RESUMEN
The detection of specific DNA sequences and the identification of single nucleotide polymorphisms are important for disease diagnosis. Herein, by combining the high specificity of the base-stacking effect with the high reproducibility of bovine serum albumin (BSA) modified electrodes and the high loading performance of DNA nanoclews (DNA NCs), a novel sandwich-type electrochemiluminescence (ECL) biosensor is reported for the highly specific detection of HPV16 (chosen as the model target). The capture probes are loaded by BSA carrier platforms modified on the gold electrode surface to improve reproducibility. DNA NCs loaded with a large amount of Ru(phen)32+ worked as signal probes. The template probe is composed of the complementary strand of the target and two free nucleic acid anchors at the head and tail. In the presence of the target DNA, the template probes can form stacked base pairs with target, generating high base-stacking energy. This results in the shorter free anchors of template probes being able to bind to the capture and signal probes. This eventually forms a sandwich structure that allows Ru(phen)32+ to be near the electrode surface, producing an ECL signal. There is a linear relationship between the signal and the target concentration range from 10 fM to 100 pM, with a detection limit of 5.03 fM (S/N=3). Moreover, the base-stacking effect has single base recognition ability for base pairs, effectively avoiding false positive signals. The results of this strategy for clinical samples are consistent with classical methods.
Asunto(s)
Técnicas Biosensibles , Técnicas Biosensibles/métodos , Papillomavirus Humano 16/genética , Albúmina Sérica Bovina , Reproducibilidad de los Resultados , Mediciones Luminiscentes/métodos , ADN/química , Técnicas Electroquímicas/métodos , Límite de Detección , Oro/químicaRESUMEN
Advanced derivatives of the Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) have shown therapeutic efficacy upon topical administration in animal models of asthma and dermatitis. Here, we studied the plasma stability of the EPI-X4 lead compounds WSC02 and JM#21, using mass spectrometry to monitor the chemical integrity of the peptides and a functional fluorescence-based assay to determine peptide function in a CXCR4-antibody competition assay. Although mass spectrometry revealed very rapid disappearance of both peptides in human plasma within seconds, the functional assay revealed a significantly higher half-life of 9 min for EPI-X4 WSC02 and 6 min for EPI-X4 JM#21. Further analyses demonstrated that EPI-X4 WSC02 and EPI-X4 JM#21 interact with low molecular weight plasma components and serum albumin. Albumin binding is mediated by the formation of a disulfide bridge between Cys10 in the EPI-X4 peptides and Cys34 in albumin. These covalently linked albumin-peptide complexes have a higher stability in plasma as compared with the non-bound peptides and retain the ability to bind and antagonize CXCR4. Remarkably, chemically synthesized albumin-EPI-X4 conjugates coupled by non-breakable bonds have a drastically increased plasma stability of over 2 h. Thus, covalent coupling of EPI-X4 to albumin in vitro before administration or in vivo post administration may significantly increase the pharmacokinetic properties of this new class of CXCR4 antagonists.
Asunto(s)
Receptores CXCR4 , Albúmina Sérica Humana , Animales , Humanos , Receptores CXCR4/metabolismo , Péptidos/química , Semivida , Albúmina Sérica/metabolismoRESUMEN
16S ribosomal-RNA (16S rRNA) is often used as an ultrasensitive marker for Chlamydia trachomatis (CT) detection because of its species specificity and high copy number in CT. Robust methods for 16S rRNA detection must be developed to realize the early diagnosis of CT infections. In this work, a highly reproducible and sensitive electrochemical biosensor based on duplex-specific nuclease (DSN)-assisted target-responsive DNA hydrogels and bovine serum albumin (BSA) carrier platform for CT detection was developed. Target rRNA can trigger the DNA hydrogel response, which causes it to be repeatedly cleaved by DSN, ultimately leading to the release of a large amount of horseradish peroxidase-labelled streptavidin (SA-HRP) embedded in the hydrogel beforehand. The released SA-HRP was stably captured by the capture probes that were orderly loaded at the gold electrode with the help of a BSA layer. Then, SA-HRP catalyzed the redox reaction of 3,3',5,5'-tetramethylbenzidine and H2O2, producing a current signal that can be detected. The current signal was proportional to the concentration of CT 16S rRNA from 10 fM to 25 pM with a detection limit of 5.8 fM (S/N = 3). The signal conversion function of the DNA hydrogel avoids the instability of nonhomogeneous nucleic acid hybridization on the gold electrode surface, and combined with optimization by BSA for capture probe modification, this electrochemical biosensor is highly reproducible with a relative standard deviation of 4.3% for the detection of 10 samples of the same concentration. The proposed strategy provides a highly reproducible and sensitive detection method for the extensive screening of CT.
