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Convenient Construction of Orthogonal Dual Aptamer-Based Plasmonic Immunosandwich Assay for Probing Protein Disease Markers in Complex Samples and Living Animals
摘要： Aptamers, due to their outstanding merits including simple synthesis and easy modification, have been widely used as antibody alternatives to construct novel immunosandwich assays. Dual aptamer-based sandwich assays exhibit multiple advantages over conventional immunosandwich assays and single aptamer-based sandwich assays. However, their construction is hampered by the limited knowledge of binding orthogonality of aptamers reported in literature. Herein we present a new strategy for conveniently constructing an orthogonal dual aptamer-based plasmonic immunosandwich assay (odA-PISA) for probing proteins in complex samples and living animals. An orthogonal aptamer pair was first efficiently selected from aptamers reported in literature by affinity capillary electrophoresis (ACE). Then, a target protein-capturing gold thin layer-coated probe and silver nanoparticle-based Raman labeling nanotags were conveniently prepared with the selected aptamers and used to construct the assay. The double aptamers used ensured the specificity while the plasmonic coupling effect between the target-capturing probe and Raman nanotags generated significantly enhanced Raman signal intensity, providing high sensitivity. As a proof of principle, alkaline phosphatase (ALP) was used as the target. The constructed odA-PISA exhibited high specificity and high sensitivity toward ALP, giving cross-reactivity ≤ 4.2% and limit of detection (LOD) of 3.8 pM (S/N = 4). Quantitative determination of ALP in human serum and probing ALP in tumor-bearing mice were achieved, showing the great application potential of the method. This strategy is widely applicable to other protein disease markers. Therefore, it opened a new access to the construction of sensitive dual aptamer-based sandwich assays for real-world applications particularly disease diagnosis.
关键词： aptamer,binding orthogonality,Raman spectrometry,plasmonic immunosandwich assay,disease marker
更新于2025-09-23 15:19:57
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Target-responsive ratiometric fluorescent aptasensor for OTA based on energy transfer between [Ru(bpy)3]2+ and silica quantum dots
摘要： A ratiometric fluorescent aptasensor based on energy transfer between [Ru(bpy)3]2+ and silica quantum dots (silica QDs) for assaying OTA was fabricated. The aptamer for OTA was used as the gate to shield the fluorescent reagent [Ru(bpy)3]2+ into mesoporous silica nanoparticle (MSN). In the presence of OTA, the constrained [Ru(bpy)3]2+ was released from MSN due to a target-induced aptamer conformational change. The released [Ru(bpy)3]2+ adsorbed onto the negatively charged silica QDs through electrostatic interaction. This creates appearance of fluorescence from [Ru(bpy)3]2+ at 625 nm and decrease of the fluorescence from silica QDs at 442 nm owing to the energy transfer. The value of FL625nm/FL442nm was in proportion to the concentration of OTA in the range 0.5~100 ng mL?1 with a LOD of 0.08 ng mL?1. Practical applicability of this method was validated by the determination of OTA in flour samples.
关键词： Ochratoxin A,Ratiometric fluorescent sensor,Aptamer,Silica quantum dots,[Ru(bpy)3]2+
更新于2025-09-23 15:19:57
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Development of an Aptamer Based Luminescent Optical Fiber Sensor for the Continuous Monitoring of Hg2+ in Aqueous Media
摘要： A ?uorescent optical ?ber sensor for the detection of mercury (Hg2+) ions in aqueous solutions is presented in this work. The sensor was based on a ?uorophore-labeled thymine (T)-rich oligodeoxyribonucleotide (ON) sequence that was directly immobilized onto the tip of a tapered optical ?ber. In the presence of mercury ions, the formation of T–Hg2+-T mismatches quenches the ?uorescence emission by the labeled ?uorophore, which enables the measurement of Hg2+ ions in aqueous solutions. Thus, in contrast to commonly designed sensors, neither a ?uorescence quencher nor a complementary ON sequence is required. The sensor presented a response time of 24.8 seconds toward 5 × 10?12 M Hg2+. It also showed both good reversibility (higher than the 95.8%) and selectivity: the I0/I variation was 10 times higher for Hg2+ ions than for Mn2+, and Cu2+, Fe3+, Cd2+ contaminants examined (Co2+, Mn2+, Zn2+, Ca2+, Pb2+, Ni2+, Ag+, and Cu2+) presented an even lower interference. The limit of detection of the sensor was 4.73 × 10?13 M Hg2+ in ultrapure water, and was also able to detect 5 × 10?12 M Hg2+ in buffer solution and 9.03 × 10?13 M Hg2+ in tap water.
