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Split aptamer based sensing platform for adenosine deaminase detection by fluorescence resonance energy transfer
摘要： In this paper, a split aptamer based fluorescence resonance energy transfer (FRET) platform was constructed for the determination of adenosine deaminase (ADA) activity by using gold nanoclusters (AuNCs) and gold nanoparticles (AuNPs). A single adenosine triphosphate (ATP) aptamer was split into two fragments (referred to as P1 and P2). P1 was covalently attached to the AuNCs at the 5′ end (P1-AuNCs), and P2 was labeled with AuNPs at the 3′ end (P2-AuNPs). In the presence of ATP, ATP bound with the two fragments with high affinity to link P1-AuNCs and P2-AuNPs together, thus the fluorescence of P1-AuNCs was quenched via FRET from P1-AuNCs to P2-AuNPs. With the addition of ADA, ATP was transformed into inosine triphosphate (ITP), and then P1 and P2 were released to cause the fluorescence recovery of the system. So a split aptamer based FRET platform for ADA detection can be established via the fluorescence intensity change of the system. This platform showed a good linear relationship between the fluorescence intensity and ADA concentration in the range of 2-120 U L-1, and the limit of detection (LOD) was 0.72 U L-1. Moreover, the detection of ATP in human serum sample demonstrated the accuracy and applicability of the method for ADA detection in real sample.
关键词： Split aptamer,Gold nanoclusters,Adenosine deaminase,Fluorescence,Gold nanoparticles
更新于2025-09-19 17:15:36
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Green fluorescent carbon quantum dots functionalized with polyethyleneimine, and their application to aptamer-based determination of thrombin and ATP
摘要： Brightly fluorescent carbon quantum dots coated with polyethylenimine (PEI-CDs) were prepared using malic acid and PEI as the precursors. The PEI-CDs have a high quantum yield (41%) and green emission (peaking at 502 nm under 430 nm excitation), both of which are not affected by high ionic strength. The PEI-CDs have a positive charge at physiological pH values and can electrostatically bind aptamers with their negative charge. This is shown for aptamers binding thrombin or ATP. Binding of aptamers results in quenching of fluorescence. If thrombin or ATP are introduced, the respective aptamer will bind them, and the complex is then released from the PEI-CDs. Fluorescence increases in proportion to the analyte concentration. Under optimized conditions, thrombin and ATP can be sensitively and selectively detected by fluorometry with lower detection limits of 1.2 and 13 nM, respectively. The assay was successfully applied to the determination of thrombin and of ATP in spiked serum samples.
关键词： Aptamer sensor,Thrombin detection,Serum analysis,Green luminescent,PEI-CDs,Malic acid,Polyethylenimine,Fluorescence assay,ATP detection,Hydrothermal reaction
更新于2025-09-19 17:13:59
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A label-free RTP sensor based on aptamer/quantum dot nanocomposites for cytochrome <i>c</i> detection
摘要： Given the outstanding room-temperature phosphorescence (RTP) of Mn–ZnS quantum dots (QDs) and the specific recognition performance of the aptamer, we built phosphorescent composites from aptamers conjugated with polyethyleneimine quantum dots (PEI-QDs) and applied them to cytochrome c (Cyt c) detection. Specifically, QDs/CBA composites were generated from the electrostatic interaction between the positively-charged PEI-QDs and the negatively-charged Cyt c binding aptamer (CBA). With the presence of Cyt c, the Cyt c can specifically bind with the QDs/CBA composites, and quench the RTP of QDs through photoinduced electron-transfer (PIET). Thereby, an optical biosensor for Cyt c detection was built, which had a detection range of 0.166–9.96 mM and a detection limit of 0.084 mM. This aptamer-mediated phosphorescent sensor with high specificity and operational simplicity can effectively avoid the interference of scattering light from complex substrates. Our findings offer a new clue for building biosensors based on QDs and aptamers.
