Assisted Recycling Amplification Research Articles

Simultaneous detection of multiplex biomarkers related with hepatocellular carcinoma by coupling DNase I-assisted recycling amplification and microfluidic electrokinetic stacking chip with parallel multi-channels

Hepatocellular cancer (HCC) is the fourth most common death related cancer in worldwide. Given that simultaneous multiplex biomarker detection might significantly increase the sensitivity and specificity of HCC diagnosis. Herein, we developed a strategy by coupling 6-carboxyfluorescein labelled aptamer (FAM-Apt), reduced graphene oxide (rGO), deoxyribonuclease I (DNase I)-assisted recycling amplification and microfluidic electrokinetic stacking chip (FAM-Apt/rGO/DNase I-MESC) for detecting multiple HCC biomarkers including α-fetoprotein (AFP), carcinoembryonic antigen (CEA) and microRNA-21 (miR-21). Thereinto, FAM-Apts were first adsorbed by rGO, resulting in fluorescence quenching, and then the FAM-Apts dissociated from rGO after binding the target. Subsequently, the FAM-Apts were degraded by DNase I and the targets were released and recognized by the FAM-Apts on the surface of rGO again. The cyclic dissociation and degradation of FAM-Apts produced FAMs and achieved primary signal amplification. Within 30 min, the released FAMs were accumulated and detected in MESC with three parallel micro-channels, resulting in secondary signal amplification. Based on FAM-Apt/rGO/DNase I-MESC, the limit of detection (LOD) for AFP, CEA and miR-21 were 37.0 pg/mL, 4.5 pg/mL and 1.3 fM, respectively. Remarkably, our method was successfully applied to detect AFP, CEA, and miR-21 in clinical serum samples. The levels of AFP and CEA achieved by our method had a good agreement with the enzyme-linked immunosorbent assay (ELISA) results. The sensitivities of AFP, CEA, miR-21 and the combination of the three targets for differentiating HCC patients from healthy individuals were 71.43%, 57.14%,85.71% and 100%, respectively, indicating the significance of the simultaneous detection of multiple indicators in clinic.

A novel split-type photoelectrochemical biosensor based on double-strand specific nuclease-assisted cycle amplification coupled with plasmonic Ag enhanced BiVO4 performance for sensitive detection of microRNA-155

A novel split-type and signal-on photoelectrochemical (PEC) biosensor was proposed based on double-strand specific nuclease (DSN)-assisted recycling amplification coupled with photodeposited Ag enhanced BiVO4 photoanode for sensitive detection of microRNA (miRNA)− 155. In the presence of target miRNA, the capture DNA (cDNA) modified with silver nanocube（AgNC）would hybridize with the miRNA, then DSN-assisted cycle amplification was triggered, leading to the release of AgNCs. The released AgNCs were acidolyzed and photodeposited on the BiVO4 electrode. Compared with the BiVO4 electrode, an enhanced photocurrent could be achieved at this obtained BiVO4/Ag electrode. And the morphology change from worm-like nanoparticles to nanosheets after Ag photodeposition was observed for the first time. This PEC signal enhancement was mainly contributed to the synergistic action of localized surface plasmon resonance (LSPR) effect of Ag, Schottky junction formed between BiVO4 and Ag, and the formation of BiVO4 nanosheets with larger specific surface area. The PEC signal enhancement was positively related to the miRNA-155 concentration, so this method could be used to sensitively and selectively detect miRNA-155. A limit of detection of 8 aM could be achieved. This strategy does not require layer-by-layer modification of the electrode, so it is easy to operate, and owns good stability.

A FRET-based detection of N-acetylneuraminic acid using CdSe/ZnS quantum dot and exonuclease III-assisted recycling amplification strategy

N-acetylneuraminic acid (Neu5Ac) is widely spread in many biologically significant glycans of mammals, commonly as a terminal α-glycoside. It is of great significance to develop analytical techniques for detection of Neu5Ac. Herein, a high-sensitive fluorescent biosensor for Neu5Ac has been developed based on FRET between CdSe/ZnS quantum dots (QDs) and BHQ2, as well as exonuclease III (Exo III)-assisted recycling amplification strategy. Employing the specially designed three-level FRET systems and fluorescent signal recovery mechanism, together with five-step recycling signal amplification chain reactions, an ultralow detection limit of 24 fM was achieved. Meanwhile, good linear response ranges within 0.2–12.5 pM and 12.5–1000 pM were founded. The assay has excellent performance in real sample detection, and thus offers great potential for detection of sialic acids modified glycans/lipids in the fields of medical diagnosis and food testing.

