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Amplified Split Aptamer Sensor Delivered Using Block Copolymer Nanoparticles for Small Molecule Imaging in Living Cells
摘要： We develop a novel amplified split aptamer sensor for highly sensitive detection and imaging of small molecules in living cells by using cationic block copolymer nanoparticles (BCNs) with entrapped fluorescent conjugated polymer as a delivery agent. The design of split aptamer as the initiator of hybridization chain reaction (HCR) affords the possibility of enhancing the signal-to-background ratio and thus allows high-contrast imaging for small molecules with relatively weak interactions with their aptamers. The novel design of using fluorescent cationic BCNs as the nanocarrier enables efficient and self-tracking transfection of DNA probes. Results reveal that BCNs exhibit high fluorescence brightness allowing direct tracking of the delivery location. The developed amplified split aptamer sensor is shown to have high sensitivity and selectivity for in vitro quantitative detection of ATP with a detection limit of 30 nM. Live cell studies show that the sensor provides a "signal on" approach for specific, high-contrast imaging of ATP. The DNA sensor based HCR system may provide a new generally applicable platform for detection and imaging of low-abundance biomarkers.
关键词： sensor,small molecule imaging,enzyme-free amplification,block copolymer nanoparticles,split aptamer
更新于2025-09-10 09:29:36
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Transition metal complexes based aptamers as optical diagnostic tools for disease proteins and biomolecules
摘要： Aptamers are powerful recognition elements that can bind a large number of target molecules, including metal ions, small molecules, proteins, enzymes, even complex targets like cancer cells, etc., with high affinity and specificity. Hence, aptamer-based biosensors (hereafter named "aptasensors") have been extensively utilized in the field of clinical diagnostics and biomedical applications. In contrast to organic luminophores and quantum dots, luminescent transition metal complexes offer many desirable and wide-ranging properties, including tunable emission throughout the visible to NIR regions, long lifetime with a large Stockes shift, high quantum yield, good thermal, chemical and photochemical stability and metabolic inertness for biosensing applications. The incorporation of biomolecules or lipophilic entities into the metal complexes could overcome problems associated with membrane permeability and uptake by cells. Especially, Ru(II) and Ir(III) complexes are promising candidates for these potential applications. This review describes an overview of recent progress in the emerging area of aptasensors utilizing Ru(II) and Ir(III) transition metal complexes. To date, though aptasensors have been used in a wide variety of detection techniques, we have focused mainly on the luminescence approach in this article. Numerous aptasensors have illustrated promising detection results, even in complicated biological environments. If more rigorous research is continued on this area, it is hoped that in the future transition metal complexes based aptamers may show tremendous applications in biomedical research, especially diagnostics, imaging and drug delivery.
关键词： Proteins,Biosensor,Metal complexes,Luminescence,Aptamer
更新于2025-09-10 09:29:36
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Imaging of Receptor Dimers in Zebrafish and Living Cells via Aptamer Recognition and Proximity-induced Hybridization Chain Reaction
摘要： On cell membrane surfaces, receptor protein dimers play a fundamental role in many signaling pathways, which are crucial for normal biological processes and cancer development. Efficient and sensitive analysis of receptor dimers in the native environment is highly desirable. Herein, we present a strategy for amplified imaging of receptor dimers in zebrafish and living cells, which relies on aptamer recognition and proximity-induced hybridization chain reaction. Taking advantages of specific aptamer recognition and enzyme-free signal amplification, this strategy is successfully applied to amplified visualize c-Met receptor dimers in the HGF-independent or -dependent manner. Therefore, the developed imaging strategy paves the way for further investigation of protein dimerization or oligomerization state of cell surface receptors and corresponding activation processes in zebrafish and living cells.
关键词： aptamer recognition,zebrafish,living cells,receptor dimers,hybridization chain reaction
更新于2025-09-10 09:29:36
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Aptamers Improving Fluorescence Anisotropy and Fluorescence Polarization Assays for Small Molecules
摘要： Nucleic acid affinity probes, such as aptamers, have been combined with fluorescence anisotropy (FA) / fluorescence polarization (FP) technology for the development of a diverse range of assays. Formation of a complex between a small fluorescent molecule and its binding partner usually increases the overall size of the fluorescent molecule and decreases its rate of rotation, resulting in increases in fluorescence anisotropy/polarization. Structure-switching of the fluorescently labeled aptamers arising from target binding can also affect molecular volume, local rotation of the fluorophore, and/or fluorescence lifetime, causing changes in anisotropy/polarization. Incorporation of the unique adsorptive properties of single-stranded nucleic acid aptamers on nanomaterials, hybridization of aptamers with complementary sequences, and the amplifiable ability of nucleic acid aptamers have broadened the applications of fluorescence anisotropy assays and enhanced their sensitivity. This review focuses on aptamer-based fluorescence anisotropy assays for the detection of small molecules, such as therapeutic drugs, environmental contaminants, natural toxins, and metabolites.
