based on Au NPs@g-C3N4 coupled with exonuclease-involved target cycle amplification.

Herein, a sensitive photoelectrochemical (PEC) biosensing platform was designed for quantitative monitoring of microRNA-141 (miRNA-141) based on Au nanoparticles@graphitic-like carbon nitride (Au NPs@g-C3N4) as the signal generator accompanying with T7 exonuclease (T7 Exo)-involved target cycle amplification process. Initially, the prepared Au NPs@g-C3N4 as the signal generator was coated on the electrode surface, which could produce a strong PEC signal due to the unique optical and electronic properties of g-C3N4 and the surface plasmonic resonance (SPR) enhanced effect of Au NPs. Meanwhile, the modified Au NPs@g-C3N4 was also considered as the fixed platform for immobilization of S1-S2 through Au-N bond. Thereafter, the T7 Exo-involved target cycle amplification process would be initiated in existence of miRNA-141 and T7 Exo, leading to abundant single chain S1 exposed on electrode surface. Ultimately, the S3-SiO2 composite was introduced through DNA hybridization, thereby producing high steric hindrance to block external electrons supply and light harvesting, which would further cause a significantly quenched PEC signal. Experimental results revealed that the PEC signal was gradually inhibited with the raising miRNA-141 concentration in the range from 1 fM to 1 nM with a detection limit of 0.3 fM. The PEC biosensor we proposed here provides a valuable scheme in miRNA assay for early disease diagnosis and biological research.

An ultrasensitive and specific electrochemical biosensor for DNA detection based on T7 exonuclease-assisted regulatory strand displacement amplification.

An electrochemical biosensor for determination of DNA is developed based on T7 exonuclease-assisted regulatory strand displacement dual recycling signal amplification strategy. First, the hairpin probe recognized and bound the target DNA to form a double strand nucleotide structure, and then the T7 exonuclease was introduced. After be digested by T7 exonuclease, the target DNA was released and entered the next cycle of T7 exonuclease-assisted recycle amplification, while accompanied by a large number of mimic targets (output DNAs) into another cycle. Second, the mimic target reacted with double-chain substrated DNA (CP) by a regulated toehold exchange mechanism, yielding the product complex of detection probes with the help of assisted DNA (S). Finally, after many cycles, a large number of detection probes were produced for binding numerous streptavidin-alkaline phosphatases. The electrochemical biosensor showed very high sensitivity and selectivity with a dynamic response ranged from 0.1 fM to 10 pM with a detection limit of 31.6 aM. Furthermore, this proposed biosensor was successfully applied to the detection of target DNA in 20% diluted serum. The developed strategy has been demonstrated to have the potential for application in molecular diagnostics.

Improving the sensitivity and selectivity of a DNA probe using graphene oxide-protected and T7 exonuclease-assisted signal amplification.

The accurate analysis of single-nucleotide polymorphisms is of great significance for clinical detection and diagnosis. Based on the hybridization hindrance caused by graphene oxide (GO) and hairpin probe, we report a T7 Exo-assisted cyclic amplification technique to distinguish single-base mismatch for highly sensitive and selective detection of mutant-type DNA. When the mutant-type target is completely complementary to the probe, the T7 Exo hydrolyzes the probe and releases the fluorescent molecule from the GO surface, resulting in a fluorescence signal. Conversely, when the wild-type mismatch target is present, the weak hybridization prevents the release of FAM-labeled probe from the GO surface. Therefore, the FAM-labeled probe cannot be degraded efficiently by T7 Exo, and the fluorescence is still quenched by GO. The detection limit of the proposed method can be as low as 34 fM due to the cyclic signal amplification. The experimental results showed that the established method could be used to detect single-nucleotide polymorphisms accurately and sensitively at low cost.

Ratiometric fluorescence sensor based on carbon dots as internal reference signal and T7 exonuclease-assisted signal amplification strategy for microRNA-21 detection.