关键词： luminescent biosensor,?uorophore-labelled aptamer,optical ?ber biosensor,mercury detection
更新于2025-09-23 15:19:57
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Metal-labeled Aptamers as Novel nanoprobes for Imaging Mass Cytometry Analysis
摘要： Imaging mass cytometry (IMC) is an emerging imaging technology that exploits the multiplexed analysis capabilities of the CyTOF mass cytometer to make spatially resolved measurements for tissue sections. In a comprehensive view of tissue composition and marker distribution, recent developments of IMC require highly sensitive, multiplexed assays. Approaching the sensitivity of the IMC technique, we designed a novel type of biocompatible metal-labeled aptamer nanoprobe (MAP), named 167Er-A10-3.2. The small molecular probe was synthesized by conjugating 167Er-polymeric pentetic acid (167Er-DTPA) with an RNA aptamer A10-3.2. For demonstration, 167Er-A10-3.2 was applied for observing protein spatial distribution on prostatic epithelium cell of paraffin embedded Prostatic adenocarcinoma (PaC) tissue sections by IMC technology. The 167Er-A10-3.2 capitalizes on the ability of aptamer to specifically bind target cancer cells, as well as the small size of 167Er-A10-3.2 can accommodate multiple aptamer binding antigen labeled at high density. The detection signal of 167Er-A10-3.2 probe was 3-fold higher than that of PSMA antibody probe for a targeted cell under lower temperature epitope retrieval (37°C) of PaC tissue. Furthermore, we successfully demonstrated the simultaneously staining ability of aptamer probes in IMC analysis. The successful imaging acquisition using aptamers probes in IMC technology may offer opportunity for the diagnosis of malignancies in the future.
关键词： imaging mass cytometry,prostate specific membrane antigen,molecular probe,metal labeled aptamer
更新于2025-09-23 15:19:57
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A fluorometric aptasensor for bisphenol a based on the inner filter effect of gold nanoparticles on the fluorescence of nitrogen-doped carbon dots
摘要： An aptamer-based fluorometric assay is described for the determination of bisphenol A (BPA). The aptamer against BPA is first attached to the surface of the red AuNPs, and this prevents the AuNPs from salt-induced formation of a blue-colored aggregate. Hence, the blue fluorescence of added nitrogen-doped carbon dots (NCDots) is quenched via an inner filter effect (IFE) caused by the red AuNPs. After addition of BPA, the BPA/aptamer complex is formed, and the AuNPs are no longer stabilized agains aggregation. This weakens the IFE and results in the recovery of the fluorescence of the NCDots which is measured best at excitation/emission wavelengths of 300/420 nm. The recovered fluorescence increases linearly in the 10 to 250 nM and 250 to 900 nM BPA concentration ranges, and the detection limit is 3.3 nM. The method was successfully applied to the determination of BPA in spiked environmental tap water samples.
关键词： Wide linear range,Quick response,Salt-induced aggregation,Tap water,Low detection limit,BPA/aptamer complexes,Aggregated AuNPs,Environmental-friendly,Fluorescence quenched,Fluorescence recovery
更新于2025-09-23 15:19:57
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Co-ordinated Split Aptamer Assembly and Disassembly on Gold Nanoparticle for Functional Detection of HIV-1 Tat
摘要： Human immunodeficiency virus (HIV) is a life threatening, weakens the immune system upon infection, thus ultimately resulting in the fatal health issues. This situation necessitates the generation of different strategies for HIV detection. HIV-1 Tat, a transactivator of HIV gene expression, was chosen in this study as the target of a non-functional split aptamer. Implementation of split aptamer has been demonstrated in this work for colorimetric detection of HIV-1 Tat. An unmodified gold nanoparticle (GNP)-based colorimetric assay was used for the visible detection of the proof, displays color transitions from red to purple in relation to the dose-dependency of HIV-1 Tat against the split aptamer in ionic solutions. The visible color transition was characterized using UV-Vis spectrophotometer showing spectrum shift and supported by Scanning Electron Microscopy observation. With addition of sodium chloride, the color of the solution started to change to purple and spectrum started to shift to higher wavelength due to aggregation at HIV-1 Tat concentration as low as 10 nM. Specificity test was conducted with duplexed split aptamer and HIV-1 p24 has shown slight color changes. With HIV-1 Nef, GNP solution retains the color similar to the control, which indicated the specific split aptamer interaction to HIV-1 Tat.
关键词： Colorimetry,HIV-1 Tat,Gold nanoparticle,Split aptamer
更新于2025-09-23 15:19:57
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Broccoli Fluorets: Split Aptamers as a User-Friendly Fluorescent Toolkit for Dynamic RNA Nanotechnology
摘要： RNA aptamers selected to bind fluorophores and activate their fluorescence offer a simple and modular way to visualize native RNAs in cells. Split aptamers which are inactive until the halves are brought within close proximity can become useful for visualizing the dynamic actions of RNA assemblies and their interactions in real time with low background noise and eliminated necessity for covalently attached dyes. Here, we design and test several sets of F30 Broccoli aptamer splits, that we call fluorets, to compare their relative fluorescence and physicochemical stabilities. We show that the splits can be simply assembled either through one-pot thermal annealing or co-transcriptionally, thus allowing for direct tracking of transcription reactions via the fluorescent response. We suggest a set of rules that enable for the construction of responsive biomaterials that readily change their fluorescent behavior when various stimuli such as the presence of divalent ions, exposure to various nucleases, or changes in temperature are applied. We also show that the strand displacement approach can be used to program the controllable fluorescent responses in isothermal conditions. Overall, this work lays a foundation for the future development of dynamic systems for molecular computing which can be used to monitor real-time processes in cells and construct biocompatible logic gates.
