Amplification Strategy For Ultrasensitive Detection Research Articles

An electrochemical aptasensor based on AuPt@PEI-g-C3N4 combined with a rolling circle amplification strategy for ultrasensitive detection of ciprofloxacin

The precise and sensitive quantification of ciprofloxacin (CIP) is imperative due to its prevalent misapplication and overutilization, which can elicit detrimental side effects. Here, an electrochemical aptasensor based on PEI-g-C3N4 compounded with gold-platinum nanoshell nanomaterials (AuPt@PEI-g-C3N4) combined with a rolling circle amplification (RCA) strategy for an ultrasensitive detection of CIP. Physicochemical properties of nanoparticles and aptamers and analytical parameters of aptasensor were determined by Fourier Transform Infrared, scanning and transmission electron microscopy, gel electrophoresis, cyclic voltammetry, and electrochemical impedance spectroscopy. Furthermore, the RCA strategy was employed to generate long G-rich sequences to load an amount of methylene blue (MB) as signal probes in a potassium ions (K+) environment for signal amplification. In the presence of CIP, the formation of the Apt-CIP complex will cause the dissociation of signal probes from gold electrodes (AuE), reducing electrochemical signals significantly. Under optimal experimental conditions, the designed aptasensor showed excellent comprehensive performance with a good linear relationship in the wide range of 1.0 × 10−4 to 1.0 × 102 ng/mL, and its detection limit was low at 1.03 × 10−5 ng/mL. The assay revealed satisfactory levels of specificity, immunity to interference, stability, and repeatability. Additionally, we successfully applied this aptasensor to spiked milk and surface water samples from Dongfeng Canal (China) for CIP detection.

In situ amplification based on assembly of aptamer sandwiches on nanochannels for ultrasensitive detection of exosomes

Exosomes hold tremendous potential as promising biomarkers for the early diagnosis of cancer. The urgent need for sensitive identification of cancer cell-derived exosomes has emphasized the importance of fabricating innovative approaches. In this work, we propose a novel in situ amplification strategy for ultrasensitive detection of cancer cell-derived exosomes based on the assembly of aptamer sandwiches on nanochannels. Specifically, the nanochannels are functionalized with aptamers targeting the transmembrane receptor protein CD63 to enable selective capture of exosomes. Meanwhile, the aptamers specific to membrane protein EpCAM are employed as anchors for binding with the targeted exosomes. This orchestrated interaction leads to the assembly of aptamer sandwiches, initiating a TdT-mediated polymerization reaction, thereby facilitating in situ amplification. Consequently, ultrasensitive, highly specific, and accurate detection of exosomes can be achieved, with a linear detection range from 2.06 × 103 to 2.06 × 108 particles/mL and a detection limit as low as 6.87 × 102 particles/mL. Moreover, the method shows satisfactory anti-interference capacity for the detection of exosomes in real serum samples, indicating potential applications in early clinical diagnosis of cancer.

Dual-responsive amplification strategy for ultrasensitive detection of norovirus in food samples: Combining magnetic relaxation switching and fluorescence assay

Ultra-sensitive, accurate, and specific diagnostic probes are urgently developed to overcome the limitations of conventional norovirus (NV) probes. Herein, we presented the fabrication of a dual-signal probe using multifunctionalized gold and magnetic nanoparticles hybrid composite (Fe3O4 @Au NPs) carriers for the specific detection of NV by magnetic relaxation-fluorescence bimodal sensing. The dual-specific probes were created by modifying Fe3O4 @Au NPs with antibody (Ab) and aptamer-FAM. The antigen-Ab immunoreaction caused aggregation of Fe3O4 @Au NPs, altering the magnetic relaxation signal; simultaneously, the aptamer-FAM that were originally adsorbed on the Au surface were desorbed and cleaved into the solution in the presence of NV, causing a change in the fluorescence signal. Ultrahigh detection sensitives of 0.38 and 3.45 pg/mL were obtained for the dual-modality detection platform, respectively. Furthermore, two quantitative methods obtained satisfactory spiked recoveries for NV-spiked oyster, scallop, strawberry, and tomato samples, consistent with the results of commercial ELISA kits. The results indicated that this dual-specificity assay not only was ultrasensitive but also markedly boosted analysis reliability and precision. Hence, the dual-mode biosensor exhibits high potential for NV detection in the field of food safety.