关键词： aptamer,quantum dots,biosensor,room-temperature phosphorescence,cytochrome c
更新于2025-09-16 10:30:52
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Simultaneous detection of mercury (II), lead (II) and silver (I) based on fluorescently labelled aptamer probes and graphene oxide
摘要： We have developed a fluorescence quantitative analysis method for the simultaneous detection of Hg2+, Pb2+ and Ag+ based on fluorescently labeled nucleic acid aptamer probes and graphene oxide. By this method, three nucleic acid aptamer probes (PHg, PPb, PAg) were designed. The carboxyl fluorescein (FAM), tetramethyl-6-carboxyrhodamine (TAMRA) and cyanine-5 (Cy-5) were respectively selected as fluorophore of aptamer probes, and graphene oxide (GO) was chosen as quencher. In general, these probes were on free single-stranded state and adsorbed on the surface of GO via π-π interactions, which brought fluorophores of probes and GO into close proximity. Due to the fluorescence resonance energy transfer (FRET) occurred between fluorophores and GO, the fluorescence was quenched and fluorescence signals were all weak. Under the optimal condition, fluorescence intensities of three fluorophores exhibited a good linear dependence on corresponding ions concentration. The detection limit for Hg2+, Pb2+ and Ag+ were 0.2 nmol/L, 0.5 nmol/L and 2 nmol/L (3σ, n=11). Average recoveries of this method were 97.56 - 104.92%, which indicated the method had a high accuracy and low detection limit. In addition, this proposed method has good selectivity, and there was no crosstalk effect among these probes.
关键词： Graphene oxide,Simultaneous detection,Metal ions,Fluorescence,Aptamer
更新于2025-09-16 10:30:52
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Conformational flexibility of adenine riboswitch aptamer in apo and bound states using NMR and an X-ray free electron laser
摘要： Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s?1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s?1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s?1. The μs–ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.
关键词： Adenine riboswitch,B-factor,CPMG,Aptamer,Conformational exchange
更新于2025-09-16 10:30:52
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A system composed of vanadium(IV) disulfide quantum dots and molybdenum(IV) disulfide nanosheets for use in an aptamer-based fluorometric tetracycline assay
摘要： A system composed of vanadium(IV) disulfide quantum dots (VS2 QDs) and molybdenum(IV) disulfide (MoS2) nanosheets for use in an aptamer-based fluorometric tetracycline assay was developed. The tetracycline (TET) aptamer was first immobilzed on the VS2 QDs with a typical size of 3 nm. The blue fluorescence of the VS2 QDs (labeled with aptamer) with emission maxima at 448 nm (under excitation at 360 nm) was subsequently quenched by MoS2 nanosheets. If TET is recognized by the aptamer, the VS2 QDs drift away from the basal plane of the MoS2 nanosheets. This generated “turn-on” fluorescence of the VS2 QDs. AVS2 QD/MoS2 nanosheet-based fluorometric TET aptasensor was thus constructed. Selective and sensitive TET bioanalysis was realized in a linear range of 1 to 250 ng mL?1. The detection limit was 0.06 ng mL?1. Its applicability of determination of TET in milk samples has been demonstrated.
关键词： MoS2,Aptamer,VS2 quantum dots,Fluorometric assay,Antibiotic,Turn-on fluorescence
更新于2025-09-11 14:15:04
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Design of Refolding DNA Aptamer on Single-Walled Carbon Nanotubes for Enhanced Optical Detection of Target Proteins
摘要： DNA aptamer conjugated single-walled carbon nanotube (Aptamer-SWNT) hybrids have demonstrated effective optical biosensors because of its high selectivity and specificity to a target protein, however, the understanding of SWNT hybridization with an aptamer forming a secondary or tertiary structure is still lacking. We study wrapping methods dependent optical biosensing modulation by insulin and platelet-derived growth factor (PDGF) using the Aptamer-SWNT hybrids and the Aptamer-Anchor-SWNT hybrids with a periodically sequenced single-stranded DNA (ssDNA) as anchoring phases. We observe that the refolding nature of the aptamer and its combination with an anchoring phase are critical to the hybridization, where the remarkable optical sensing properties are attributed to the wrapping procedures including the direct wrapping with sonication and the indirect wrapping through dialysis. The tetrameric parallel-stranded G-quadruplex conformation of insulin binding aptamers (IBA) shows an enhanced fluorescence response quenching when using the directly wrapped Aptamer-Anchor-SWNT hybrids. In addition, helix-structural refolding of PDGF binding aptamers (PBA) on the SWNT vicinity under the indirect wrapping exhibits anchoring length-dependent optical modulation. Furthermore, the consecutive centrifuging processes with the indirect wrapping demonstrate fluorescence response brightening, in which the diameter dependent brightening effect is observed by aptamer-protein interactions. This study provides an understanding the underlying conjugation nature of both the Aptamer-SWNT and the Aptamer-Anchor-SWNT hybrids formation, facilitating exceptional optical sensing modules with consideration of refolding feature of aptamers, selection of anchoring phases, wrapping methods and centrifuging process, and the hybridization voyage for DNA-SWNT platforms maneuvers their outcoming optical biosensing capabilities.