The expression level of miRNA-21 is closely related to the occurrence and development of cancer, especially in gastrointestinal cancer. Monitoring miRNA-21 has clinical application in the diagnosis and evaluation of gastrointestinal cancer. A turn-on ratiometric fluorescence bioassay based on the T7 exonuclease-mediated cyclic enzymatic amplification method was developed for miRNA-21 determination by using carbon dots (CDs) and FAM-labeled ssDNA as the signal source. CDs demonstrated the triple functions of built-in internal fluorescence, probe carrier, and quencher in this strategy. In the absence of miRNA-21, FAM-labeled ssDNA would be adsorbed and quenched by CDs. The addition of miRNA-21 induced cycle hydrolysis from the 5' end by the T7 exonuclease and then released the short-cleaved FAM-labeled oligonucleotides. Then, the increased FAM signal (FFAM) and the stable CD signal (FCDs) would be tested through a ratiometric routine for the quantification of miRNA-21. The FFAM/FCDs value showed a good linear relationship with the concentration of miRNA-21 in the range of 0.05-10 nM, and the detection limit for miRNA-21 was 1 pM with excellent selectivity and reproducibility. Furthermore, this sensor successfully evaluated the expression level of miRNA-21 in clinical blood samples from healthy individuals and gastrointestinal cancer patients, and the results were highly consistent with those of qRT-PCR, suggesting the great clinical application value in the diagnosis of cancer associated with miRNA-21 expression levels.

A T7 exonuclease assisted dual-cycle signal amplification assay of miRNA using nanospheres-enhanced fluorescence polarization.

Based on streptavidin coated nanospheres and T7 exonuclease assisted dual-cycle signal amplification, we developed a novel sensitive fluorescence polarization detection method for miRNA. When target miRNA was present in the system, hairpin probe hybridized with miRNA, forming a double-stranded structure. The 5' end of hairpin probe was then recognized and digested by T7 exonuclease, releasing the non-degraded single strand DNA fragments and miRNA. The released target miRNA could trigger the next cycle of hybridization and digestion, releasing more non-degraded fragments from hairpin probe. The fragments could hybridize with a signal probe (with carboxyfluorescein modification at 5'-end and biotin modification at 3'-end). The formed blunt 5'-end of signal probe was then recognized and degraded by T7 exonuclease, releasing the fragments and the fluorophore carboxyfluorescein. The next cycle of hybridization and digestion of signal probe was triggered by the released fragment at the same time. The free carboxyfluorescein cannot connect with streptavidin coated nanospheres which were used as the fluorescence polarization signal amplifier. So, there was a big change of fluorescence polarization signal after adding miRNA into the detection system, due to the different fluorescence polarization signal between nanospheres-captured intact signal probe and free carboxyfluorescein. The detection limit of this method is about 0.001 nM, and it has a good selectivity. In addition, it was also applicable to determine target miRNA in total miRNA extracts and compare the expression level of target miRNA in different cells. Consequently, the proposed method is expected to be used for the potential cancer diagnosis and the related biomedical research.

T7 exonuclease-assisted and target-triggered cascade dual recycling signal amplification strategy for the sensitive and specific detection of adenosine.