Dual signal output ratiometric biosensor platform with regulable nanofiber-mediated signal amplification strategy for ultrasensitive detection of tumor biomarker

It is of great significance for the early diagnosis of cancer to detect the tumor biomarkers in human blood. However, it is still challenging to develop easily available and cost-effective methods to detect the ultralow-level tumor biomarkers. In this work, an innovative signal amplification strategy was developed for the ultrasensitive detection of tumor biomarkers. Amphiphilic peptides with high self-assembly performance can self-assemble into regulable nanofibers and enrich fluorophore to amplify the signal, which can improve the sensitivity of detection. Meanwhile, the design of MOFs with higher fluorescence quenching efficiency and luminescence characteristics can realize double-signal output and further improve the accuracy and sensitivity of detection. Taking alpha-fetoprotein (AFP) as a model tumor biomarker, this strategy achieved a wide linear range of 1–40 ng/mL detection, reducing the limit of detection (LOD) from 9.02 ng/mL to 0.28 ng/mL compared with no signal amplification strategy. Significantly, the results of clinical samples detected by this strategy was consistent with chemiluminescence immunoassay used in clinical practice. Our work provides a brand-new idea for signal amplification strategy and shows an important application prospect in the early diagnosis of various diseases.

Aggregation-Induced Enhancement Electrochemiluminescence of Ag Nanoribbons as a Signal Probe for Ultrasensitive Detection of the Halogenase Gene Segment from Aspergillus Ochratoxin.

Here, an electrochemiluminescence (ECL) biosensor was developed based on aggregation-induced enhancement ECL of Ag nanoribbons (Ag NRs) as a signal probe and the mismatched bases-fueled DNA walker as the amplification strategy for ultrasensitive detection of the halogenase gene segment (target) from aspergillus ochratoxin to evaluate fungi capable of producing ochratoxin in dairy products. Compared with the existing agminated NCs, the Ag NRs displayed dramatically enhanced ECL emission due to effective electron transfer and less energy dissipation of excited-state Ag NRs based on the metallophilic interaction of Ag(I)-Ag(I). Impressively, the target was conversed mismatched bases-DNA walker to trigger a significant ECL response through toehold-mediated strand displacement amplification. Thus, the strategy realized trace analysis of the halogenase gene segment with a detection limit of 45 aM, which ushered a new detection method for toxin determination in the previous stage of toxin biosynthesis to protect people from toxic damage.

A novel Aptamer-induced CHA amplification strategy for ultrasensitive detection of Staphylococcus aureus and NIR-triggered photothermal bactericidal Activity based on aptamer-modified magnetic Fe3O4@AuNRs

The development of multifunctional biosensors integrating recognition and therapeutic functions is critical for ensuring food safety and preventing foodborne diseases. Herein, an integrated design strategy was demonstrated to establish an innovative Fe3O4 @AuNRs-aptasensor for efficient detecting Staphylococcus aureus (S. aureus), which could enrich and photothermally eliminate bacteria under the radiation of near-infrared light irradiation (NIR). The detection assay relies on the binding of aptamer to Fe3O4 @AuNRs (Fe3O4 @ AuNRs-aptasensor) to induce the release of c-aptamer (c-Apt) in the presence of S. aureus, thereby triggering catalytic hairpin assembly (CHA) reaction and restoring the fluorescence of the reporter. The developed biosensor achieved direct, ultrasensitive, and specificity detection of S. aureus with a linear response ranging from 102 to 106 CFU/mL and a detection limit of 101 CFU/mL under optimal conditions. Furthermore, the aptasensor can be applied for the detection of S. aureus in milk with recoveries of 90.3–95.6% at the concentration between 1.0 × 102 and 1.0 × 104 CFU/mL. Additionally, Fe3O4 @AuNRs-aptasensor shows high photothermal sterilization performance under NIR irradiation. The study emphasizes the combination of high sensitivity base on molecular detection and photothermal antibacterial activity of novel nanomaterials activated by NIR light to design multifunctional probe, which show great potential for application in food analysis and early foodborne disease diagnosis.

A fluorescent sensor based on the cascade signal amplification strategy for ultra-sensitive detection of Cu2.