关键词： aptamer,adenosine triphosphate (ATP),nanomaterials,anisotropy,cocaine,nucleic acids,toxins,DNAzyme,polarization,aflatoxin B1,ochratoxin A (OTA),tyrosinamide
更新于2025-09-10 09:29:36
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Structure guided fluorescence labeling reveals a two-step binding mechanism of neomycin to its RNA aptamer
摘要： The ability of the cytidine analog C? m f to act as a position specific reporter of RNA-dynamics was spectroscopically evaluated. C? m f-labeled single- and double-stranded RNAs differ in their fluorescence lifetimes, quantum yields and anisotropies. These observables were also influenced by the nucleobases flanking C? m f. This conformation and position specificity allowed to investigate the binding dynamics and mechanism of neomycin to its aptamer N1 by independently incorporating C? m f at four different positions within the aptamer. Remarkably fast binding kinetics of neomycin binding was observed with stopped-flow measurements, which could be satisfactorily explained with a two-step binding. Conformational selection was identified as the dominant mechanism.
关键词： aptamer,binding mechanism,neomycin,fluorescence labeling,RNA
更新于2025-09-09 09:28:46
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Liquid crystal-based aptasensor for the detection of interferon-γ and its application in the diagnosis of tuberculosis using human blood
摘要： We report the development of a liquid crystal (LC)-based aptasensor for the detection of interferon-γ (IFN-γ) in human blood as well as the diagnosis of tuberculosis (TB). In this system, the binding of IFN-γ to an aptamer immobilized on a surface disrupts the orientation of LCs, inducing a transition from a homeotropic orientation to a random one. This change in the orientation of the LCs can easily be converted and observed as a shift from a dark optical LC image to a bright one under a polarized light microscope. Through this sensing mechanism, IFN-γ levels as low as 1 pM (17 pg/ml) could be detected. This LC-based aptasensor was employed for the diagnosis of TB using blood samples from patients with latent TB. With this LC-based approach, not only could IFN-γ readily be detected, but also latent TB could be diagnosed using human blood simply and effectively without any intricate processes or instrumentation. Therefore, our present research provides a promising IFN-γ sensor with applications in clinical diagnosis of various infectious diseases, and in immunological research.
关键词： Tuberculosis (TB),Interferon-γ (IFN-γ),4-cyano-4′-pentylbiphenyl (5CB),Liquid crystal (LC),Aptamer,Human blood sample
更新于2025-09-09 09:28:46
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High-performance interactive analysis of split aptamer and HIV-1 Tat on multiwall carbon nanotube-modified field-effect transistor
摘要： Interaction between split RNA aptamer and the clinically important target, HIV-1 Tat was investigated on a biosensing surface transduced by functionally choreographed multiwall carbon nanotubes (MWCNTs). Acid oxidation was performed to functionalize MWCNTs with carboxyl functional groups. X-ray photoelectron spectroscopy analysis had profound ~2.91% increment in overall oxygen group and ~1% increment was noticed with a specific carboxyl content owing to C=O and O–C=O bonding. The interaction between split RNA aptamer and HIV-1 Tat protein was quantified by electrical measurements with the current signal (Ids) over a gate voltage (Vgs). Initially, 34.4 mV gate voltage shift was observed by the immobilization of aptamer on MWCNT. With aptamer and HIV-1 Tat interaction, the current flow was decreased with the concomitant gate voltage shift of 23.5 mV. The attainment of sensitivity with split aptamer and HIV-1 Tat interaction on the fabricated device was 600 pM. To ensure the genuine interaction of aptamer with HIV-1 Tat, other HIV-1 proteins, Nef and p24 were interacted with aptamer and they displayed the negligible interferences with gate voltage shift of 3.5 mV and 5.7 mV, which shows 4 and 2.5 folds lesser than HIV-1 Tat interaction, respectively.