Adenosine is closely related to the development of cancer, and it can be regarded as a biomarker for cancer diagnosis and therapy. Here, a T7 exonuclease (T7 Exo)-assisted and target-triggered cascade dual recycling signal amplification strategy was developed for the sensitive and specific detection of adenosine. In this strategy, a capture strand (Cap)-inhibit strand (Inh) duplex and a DNA hairpin with intact G-quadruplex sequence are designed. In the presence of adenosine, Cap binds with adenosine specifically to form Cap-adenosine complex with recessed 5'-hydroxyl termini, causing the release of Inh. Under the action of T7 Exo, the adenosine is released from Cap-adenosine complex. Then the released adenosine interacts with the next Cap-Inh duplex to promote the release of a new Inh and the recycle I is completed. After that, the released Inh hybridizes with hairpin to form Inh-hairpin duplex with blunt 5' -hydroxyl termini. Under the same action of T7 Exo, the Inh gets released and a G-quadruplex sequence is obtained. Subsequently, the released Inh continues opening hairpin and the recycle II is accomplished. Finally, plenty of G-quadruplex sequences are generated and then interact with N-methyl-mesoporphyrin IX (NMM) to obtain enhanced fluorescence signal. The limit of detection (LOD) of the proposed strategy is estimated to be 9.8 × 10-7 mol L-1, and the linear range of the strategy is from 5.0 × 10-6 mol L-1 to 7.0 × 10-4 mol L-1. Besides, the proposed strategy is capable of distinguishing adenosine from its analogues. This strategy holds promise in adenosine related biomedical research, clinical diagnosis and disease treatment.

Integration of T7 exonuclease-triggered amplification and cationic conjugated polymer biosensing for highly sensitive detection of microRNA.

A novel and highly sensitive method for detection of microRNA (miRNA) was developed by integration of T7 exonuclease-triggered amplification and cationic conjugated polymer (CCP) biosensing. First, a fluorescein-labeled probe was designed with the complementary sequence to the target miRNA. When target miRNA was absent in the solution, the fluorescence probe interacted with CCP through the strong electrostatic interactions, leading to the highly efficient fluorescence resonance energy transfer (FRET) from CCP to fluorescein. In the presence of target miRNA, the probe hybridized with the miRNA to form DNA/miRNA duplex hybrids. Then, T7 exonuclease digested cyclically the fluorescence probes in hybrids and triggered the enzyme amplification reaction, generating a large number of single nucleotides. Owing to the weak electrostatic interaction between CCP and the single nucleotide, the FRET from CCP to fluorescein would not take place, which effectively reduced the background and significantly enhanced the sensitivity and the dynamic range of miRNA detection. The linear range of the assay was 0.2-100 pM and the detection limit 0.08 pM was 58 times lower than that of the endonuclease-based assay. The method is simple, cost-effective, and with no need for the sophisticated instrument, and has broad application prospects for miRNA detection and early diagnosis.

RNase H meets molecular beacons: an ultrasensitive fluorometric assay for nucleic acids.

An innovative signal amplification strategy assisted by RNase H is described here for the detection of DNA targets in a universal fashion. A tailor-made RNA molecular beacon (RMB) conjugated with a fluorescence resonance energy transfer (FRET) pair (fluorophore and quencher) was designed, characterized, and combined with the employment of RNase H. Its performance is compared to that of other nucleases including Exonuclease III and T7 exonuclease. Fluorometry, performed best at excitation/emission wavelengths of 490/520 nm, gives an amazingly low detection limit of 23 fM for target DNA. The method was verified by the determination of human hemochromatosis (HFE) gene. It is perceived that the method represents a versatile tool for the detection of a wide range of targets. Graphical Abstract An RNase H-assisted signal amplification (RASA) method for the fluorometric assay of nucleic acids has been developed by using a unique RNA molecular beacon (RNA MB) conjugated with a fluorophore (F) and quencher (Q) pair for signal generation.

Multiplex detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

A simple, highly sensitive, and specific assay was developed for the homogeneous and multiplex detection of microRNAs (miRNAs) by combining molecular beacon (MB) probes and T7 exonuclease-assisted cyclic amplification. An MB probe with five base pairs in the stem region without special modification can effectively prevent the digestion by T7 exonuclease. Only in the presence of target miRNA is the MB probe hybridized with the target miRNA, and then digested by T7 exonuclease in the 5' to 3' direction. At the same time, the target miRNA is released and subsequently initiates the nuclease-assisted cyclic digestion process, generating enhanced fluorescence signal significantly. The results show that the combination of T7 exonuclease-assisted cyclic amplification reaction and MB probe possesses higher sensitivity for miRNA detection. Moreover, multiplex detection of miRNAs was successfully achieved by designing two MB probes labeled with FAM and Cy3, respectively. As a result, the method opens a new pathway for the sensitive and multiplex detection of miRNAs as well as clinical diagnosis. Graphical Abstract A simple, highly sensitive, and specific assay was developed for the detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

A T7exonuclease-assisted target recycling amplification with graphene oxide acting as the signal amplifier for fluorescence polarization detection of human immunodeficiency virus (HIV) DNA.