Copper is an essential element in the human body, participating in various physiological activities in the bodies of organisms. However, an excessive load of Cu2+ is associated with several neurodegenerative diseases and prion diseases, also identified as a symptom of Wilson's disease (WD). A straightforward, rapid, sensitive, and specific copper sensor is highly required but remains a challenge. In this study, guided by the simulation, we developed a chemical sensor using a cascade signal amplification strategy based on the Cu-catalyzed click reaction, combined with a fluorescence-enhanced substrate with gold nanorods coupled with silver nanoislands. The sensor can selectively detect Cu2+ as low as 3.87 nM within 10 min. We have demonstrated that this method can be directly employed for WD diagnosis in urine samples. In addition, using antibiotic susceptibility testing (AST) as an example, we verify whether this assay can be adapted to other targets where Cu is designed as an indirect indicator.

Plasmon-Enhanced Bimodal Nanosensors: An Enzyme-Free Signal Amplification Strategy for Ultrasensitive Detection of Pathogens.

Increasing foodborne illnesses have led to global health and economic burdens. E. coli O157:H7 is one of the most common disease-provoking pathogens and known to be lethal Shiga toxin-producing E. coli (STEC) strains. With a low infection dose in addition to person-to-person transmission, STEC infections are easily spread. As a result, specific and rapid testing methods to identify foodborne pathogens are urgently needed. Nanozymes have emerged as enzyme-mimetic nanoparticles, demonstrating intrinsic catalytic activity that could allow for rapid, specific, and accurate pathogen identification in the agrifood industry. In this study, we developed a sensitive nanoplatform based on the traditional ELISA assay with the synergistic properties of gold and iron oxide nanozymes, replacing the conventional enzyme horseradish peroxidase (HRP). We designed an easily interchangeable sandwich ELISA composed of a novel, multifunctional magneto-plasmonic nanosensor (MPnS) with target antibodies (MPnS-Ab). Our experiments demonstrate a 100-fold increase in catalytic activity in comparison to HRP with observable color changes within 15 min. Results further indicate that the MPnS-Ab is highly specific for E. coli O157:H7. Additionally, effective translatability of catalytic activity of the MPnS technology in the lateral flow assay (LFA) platform is also demonstrated for E. coli O157:H7 detection. As nanozymes display more stability, tunable activity, and multi-functionality than natural enzymes, our platform could provide customizable, low-cost assay that combines high specificity with rapid detection for a variety of pathogens in a point-of-care setup.

A self-assembly amplification strategy for ultra-sensitive detection of microRNA based on phosphorothioated probes

Based on self-assembly amplification, we designed a novel microRNA (miRNA)-detection method with high specificity and sensitivity. Two unique DNA probes named Linker A and Linker B were modified with phosphorothioate (PS) at both ends. In the presence of the target miRNA, these two DNA probes were ligated together by T4 DNA ligase enzyme to form a dumbbell-shaped DNA. Then the dumbbell-shaped structure would be extended with Bst 2.0 DNA polymerase enzyme, triggering the strand displacement activity without using additional primers. These results revealed our method's ultralow detection limit (300 fM), excellent selectivity, simple operation, and capability to discriminate single-base mismatches. It is believed that this proposed approach would have great application potential in clinical diagnosis and other involved fields.

A Self-Assembly Amplification Strategy for Ultra-Sensitive Detection of Microrna Based on Phosphorothioated Probes

A Self-Assembly Amplification Strategy for Ultra-Sensitive Detection of Microrna Based on Phosphorothioated Probes

The PCR-HCR dual signal amplification strategy for ultrasensitive detection of Escherichia coli O157:H7 in milk

In this study, we established a novel polymerase chain reaction-hybridization chain reaction (PCR-HCR) dual signal amplification assay for sensitive detection of Escherichia coli O157:H7 in milk. In this assay, a blocker forward primer and a biotin reverse primer based on E. coli O157:H7 specific fliCh7 gene were specifically designed for PCR amplification resulting in a single-double stranded DNA with biotin (ss-dsDNA~biotin). These PCR products, ss-dsDNA~biotin, were captured by magnetic beads (MBs) decorated with streptavidin (SA) forming the ss-dsDNA~biotin+SA~MBs. The ssDNA on the PCR products opened the hairpin probe (H1 and H2) to trigger the HCR for secondary signal amplification. This is the first to use HCR to directly amplify PCR products through a novel blocker primer. With the help of two nucleic acid dual signal amplification techniques, PCR-HCR analysis can effectively improve the detection sensitivity and the limit of detection is 7.2 × 101 CFU/mL. In addition, this assay has been successfully used to detection target bacteria in skim milk spiked samples.