关键词： Broccoli,RNA nanotechnology,dynamic nanoparticles,aptamer,conditional activation
更新于2025-09-19 17:15:36
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Simultaneous detection of fumonisin B1 and ochratoxin A using dual-color, time-resolved luminescent nanoparticles (NaYF4: Ce, Tb and NH2-Eu/DPA@SiO2) as labels
摘要： A rapid and highly sensitive time-resolved fluorescence (TRF)-based aptasensor for simultaneous recognition of mycotoxins ochratoxin A (OTA) and fumonisin B1 (FB1) was developed using multi-color, Ln3+-doped time-resolved fluorescence nanoparticles (TRF-NPs) (NaYF4: Ce, Tb and NH2-Eu/DPA@SiO2 NPs) coupled with complementary strand DNA (cDNA) as luminescence probe and aptamers-conjugated amine-functionalized Fe3O4 magnetic nanoparticles (MNPs) act as a capture probe. Under the optimized conditions, the time-resolved fluorescence intensities at 544 and 618 nm corresponded with Tb3+ and Eu3+, respectively, were used to measure FB1 (Y = 19,177.1 + (? 12,054.4)x, R2 = 0.9917) and OTA (Y = 4138.8 + (? 11,182.6)x, R2 = 0.9924), respectively. The limits of detection (LODs) for FB1 and OTA were 0.019 pg mL?1 and 0.015 pg mL?1, respectively, which were much lower than previously described methods for simultaneous recognition of mycotoxins OTA and FB1 while detection range varied from 0.0001–0.5 ng mL?1. This aptasensor was effectively applied to quantity FB1 and OTA in maize samples and results were compared with ELISA method. This is the first reported time-resolved fluorescence (TRF)-based aptasensor to detect two agriculturally important toxins in the maize. The developed aptasensor has potential to be used for detection of toxins in food safety fields.
关键词： Simultaneous detection,Aptamer,Time-resolved fluorescence nanoparticles,Mycotoxins
更新于2025-09-19 17:15:36
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Proteomic Profiles in Advanced Age-Related Macular Degeneration Using an Aptamer-Based Proteomic Technology
摘要： Purpose: To explore top-ranked plasma proteins related to neovascular age-related macular degeneration (AMD) and geographic atrophy (GA), and explore pathways related to neovascular AMD and GA. Methods: We conducted a pilot study of patients with neovascular AMD (n = 10), GA (n = 10), and age-matched cataract controls (n = 10) who were recruited into an AMD registry. We measured 4001 proteins in ethylenediaminetetraacetic acid plasma samples using an aptamer-based proteomic technology. Relative concentrations of each of 4001 proteins were log (base 2) transformed and compared between cases of neovascular AMD and GA versus controls using linear regression. Pathway analysis was conducted using pathways downloaded from Reactome. Results: In this pilot study, higher levels of vinculin and lower levels of CD177 were found in patients with neovascular AMD compared with controls. Neuregulin-4 was higher and soluble intercellular adhesion molecule-1 was lower in patients with GA compared with controls. For neovascular AMD, cargo trafficking to the periciliary membrane, fibroblast growth factor receptor 3b ligand binding and activation, and vascular endothelial growth factor–related pathways were in the top ranked pathways. The top-ranked pathways for GA included several related to ErbB4 signaling. Conclusions: We found different proteins and different pathways associated with neovascular AMD and GA. Vinculin and some of the top-ranked pathways have been previously associated with AMD, whereas others have not been described.
关键词： neovascular AMD,aptamer-based technologies,geographic atrophy,proteomics
更新于2025-09-19 17:15:36
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A highly sensitive DNA aptamer-based fluorescence assay for sarcosine detection down to picomolar levels
摘要： Sarcosine is an amino acid derivative, which is considered as a key metabolite in various metabolic processes. Therefore, simple and sensitive detection methods are needed for further understanding its metabolic role and diagnostic value. In this study, we developed a novel method that meets the need for practical and sensitive detection in a complex medium mimicking urine conditions. For this aim, we selected sarcosine-specific DNA aptamers using graphene oxide-assisted systemic evolution of ligands by exponential enrichment (GO-SELEX). The candidate aptamers were labeled with 6-carboxyfluorescein (6-FAM) at their 5' ends. Two aptamers, namely 9S and 13S produced a significant fluorescence signal upon sarcosine binding. Both aptamers enabled a sensitive analysis with a detection limit of 0.5 pM. The linear detection ranged between 5 pM and 50 μM for 9S aptamer, while 13S aptamer enabled a wider linear detection range between 5 pM and 500 μM. The aptamer-based assay allowed rapid detection with no need for chemical derivatization of sarcosine and sophisticated instruments. Moreover, the aptamer-based assay was free of interference from urea and human serum albumin.
关键词： DNA aptamer,GO-SELEX,sarcosine
更新于2025-09-19 17:15:36