We report a fluorescence polarization (FP) platform for human immunodeficiency virus (HIV) DNA detection based on T7exonuclease-assisted target recycling amplification with graphene oxide (GO) acting as a FP signal amplifier. In the sensing method, the presence of the target DNA leads to target recycling with the assistance of T7exonuclease, furthermore, the amplification products are absorbed onto the surface of GO, so the all FP values are enhanced by GO. More importantly, this FP sensor exhibits high detection sensitivity; under optimal conditions, the change in FP is linear with the concentration of the target DNA within a concentration range of 50-2000 pmol/L, and the detection limit of this method is as low as 38.6 pmol/L. This FP sensor also exhibits high selectivity, even single-base mismatched DNA can be effectively discriminated from complementary target DNA. Above all, the proposed FP sensor may serve as a general platform for the sensitive assay of disease-related genes.

Two-stage cyclic enzymatic amplification method for ultrasensitive electrochemical assay of microRNA-21 in the blood serum of gastric cancer patients.

MicroRNA (miRNA) is riveting nowadays due to its close relevance to human malignancies. Exploiting a fast and convenient biosensor for sensitive and specific detecting miRNA is necessary and it has significant meaning for oncology. In this work, to understand the relationship between miRNA-21 and gastric cancer, we employ T4 RNA ligase 2 to initiate specific ligation reaction depending on the sequence of target RNA, and T7 exonuclease to catalyze the first stage cyclic enzymatic amplification method (CEAM) specifically, which also resting on the target RNA sequence, and the second stage CEAM to further amplify the response resulting from the initial target RNA. Our two-stage CEAM strategy can detect miRNA-21 as low as 0.36fM with remarkable specificity, and most importantly, it can be used to inspect the expression level of miRNA-21 in blood serum of gastric cancer patients. This will offer new perspective for figuring out the pathways of miRNA-21 involving in cancer.

Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel.

T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.

Detection of 3'→5' exonuclease activity using a metal-based luminescent switch-on probe.

A luminescent iridium(III) complex has been discovered to be selective for G-quadruplex DNA, and was employed in a label-free G-quadruplex-based detection assay for 3'→5' exonuclease activity in aqueous solution. A proof-of-concept of this assay has been demonstrated by using prokaryotic exonuclease III (ExoIII) as a model enzyme. In this assay, a G-quadruplex-forming hairpin oligonucleotide (hairpin-G4 DNA, 5'-GAG3TG4AG3TG4A2GCAGA2G2ATA2CT2C4AC3TC4AC3TC-3') initially exists in a duplex conformation, resulting in a low luminescence signal due to the weak interaction between the iridium(III) complex and duplex DNA. Upon digestion by ExoIII, the guanine-rich sequence is released and folds into a G-quadruplex, which greatly enhances the luminescence emission of the iridium(III) probe. This method was highly sensitive for 3'→5' exonuclease over other DNA-modifying enzymes.

A highly sensitive G-quadruplex-based luminescent switch-on probe for the detection of polymerase 3'-5' proofreading activity.

We report herein a luminescent switch-on label-free G-quadruplex-based assay for the rapid and sensitive detection of polymerase proofreading activity using a novel iridium(III) complex as a G-quadruplex-selective probe. The interaction of the iridium(III) complex with the G-quadruplex motif facilitates the highly sensitive switch-on detection of polymerase proofreading activity. Using T4 DNA polymerase (T4 pol) as a model enzyme, the assay achieved high sensitivity and selectivity for T4 pol over other tested enzymes.