Reduced graphene oxide nanosheets and gold nanoparticles covalently linked to ferrocene-terminated dendrimer to construct electrochemical sensor with dual signal amplification strategy for ultra-sensitive detection of pesticide in vegetable

We report herein a novel electrochemical signal amplification strategy for ultra-sensitive detection of pesticide by covalently linking nanoparticles (AuNPs) and reduced graphene oxide (rGO) nanosheets to ferrocene units of ply (amine) ester based ferrocene dendrimer(FcDr).The FcDr dendrimers with the native redox signaling center (Fe3+/Fe2+) provide a large number of active terminal carboxyl groups as well as a compact matrix for the covalent linking of rGO nanosheets and AuNPs. AuNPs has superior electrical conductivity and catalytic activity, while rGO nanosheets have a larger surface area that allow more deposition of AuNPs. In this way, AuNPs and rGO nanosheets can synergistically accelerate the electron transfer from FcDr to glassycarbonelectrode (GCE), which will amplify the electrochemical signal of AuNPs/FcDr/rGO/GCE sensor and then improve the sensing performance. The fabricated electrode demonstrates an admirable electrochemical sensing performance towards dichlorvos in terms of low detection limit (0.21 μM), wide concentration range (0.43–218.4 μM) and rapid response time (1.8 s). The practical applicability of the sensor was successfully evaluated in real vegetable sample and achieved satisfactory accuracy. This designed approach provides a facile, efficient strategy for timely detection of dichlorvos.

Anode-Driven Controlled Release of Cathodic Fuel via pH Response for Smart Enzymatic Biofuel Cell.

SummaryEnzymatic biofuel cells (EBFCs) with or without a membrane to separate the anodic and cathodic compartments generally suffered from high internal resistance or interactive interference, both of which restricted the improvement of their performance. Herein, a smart membrane-less EBFC was engineered based on anode-driven controlled release of cathodic acceptor via pH-responsive metal-organic framework ([Fe(CN)6]3-@ZIF-8) nanocarriers. The glucose anodic oxidation would produce gluconic acid accompanied by the change in pH value from neutral to the acidic case, which could drive the degradation of [Fe(CN)6]3-@ZIF-8 nanocarriers and further realize the controlled release of cathodic acceptor [Fe(CN)6]3-. More importantly, compared with controlled EBFC with or without membrane, the power output of the as-proposed EBFC enhanced at least 700 times due to the seamless electronic communication. Therefore, the ingenious strategy not only realized the successful engineering of the membrane-less EBFC but also provided an appealing idea for constructing smart devices.

Enzymatic Biofuel-Cell-Based Self-Powered Biosensor Integrated with DNA Amplification Strategy for Ultrasensitive Detection of Single-Nucleotide Polymorphism.

Enzymatic biofuel cell (EBFC)-based self-powered biosensors could offer significant advantages: no requirement for an external power source, simple instruments, and easy miniaturization. However, they also suffered from the limitations of lower sensitivity or specific targets. In this study, a self-powered biosensor for the ultrasensitive and selective detection of single nucleotide polymorphisms (SNPs) produced by combining the toehold-mediated strand displacement reaction (SDR) and DNA hybridization chain reaction (HCR) was proposed. Herein, the capture probe (CP) with an external toehold was designed to switch on the sensing system. In the presence of target sequence, both SDR and DNA HCR reaction would happen to produce a long double-helix chain. Because of the electrostatic interaction between [Ru(NH3)6]3+ and the double-helix chain described above, the open circuit voltage ( EOCV) of the as-proposed biosensor was significantly elevated, thus realizing the detection of SNPs. Overall, in this work, an ingeniously constructed self-powered biosensor for the detection of SNPs was created by integrating EBFCs with a DNA amplification strategy. Furthermore, the as-proposed self-powered biosensor not only showed prominent specificity to distinguish the p53 gene fragment from random sequences (e.g., single-base mutant sequences) but exhibited excellent sensitivity with the detection limit of 20 aM. More importantly, the results obtained from the real cell lysate sample have laid a strong foundation for disease diagnostics and, potentially, as a powerful tool for even more fields.