关键词： insulin,single-walled carbon nanotubes,PDGF,wrapping methods,optical biosensing,DNA aptamer
更新于2025-09-11 14:15:04
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Aptamer-gold nanoparticle doped covalent organic framework followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for selective enrichment and detection of human insulin
摘要： In this work, we introduced an aptamer modified Au nanoparticles doped covalent organic frameworks composite (IBAs-AuNPs/COF) to improve the property of selective enrichment of insulin from serum samples. The Au nanoparticles were immobilized on imine-based COF by in-situ reduction reaction via mussel inspired polydopamine coating, and then sulfhydryl-containing aptamers were bonded to the surface of AuNPs through an Au-S linkage. Due to the excellent adsorption property of COF and specific recognition between insulin and IBAs, the IBAs-AuNPs/COF composites show selective and satisfactory extraction property to insulin in serum samples. Excellent specificity was obtained for insulin in the presence of 50-fold interfering substances including human immunoglobulin, lysozyme and biotin. The concentrations of insulin in the range of 1.0 to 50.0 μg L ?1 show good linear relationship ( R 2 = 0.9917) with limit of detection and limit of quantitation of 0.28 μg L ?1 and 0.93 μg L ?1 , respectively. Then, the IBAs-AuNPs/COF composites were applied to enrich insulin in serum samples followed by analysis with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). After the recovery experiment, the developed method shows good recoveries in range of 91.6%–112.4% with low RSD value (2.4%–9.4%, n = 3) for diabetic and healthy serum samples. The developed IBAs-AuNPs/COF composites propose a new perspective for selective and efficient enrichment of biomarkers in serum samples by functionalized COF.
关键词： Composites,Insulin aptamer,Covalent organic frameworks,Serum samples,Biomolecule,MALDI-TOF-MS
更新于2025-09-11 14:15:04
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A Fluorescent Aptamer/Carbon Dots based Assay for Cytochrome c Protein Detection as a Biomarker of Cell Apoptosis
摘要： Cytochrome c (Cyt c), a heme protein, can be a potential biomarker for cell-apoptosis or even cancer diagnosis. In this work, a simple, rapid, sensitive and selective label-free assay for Cytochrome c (Cyt c) detection is introduced based on an interaction between nucleic acid aptamer biomolecules and surfaces of Carbon Dots (CDs). CDs are used as a fluorescent probes and Cyt c-aptamers as a sensing materials. Interactions of aptamers with CDs quench the fluorescent intensity of CDs. By addition of Cyt c biomolecule as an analyte to the solution and binding to the aptamers, CDs fluorescence turns on. Stronger binding affinity of the aptamers toward Cyt c than CDs, causes they leave the CDs surfaces and the fluorescence is recovered. The amount of recoveries corresponds linearly to the concentration of Cyt c and be used as the basis of detection. The method exhibited high sensitivity to Cyt c with a detection limit of 25.90 nM and a linear range from 40 nM to 240 nM.
关键词： Fluorimetric Detection,Aptamer,Cytochrome c,Carbon dots
更新于2025-09-10 09:29:36
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A multicolor large Stokes shift fluorogen-activating RNA aptamer with cationic chromophores
摘要： Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of 4-hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes-shifted emission from several new green and red HBI analogs that are non-fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functionalization at the C2 position of the imidazolone, this strategy yielded DMHBO+, which binds to the Chili aptamer with a low-nanomolar KD. Because of its highly red-shifted fluorescence emission at 592 nm, the Chili–DMHBO+ complex is an ideal fluorescence donor for F?rster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET-based analytical RNA systems.
关键词： RNA aptamer,fluorescence,fluorescence resonance energy transfer,fluorescent protein,large Stokes shift
更新于2025-09-10 09:29:36