Functionalized europium-porphyrin coordination polymer: Rational design of high performance electrochemiluminescence emitter for mucin 1 sensing

The excellent characteristics of porphyrins have inspired widespread interest in electrochemiluminescence (ECL). However, the limited ECL intensity and poor stability of porphyrins in aqueous solution are still severely restricted further biological application. Here, we subtly synthesized a functionalized europium and 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin (TCPP) coordination polymer (Eu-PCP) by a one-step solvothermal method. In sharp contrast to the pristine TCPP, Eu-PCP showed a higher and more stable ECL red-light emission (673 nm) at low potential (−1.1 V, vs Ag/AgCl), which was 7.7-fold higher ECL intensity and 4.6-fold efficiency. In view of the crucial role of mucin 1 (MUC1) in tumor overexpression, it was selected as the target molecule. Combined with exonuclease III (Exo III)-assisted recycling amplification strategy, a ternary ECL biosensor was constructed for the MUC1 detection based on Eu-PCP as a satisfied ECL emitter, gold nanoparticles capped CeO2 (CeO2@Au) as the coreactant accelerator and peroxydisulfate as coreactant. Meanwhile, gold nanoparticles capped MnO2 (MnO2@Au) was used as the quenching probe to achieve a highly sensitive detection of MUC1. The proposed biosensor exhibited a wide linear range from 1 fg mL−1 to 10 ng mL−1 with a low limit of detection (0.32 fg mL−1). By changing the corresponding target recognition DNA, this strategy could be expanded to detect other biomarkers.

Highly sensitive microRNA detection by a duplex-specific nuclease amplification triggered three-dimensional DNA machine.

MicroRNAs play important roles in disease diagnosis and therapy. However, current methods for microRNA detection suffer from low sensitivity and cannot directly detect short microRNAs. Herein, we have developed a highly sensitive and selective fluorescent method for direct microRNA detection by combining the duplex-specific nuclease-assisted recycling amplification and the nicking enzyme-powered three-dimensional DNA walker. Target microRNA initiates duplex-specific nuclease-assisted recycling amplification, releasing numerous bipedal walking strands. The released bipedal walking strands hybridize with carboxyfluorescein-labeled track DNA and form nicking recognition site. Driven by the hydrolysis of the nicking enzyme, the bipedal walking strand autonomously moves along the track strand, releasing a large number of carboxyfluorescein-labeled DNA fragments and generating obvious fluorescence signals. This dual-signal amplification method can directly detect microRNA 21 as low as 130 fM and has good selectivity. The proposed method is not only simple for nucleic acid design, but also can be used as a universal method for the highly sensitive detection of all RNAs.

G-triplex molecular beacon‒based fluorescence biosensor for sensitive detection of small molecule-protein interaction via exonuclease III‒assisted recycling amplification

Small molecule-protein interaction plays a significant role in disease diagnosis and biomedical research. Most of the reported fluorescence strategies for small molecule-protein interaction still involve the use of costly fluorophore-labelled probes. In this work, a label-free fluorescent method for sensitive detection of small molecule-protein interaction is proposed. It is based on G-triplex‒based molecular beacon (G3-MB) and exonuclease III (Exo III)‒aided DNA recycling amplification. Biotin-streptavidin interaction is selected as one model for proof-of-concept. The biotin-linked double-strand DNA (dsDNA) is used as the binding probe. In the presence of streptavidin, Exo III hydrolyzes the binding probe to release the trigger DNA, and the released trigger DNA can hybridize G3-MB to trigger the Exo III cleavage process, accompanied by releasing the trigger DNA and G3 sequences. The trigger DNA can recycle, generating a large number of G3 sequences. The generated G3 sequences bind thioflavin T (ThT) to produce strong fluorescence emission. The interaction between small molecule–protein can be detected by the fluorescence reader. This method exhibits low detection limit (5.6 pg/mL), and owns the merits of the simplicity, high specificity and satisfactory applicability in serum sample.

Matrix Coordination-Induced Electrochemiluminescence Enhancement of Tetraphenylethylene-Based Hafnium Metal-Organic Framework: An Electrochemiluminescence Chromophore for Ultrasensitive Electrochemiluminescence Sensor Construction.