Palindromic molecular beacon-based intramolecular strand-displacement amplification strategy for ultrasensitive detection of K-ras gene

The sensitive detection of tumor proto-oncogenes is indispensable because the early diagnosis and accurate treatment of genetic diseases is the key guarantee of patients' health. In this study, we proposed a novel palindromic molecular beacon (PMB) that it bases on the signal amplification strategy for ultrasensitive detection of Kras gene codon 12. PMB is designed to have two palindromic fragments at its two ends, one of which is locked via folding into a hairpin structure and the other promotes the formation of PMB duplex via intermolecular self-hybridization. Target DNA can hybridize to the loop portion of PMB and release the palindromic fragment at the 3’ end. Within the PMB duplex, the two palindromic fragments released hybridize with each other and serve as polymerization primer responsible for the strand-displacement amplification (SDA). Namely, hybridized target DNA can be displaced and initiates the next round of reactions, making the polymerization/displacement/hybridization process go forward circularly. As a result, a large number of polymerization products are produced, dramatically enhancing optical signal. Because primer hybridization and polymerization-based displacement occur within PMB duplex, the reaction process is called intramolecular strand-displacement amplification (ISDA). Via utilizing the newly-proposed PMB-based ISDA strategy, the target K-ras gene could be detected down to 10 pM with a wide response range of 1 × 10−11-1.5 × 10−7 M, and point mutations are easily distinguished, realizing the ultrasensitive, highly selective detection of K-ras gene. This impressive sensing paradigm demonstrates a new concept of signal amplification for the detection of disease-related genes only via using a simple way to efficiently amplify optical signal.

An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity.

DNA methylation is a significant epigenetic mechanism involving processes of transferring a methyl group onto cytosine or adenine. Such DNA modification catalyzed by methyltransferase (MTase) plays important roles in the modulation of gene expression and other cellular activities. Herein, we develop a simple and sensitive biosensing platform for the detection of DNA MTase activity by using only two oligonucleotides. The fluorophore labeled molecular beacon (MB) can be methylated by MTase and subsequently cleaved by endonuclease DpnI at the stem, giving a shortened MB. The shortened MB can then hybridize with a primer DNA, initiating a cycle of strand displacement amplification (SDA) reactions. The obtained SDA products can unfold new MB and initiate another cycle of SDA reaction. Therefore, continuous enlargement of SDA and exponential amplification of the fluorescence signal are achieved. Because the triple functions of substrate, template and probe are elegantly integrated in one oligonucleotide, only two oligonucleotides are necessary for multiple amplification cycles, which not only reduces the complexity of the system, but also overcomes the laborious and cumbersome operation that is always a challenge in conventional methods. This platform exhibits an extremely low limit of detection of 3.3 × 10-6 U mL-1, which is the lowest to our knowledge. The proposed MTase-sensing platform was also demonstrated to perform well in a real-time monitoring mode, which can achieve a further simplified and high-throughput detection. The sensing strategy might be extended to the activity detection of other enzymes, thus showing great application potential in bioanalysis and clinical diagnosis.

Perylene Derivative/Luminol Nanocomposite as a Strong Electrochemiluminescence Emitter for Construction of an Ultrasensitive MicroRNA Biosensor.

In this work, an electrochemiluminescent (ECL) biosensor was constructed on the basis of amino-modified 3,4,9,10-perylenetetracarboxylic dianhydride/luminol (PTC-NH2/Lu) nanocomposite as emitter and bipedal DNA walker signal amplification strategy for ultrasensitive detection of microRNA-21 (miRNA-21). The PTC-NH2/Lu nanocomposite was prepared as signal tag via π-π stacking molecular assembly, in which amino-modified 3,4,9,10-perylenetetracarboxylic dianhydride (PTC-NH2) as a novel coreaction accelerator significantly enhanced the ECL emission of luminol-H2O2 system. Moreover, target miRNA-21 triggered bipedal DNA walker was powered by toehold-mediated strand displacement reaction (TSDR) for signal amplification. Consequently, the proposed ECL biosensor achieved ultrasensitive detection of miRNA-21 with a linear range from 100 aM to 100 pM and a limit of detection of 33 aM. Simultaneously, the biosensor was also successfully applied to detect target miRNA-21 in lysates from human cancer cells. As a result, this work constructed a new signal amplification platform, exhibiting great application potential in biomedical analysis and early clinical diagnostics.