关键词： Field effect transistor,Multiwall carbon nanotube,Split aptamer,HIV-1 Tat
更新于2025-09-09 09:28:46
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A light-responsive RNA aptamer for an azobenzene derivative
摘要： Regulation of complex biological networks has proven to be a key bottleneck in synthetic biology. Interactions between the structurally flexible RNA and various other molecules in the form of riboswitches have shown a high-regulation specificity and efficiency and synthetic riboswitches have filled the toolbox of devices in many synthetic biology applications. Here we report the development of a novel, small molecule binding RNA aptamer, whose binding is dependent on light-induced change of conformation of its small molecule ligand. As ligand we chose an azobenzene because of its reliable photoswitchability and modified it with chloramphenicol for a better interaction with RNA. The synthesis of the ligand ‘azoCm’ was followed by extensive biophysical analysis regarding its stability and photoswitchability. RNA aptamers were identified after several cycles of in vitro selection and then studied regarding their binding specificity and affinity toward the ligand. We show the successful development of an RNA aptamer that selectively binds to only the trans photoisomer of azoCm with a KD of 545 nM. As the aptamer cannot bind to the irradiated ligand (λ = 365 nm), a light-selective RNA binding system is provided. Further studies may now result in the engineering of a reliable, light-responsible riboswitch.
关键词： synthetic biology,photoswitchability,riboswitch,azobenzene,RNA aptamer
更新于2025-09-09 09:28:46
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SERS-Based Quantification of PSMA in Tissue Microarrays Allows Effective Stratification of Patients with Prostate Cancer
摘要： Prostate specific membrane antigen (PSMA), a type II membrane protein, is an attractive biomarker that has been validated clinically for the diagnosis of prostate cancer. In this study, we developed surface-enhanced Raman scattering (SERS) nanoprobes for PSMA detection and quantification at the single-cell level on prostate cancer cells. The cells were targeted employing SERS nanoprobes that consisted of gold nanostars functionalized with PSMA aptamer molecules. We were able to quantify picomolar concentrations of soluble PSMA protein and used the resulting calibration curve to estimate the expression of PSMA on the surface of the prostate cancer cell, LNCaP, at the single-cell level. Importantly, we employed these SERS tags to stratify prostate cancer patients by assessing PSMA expression in tissues contained in a prostate tissue microarray. The stratification results clearly correlated PSMA expression to recommended therapy groups, rendering the described method as an effective tool to aid in designing personalized therapeutic protocols. Benchmarking detection sensitivity against immunofluorescence staining and comparing stratification results obtained with the two methods allowed us to validate our novel approach against standard practices. On the basis of these results, we confirm the validity of PSMA as an effective biomarker for prostate cancer patient evaluation and propose SERS-based diagnostic techniques as integrative methods for the assessment of disease stage and the identification of effective therapeutic protocols.
关键词： aptamer,tissue microarray,surface-enhanced Raman scattering,PSMA,Prostate specific membrane antigen,SERS,nanoprobes,prostate cancer,biomarker,gold nanostars
更新于2025-09-04 15:30:14
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Hybridization induced fluorescence enhanced DNA-Ag nanocluster/aptamer probe for detection of prostate-specific antigen
摘要： In this work, a label-free Ag nanocluster (AgNC)-based fluorescent probe is proposed to detect tumor marker, prostate-specific antigen (PSA). In the experiments, DNA sequences containing segments complemented to different parts of PSA aptamer were used to synthesize DNA-Ag nanoclusters (DNA-AgNC). Some of the obtained specific DNA-AgNC exhibited significant fluorescence increase after hybridization with PSA aptamer. Based on this, a simple DNA-AgNC/aptamer hybridization probe was fabricated for PSA detection using fluorescence quenching, because competitively specific binding between PSA and its aptamer inhibited the fluorescence enhancement effect of PSA aptamer on DNA-AgNC. The sequence of template DNA, pH and salt concentration of binding buffer, and the concentration of aptamer were optimized. Under optimum conditions, the concentration of PSA within the range of 2–150 ng mL?1 with the detection limit of 1.14 ng mL?1 was detected (3σ; n = 7). This approach was also successfully applied to determine PSA in spiked serum samples. As is well known, this was the first report to realize PSA detection using fluorescent AgNC-based probe. This work would provide reference for construction of AgNC-based probes for detecting other proteins.
关键词： DNA-AgNC fluorescent probe,Ag nanocluster,Prostate-specific antigen,Aptamer
更新于2025-09-04 15:30:14