Here, we discovered that rigidifying the tetraphenylethylene (TPE)-based ligand H4TCBPE (H4TCBPE = 1,1,2,2-tetra(4-carboxylbiphenyl)ethylene) into Hf-based metal-organic framework (Hf-TCBPE) could lead to a stronger electrochemiluminescence (ECL) emission in comparison to H4TCBPE aggregates and H4TCBPE monomers. Due to the lack of close-packed TCBPE chromophores in Hf-TCBPE, which was required for aggregation-induced ECL (AI-ECL) enhancement, we defined this unprecedented phenomenon as matrix coordination-induced ECL (MCI-ECL) enhancement. The strong ECL intensity of Hf-TCBPE not only originated from the fixation of the TCBPE ligand between Hf6 clusters that restricted the intramolecular free motions of TCBPE and suppressed the nonradiative relaxation but also stemmed from the high porosity of Hf-TCBPE that rendered both internal and external TCBPE chromophores able to be excited. Considering the unique ECL characteristic of Hf-TCBPE, we combined the new ECL indicator of Hf-TCBPE as well as the phosphate-terminal ferrocene (Fc)-labeled hairpin DNA (Fc-HP3) aptamer together as a signal probe (Hf-TCBPE/Fc-HP3), which was employed to construct a novel "off-on" ECL sensor for ultrasensitive mucin 1 (MUC1) detection with the assistance of the exonuclease III (Exo III)-assisted recycling amplification strategy. As expected, the ECL sensor displayed a desirable linear response range from 1 fg/mL to 1 ng/mL and the detection limit down to 0.49 fg/mL. The MCI-ECL enhancement demonstrated by the Hf-TCBPE developed a new and promising strategy to design and synthesize high-performance metal-organic framework (MOF)-based ECL materials for constructing ultrasensitive ECL sensors.

Graphene oxide-based fluorometric determination of microRNA-141 using rolling circle amplification and exonuclease III-aided recycling amplification.

A graphene oxide-based method has been developed for ultrasensitive and selective determination of microRNA-141 by means of rolling circle amplification (RCA) and exonuclease III (Exo III)-assisted recycling amplification. The method uses (a) a padlock probe with a hybrid sequence that is complementary to the target microRNA-141 at both the 5'- and the 3'-end, and (b) a long binding region of a signalling reporter strand. On addition of microRNA-141, it acts as the primer for triggering the RCA reaction following ligation. This results in the formation of a repeatedly concatenated sequence of the padlock probe. Subsequently, the RCA product hybridizes with the fluorescein-labelled signal strand to form the duplex DNA containing the blunt 3'-termini of signal strand. Addition of Exo III causes signal strand digestion and leads to RCA product recycling and liberation of fluorescein. Added graphene oxide does not absorb the fluorescein liberated because of the poor mutual interaction. Therefore, microRNA-141 can be quantified by measurement of the green fluorescence under excitation/emission wavelengths of 470/520nm. The method has a 100 aM detection limit towards microRNA-141and works in the wide range from 1 fM to 1nM. It can discriminate even single-mismatched microRNAand shows good selectivity and sensitivity when applied to spiked human serum. Graphical abstract Schematic representation of agraphene oxide (GO)-based method for fluorometricdetermination ofmicroRNA by using rolling circle amplification and exonuclease III (Exo III)-aided recycling amplification. With microRNA, the fluorescein-labelled signal strand becomes digested, and thisgenererates a fluorescent signal.

Novel 3D Printed Device for Dual-Signaling Ratiometric Photoelectrochemical Readout of Biomarker Using λ-Exonuclease-Assisted Recycling Amplification.

A ratiometric photoelectrochemical (PEC) sensing strategy was proposed for monitoring of carcinoembryonic antigen (CEA) based on a homemade 3D printing device with dual-working photoelectrodes (PE1 and PE2), coupling λ-exonuclease (λ-Exo)-assisted recycling amplification with CdS quantum dots. Gold nanoparticles-functionalized ZnO nanorods were utilized as PEC substrate for generating initial photocurrent and immobilizing DNA probe. Upon incubation of target with DNA trigger/CEA aptamer-modified magnetic bead (tri/apt-MB), DNA trigger dissociated from magnetic bead and then hybridized with capture probe (cp) on PE1 or opened hairpin probe (hp) on PE2 to form double-stranded DNA (dsDNA). The exonuclease could recognize and cleave two newly generated dsDNA, leading to the release of trigger. The free trigger strand continued to hybridize with the remaining cp/hp, which were cleaved by λ-Exo, and then trigger was released again and restarted next recycle with the λ-Exo. After digestion of λ-Exo, the number of capture probes on PE1 was reduced, and many short DNA fragments were produced on PE2, thereby resulting in the decreasing CdS QDs on PE1 and the increasing CdS QDs on PE2. As a result, it was observed that the ratio value of photocurrents (PE1/PE2) significantly decreased with the increasing CEA. Under optimum conditions, the sensing method showed a good linear relationship toward CEA within the dynamic range of 0.02-10 ng mL-1 and a detection limit of 6.0 pg mL-1. Moreover, the ratiometric PEC sensor exhibited good reproducibility, satisfying stability, and remarkable anti-interference performance, which suggests its promising application prospect to detect target CEA.