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1.
Mikrochim Acta ; 191(6): 334, 2024 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-38758362

RESUMO

Single nucleotide polymorphism (SNP) biosensors are emerging rapidly for their promising applications in human disease prevention diagnosis, treatment, and prognosis. However, it remains a bottleneck in equipping simple and stable biosensors with the traits of high sensitivity, non-enzyme, and low cost. Double base mismatches mediated chain displacement reactions have attracted fascinating advantages of tailorable thermodynamics stability, non-enzyme, and excellent assembly compliance to involvement in SNP identification. As the base mismatch position and amount in DNA sequence can be artificially adjusted, it provides plenty of selectivity and specificity for exploring perfect biosensors. Herein, a biosensor with double base mismatches mediated catalytic hairpin assembly (CHA) is designed via one base mismatch in the toehold domain and the other base mismatch in the stem sequence of hairpin 1 (H1) by triggering CHA reaction to achieve selective amplification of the mutation target (MT) and fluorescence resonance energy transfer (FRET) effect that is composed of Cy3 and Cy5 terminally attached H1 and hairpin 2 (H2). Depending on the rationally designed base mismatch position and toehold length, the fabricated biosensors show superior SNP detection performance, exhibiting a good linearity with high sensitivity of 6.6 fM detection limit and a broad detection abundance of 1%. The proposed biosensor can be used to detect the KRAS mutation gene in real samples and obtain good recoveries between 106 and 116.99%. Remarkably, these extendible designs of base mismatches can be used for more types of SNP detection, providing flexible adjustment based on base mismatch position and toehold length variations, especially for their thermodynamic model for DNA-strand displacement reactions.


Assuntos
Pareamento Incorreto de Bases , Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Técnicas de Amplificação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Técnicas Biossensoriais/métodos , Humanos , Transferência Ressonante de Energia de Fluorescência/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Limite de Detecção , Sequências Repetidas Invertidas , DNA/química , DNA/genética , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Catálise
2.
Biomed Mater ; 19(4)2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38772383

RESUMO

The traditional chemotherapeutic agents' disadvantages such as high toxicity, untargeting and poor water solubility lead to disappointing chemotherapy effects, which restricts its clinical application. In this work, novel size-appropriate and glutathione (GSH)-responsive nano-hydrogels were successfully prepared via the active ester method between chitosan (containing -NH2) and cross-linker (containing NHS). Especially, the cross-linker was elaborately designed to possess a disulfide linkage (SS) as well as two terminal NHS groups, namely NHS-SS-NHS. These functionalities endowed chitosan-based cross-linked scaffolds with capabilities for drug loading and delivery, as well as a GSH-responsive mechanism for drug release. The prepared nano-hydrogels demonstrated excellent performance applicable morphology, excellent drug loading efficiency (∼22.5%), suitable size (∼100 nm) and long-term stability. The prepared nano-hydrogels released over 80% doxorubicin (DOX) after incubation in 10 mM GSH while a minimal DOX release less than 25% was tested in normal physiological buffer (pH = 7.4). The unloaded nano-hydrogels did not show any apparent cytotoxicity to A 549 cells. In contrast, DOX-loaded nano-hydrogels exhibited marked anti-tumor activity against A 549 cells, especially in high GSH environment. Finally, through fluorescent imaging and flow cytometry analysis, fluorescein isothiocyanate-labeled nano-hydrogels show obvious specific binding to the GSH high-expressing A549 cells and nonspecific binding to the GSH low-expressing A549 cells. Therefore, with this cross-linking approach, our present finding suggests that cross-linked chitosan nano-hydrogel drug carrier improves the anti-tumor effect of the A 549 cells and may serve as a potential injectable delivery carrier.


Assuntos
Antineoplásicos , Quitosana , Reagentes de Ligações Cruzadas , Doxorrubicina , Glutationa , Hidrogéis , Quitosana/química , Humanos , Doxorrubicina/farmacologia , Doxorrubicina/química , Glutationa/química , Glutationa/metabolismo , Hidrogéis/química , Reagentes de Ligações Cruzadas/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Linhagem Celular Tumoral , Células A549 , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Dissulfetos/química , Preparações de Ação Retardada/química
3.
Anal Chem ; 96(1): 554-563, 2024 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-38112727

RESUMO

The efficiency of the enzyme-free toehold-mediated strand displacement (TMSD) technique is often insufficient to detect single-nucleotide polymorphism (SNP) that possesses only single base pair mismatch discrimination. Here, we report a novel dual base pair mismatch strategy enabling TMSD biosensing for SNP detection under enzyme-free conditions when coupled with catalytic hairpin assembly (CHA) and fluorescence resonance energy transfer (FRET). The strategy is based on a competitive strand displacement reaction mechanism, affected by the thermodynamic stability originating from rationally designed dual base pair mismatch, for the specific recognition of mutant-type DNA. In particular, enzyme-free nucleic acid circuits, such as CHA, emerge as a powerful method for signal amplification. Eventually, the signal transduction of this proposed biosensor was determined by FRET between streptavidin-coated 605 nm emission quantum dots (605QDs, donor) and Cy5/biotin hybridization (acceptor, from CHA) when incubated with each other. The proposed biosensor displayed high sensitivity to the mutant target (MT) with a detection concentration down to 4.3 fM and led to high discrimination factors for all types of mismatches in multiple sequence contexts. As such, the application of this proposed biosensor to investigate mechanisms of the competitive strand displacement reaction further illustrates the versatility of our dual base pair mismatch strategy, which can be utilized for the creation of a new class of biosensors.


Assuntos
Técnicas Biossensoriais , Polimorfismo de Nucleotídeo Único , Pareamento Incorreto de Bases , Hibridização de Ácido Nucleico , Transferência Ressonante de Energia de Fluorescência , Biotina , Técnicas Biossensoriais/métodos
4.
Front Bioeng Biotechnol ; 11: 1201320, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37251571

RESUMO

Titanium dioxide (TiO2) materials are suitable for use as drug carriers due to their natural biocompatibility and nontoxicity. The aim of the study presented in this paper was to investigate the controlled growth of TiO2 nanotubes (TiO2 NTs) of different sizes via an anodization method, in order to delineate whether the size of NTs governs their drug loading and release profile as well as their antitumor efficiency. TiO2 NTs were tailored to sizes ranging from 25 nm to 200 nm according to the anodization voltage employed. The TiO2 NTs obtained by this process were characterized using scanning electron microscopy, transmission electron microscopy, and dynamic light scattering The larger TiO2 NTs exhibited greatly improved doxorubicin (DOX)-loading capacity (up to 37.5 wt%), which contributed to their outstanding cell-killing ability, as evidenced by their lower half-maximal inhibitory concentration (IC50). Comparisons were carried out of cellular uptake and intracellular release rates of DOX for large and small TiO2 NTs loaded with DOX. The results showed that the larger TiO2 NTs represent a promising therapeutic carrier for drug loading and controlled release, which could improve cancer treatment outcomes. Therefore, TiO2 NTs of larger size are useful substances with drug-loading potency that may be used in a wide range of medical applications.

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