Asunto(s)
Técnicas Biosensibles , Albúmina Sérica Bovina , Chlamydia trachomatis/genética , ADN , Técnicas Electroquímicas , Oro , Hidrogeles , Peróxido de Hidrógeno , Límite de Detección , ARN Ribosómico 16SRESUMEN
Most DNA-based electrochemiluminescence (ECL) biosensors are established through the self-assembly of thiolated single-stranded DNA (ssDNA) probes on the Au electrode surface. Because of this random assembly process, a significant discrepancy exists in the distribution of a modified DNA film on different electrodes, which greatly affects the reproducibility of a biosensor. In this study, a porous bovine serum albumin (BSA) layer was first modified on the electrode surface, which can improve the position distribution and spatial orientation of the self-assembly ssDNA probe. It was then coupled with hyperbranched rolling circle amplification to develop the high-reproducibility-and-sensitivity ECL biosensor for human papillomavirus 16 E6 and E7 oncogene detection. In the presence of the target DNA, the surface of the electrode accumulates abundant amplified products through reaction, which contain double-stranded DNA (dsDNA) fragments of different lengths, followed by plentiful dichlorotris (1,10-phenanthroline) ruthenium(II) hydrate (Ru(phen)32+, acting as an ECL indicator) insertion into grooves of dsDNA fragments, and a strong signal can be detected. There is a linear relationship between the signal and the target concentration range from 10 fM to 15 pM, and the detection limit is 7.6 fM (S/N = 3). After the BSA modification step, the relative standard deviation was reduced from 9.20 to 3.96%, thereby achieving good reproducibility. The proposed ECL strategy provides a new method for constructing high-reproducibility-and-sensitivity ECL biosensors.
Asunto(s)
Técnicas Biosensibles/métodos , Papillomavirus Humano 16/aislamiento & purificación , Proteínas Oncogénicas Virales/análisis , Proteínas E7 de Papillomavirus/análisis , Proteínas Represoras/análisis , Albúmina Sérica Bovina/química , Animales , Bovinos , Cuello del Útero/virología , Sondas de ADN/química , Sondas de ADN/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Técnicas Electroquímicas/métodos , Femenino , Papillomavirus Humano 16/química , Humanos , Límite de Detección , Sustancias Luminiscentes , Técnicas de Amplificación de Ácido Nucleico/métodos , Hibridación de Ácido Nucleico , Proteínas Oncogénicas Virales/genética , Compuestos Organometálicos/química , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/diagnóstico , Fenantrolinas/química , Proteínas Represoras/genética , Reproducibilidad de los Resultados , Rutenio/químicaRESUMEN
Intravesical chemotherapy for bladder cancer has limited efficacy due to the lack of specificity of drugs/drug carriers toward the cancer cells as well as inadequate drug residence time in the bladder due to urine voiding. From analyses of surface receptor expression of UMUC3 bladder cancer cells and the targeting efficacy of different peptides, we selected a peptide (txCD47) that targets the cluster of differentiation 47 (CD47) surface protein overexpressed on these cells as a targeting ligand for docetaxel (DTX) and an albumin nanocarrier of DTX. The IC50 of DTX conjugated to txCD47 (txCD47-DTX) in a 1:1 molar ratio was lowered by a factor of 3 from that of free DTX. By using the albumin molecule (txCD47-BSA) as a delivery vehicle, different amounts of txCD47 can be conjugated to investigate the effects of peptide concentration on targeting efficacy. The IC50 of DTX loaded in txCD47-BSA with 14 txCD47 per albumin molecule was 1 order of magnitude lower than that of free DTX, and a factor of 4 lower than that of txCD47-BSA with 8 txCD47 per albumin molecule. DTX was released from the albumin nanocarrier at a controlled rate, and the endocytosed carrier will release most of its payload inside the cells within 72 h. Thus, txCD47 promotes delivery of the drug/drug carrier, and the resultant enhanced killing efficacy of the drug can potentially alleviate some of the limitations of intravesical chemotherapy against bladder cancer.
RESUMEN
With the aim of increasing the accumulation of Ru anticancer agents in the tumor, a targeted delivery strategy based on a maleimide anchor for the biological vector human serum albumin (HSA) was developed. A group of piano stool Ru- and Os(η6-arene) complexes carrying a maleimide-functionalized N-phenyl-2-pyridinecarbothioamide (PCA) ligand was designed allowing for covalent conjugation to biological thiols. The complexes were characterized by NMR spectroscopy, ESI-MS, elemental analysis and single-crystal X-ray diffraction analysis. The compounds were shown to undergo halido/aqua ligand exchange reactions in aqueous solution, depending mainly on the metal center and the nature of the halide. In vitro cytotoxicity studies revealed low potency which is explained by the observed high reactivity of the maleimide to the thiol of l-cysteine (Cys), while the metal center itself shows little affinity to amino acids of the model protein lysozyme.