Ultrasensitive Detection of DNA Based on Exonuclease III-Assisted Recycling Amplification and DNAzyme Motor.

Herein, we have developed a dual amplification strategy for ultrasensitive detection of DNA combining exonuclease III (Exo III)-assisted reaction and DNAzyme motor. DNA probes are carefully designed; thus, target recognition and the first amplification cycle are accomplished simultaneously, which makes the operation very convenient. Moreover, the self-powered DNAzyme motor may translate a single binding event into cleavage of multiple fluorescence probes, which significantly heightens the signal intensity. As a result, the limit of detection as low as 21 fM is achieved. The fluorescence intensity is found to have a linear relationship with respect to the logarithm of DNA concentration in a wide range from 100 fM to 10 nM. This proposed method shows great potential for the applications of biological studies and clinical diagnosis.

A ratiometric fluorescent biosensor based on cascaded amplification strategy for ultrasensitive detection of kanamycin

Antibiotic residues in food products significantly threaten public health. In the present work, a fluorescence resonance energy transfer (FRET)-based ratiometric fluorescent biosensor was developed for high sensitive and selective detection of kanamycin by combining aptamer-recognition assay and signal amplification strategy. To improve detection sensitivity, the optimum FRET efficiency of donor-acceptor was explored by modulating the separation distance between the donor (Cy3) and the acceptor (Cy5). In the developed biosensing system, kanamycin could be trapped by its aptamer, which triggered a polymerase-catalyzed amplification (PCA) process to autonomously generate a single-strand DNA. In subsequence, the generated DNA strand initiated the second amplification process of catalyzed hairpin assembly (CHA) to cause fluorescence decreasing of Cy3 and increasing of Cy5. The signal change caused by FRET of donor (Cy3)-acceptor (Cy5) was then augmented by the PCA and CHA processes. The constructed biosensors results in high sensitive detection of kanamycin as low as 0.29 nM within a linear range from 1.0 nM to 80.0 nM. The potential application of the developed biosensing system was evaluated with milk as real sample. The provided strategy could serve as a prototype for developing FRET-based ratiometric bioassays.

Terminal Deoxynucleotidyl Transferase and T7 Exonuclease-Aided Amplification Strategy for Ultrasensitive Detection of Uracil-DNA Glycosylase.

As one of the key initiators of the base excision repair process, uracil-DNA glycosylase (UDG) plays an important role in maintaining genomic integrity. It has been found that aberrant expression of UDG is associated with a variety of diseases. Thus, accurate and sensitive detection of UDG activity is of critical significance for biomedical research and early clinical diagnosis. Here, we developed a novel fluorescent sensing platform for UDG activity detection based on a terminal deoxynucleotidyl transferase (TdT) and T7 exonuclease (T7 Exo)-aided recycling amplification strategy. In this strategy, only two DNA oligonucleotides (DNA substrate containing one uracil base and Poly dT probe labeled with a fluorophore/quencher pair) are used. UDG catalyzes the removal of uracil base from the enclosed dumbbell-shape DNA substrate to give an apyrimidinic site, at which the substrate oligonucleotide is cleaved by endonuclease IV. The released 3'-end can be elongated by TdT to form a long deoxyadenine-rich (Poly dA) tail, which may be used as a recyclable template to initiate T7 Exo-mediated hybridization-digestion cycles of the Poly dT probe, giving a significantly enhanced fluorescence output. The proposed UDG-sensing strategy showed excellent selectivity and high sensitivity with a detection limit of 1.5 × 10-4 U/mL. The sensing platform was also demonstrated to work well for UDG inhibitor screening and inhibitory activity evaluation, thus holding great potential in UDG-related disease diagnosis and drug discovery. The proposed strategy can be easily used for the detection of other DNA repair-related enzymes by simply changing the recognition site in DNA substrate and might also be extended to the analysis of some DNA/RNA-processing enzymes, including restriction endonuclease, DNA methyltransferase, polynucleotide kinase, and so on.