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1.
Adv Biochem Eng Biotechnol ; 170: 107-119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30847536

RESUMO

Aptazymes are synthetic molecules composed of an aptamer domain and a catalytic active nucleic acid unit, which may be a ribozyme or a DNAzyme. In these constructs the aptamer domain serves as a molecular switch that can regulate the catalytic activity of the ribozyme or DNAzyme subunit. This regulation is triggered by binding of the aptamers target molecule, which causes significant structural changes in the aptamer and thus in the entire aptazyme. Therefore, aptazymes function similar to allosteric enzymes, whose catalytic activity is regulated by binding of ligands (effectors) to allosteric sites due to alteration of the three-dimensional structure of the active site of the enzyme. In case of aptazymes, the allosteric site is composed of an aptamer. Aptazymes can be designed for different applications and have already been used in analytical assays as well as for the regulation of gene expression.


Assuntos
Aptâmeros de Nucleotídeos , DNA Catalítico , RNA Catalítico , Aptâmeros de Nucleotídeos/química , Catálise , DNA Catalítico/química , DNA Catalítico/metabolismo , Ligantes , Ligação Proteica , RNA Catalítico/química , RNA Catalítico/metabolismo
3.
Analyst ; 144(17): 5098-5107, 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31373344

RESUMO

Simultaneous detection and regulation of tumor-related genes presents a promising strategy for early diagnosis and treatment of cancer, but achieving this has been a huge challenge for both chemical and biomedical communities. Towards this objective, we have devised a novel aptamer-tethered, DNAzyme-embedded molecular beacon (MB) for multiple functions in cancer cells. In this design, a tumor targeting aptamer was employed to specifically deliver the sensor into cancer cells for target gene detection, and an RNA-cleaving DNAzyme was embedded to realize gene regulation. Both aptamer-tethering and DNAzyme-embedding had little influence on the sensor performance, with a detection limit of ∼2 nM and high specificity. After delivering into tumor cells, our device could monitor the tumor-related genes by producing detectable fluorescence signals, and regulate the gene expression at both mRNA and protein levels as evidenced by the RT-PCR and western blot analyses. This study provides a simple and efficient strategy to rationally combine various functional nucleic acids for multi-functional applications in living cells, which hold great potential for cancer diagnosis and therapy.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA Catalítico/química , Genes Neoplásicos , Linhagem Celular Tumoral , Humanos , Limite de Detecção
5.
Anal Chim Acta ; 1078: 125-134, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358210

RESUMO

We synthesized three kinds of nitrogen-doped nanoporous carbon nanomaterials (represented by N-mC) through a cost-effective method, that is, pyrolysis of plant biomasses (grass, flower, and peanut shells). We further explored their potential as sensitive bioplatforms for electrochemical label-free aptasensors to facilitate the early detection of alpha-fetoprotein (AFP). Chemical structure characterizations revealed that rich functional groups coexisted in as-synthesized N-mC nanomaterials, such as C-C, C-O, C=O, C-N, and COOH. Among the three kinds of N-mC nanomaterials, the one derived from grass (N-mCg) exhibited the lowest carbon defect degree, the highest ID/IG ratio in the Raman spectra, and the largest specific surface area (186.2 m2 g-1). Consequently, N-mCg displayed excellent electrochemical activity and strong affinity toward aptamer strands, further endowing the corresponding aptasensor with sensitive detection ability for AFP. Electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) were used to investigate the whole detection procedure for AFP. The EIS and DPV results showed that the fabricated N-mCg-based aptasensor possessed an extremely low limit of detection of 60.8 and 61.8 fg·mL-1 (s/n = 3), respectively, for detecting AFP within a wide linear range from 0.1 pg mL-1 to 100 ng mL-1. Moreover, the aptasensor displayed acceptable selectivity and applicability, high reproducibility, and excellent stability in serum samples of cancer patients. Therefore, the proposed cost-effective and label-free strategy based on the nitrogen-doped nanoporous carbon derived from plant biomass is a promising approach for the early detection of various tumor markers.


Assuntos
Biomassa , Técnicas Biossensoriais/métodos , Carbono/química , Técnicas Eletroquímicas/métodos , Nanoestruturas/química , alfa-Fetoproteínas/análise , Adolescente , Adulto , Idoso , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Técnicas Biossensoriais/instrumentação , DNA/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Feminino , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Nitrogênio/química , Plantas/química , Porosidade , Reprodutibilidade dos Testes , Adulto Jovem
6.
Anal Chim Acta ; 1078: 176-181, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358217

RESUMO

Intracellular microRNA (miRNA) analysis in single cell is highly informative and offers valuable insights to its physiological and pathological state, but it must confront the pivotal challenge of gene probe delivery and conditional release. Herein, we report an assembled DNA mini-hexahedron (DMH) that can selectively package and protect miRNA probe, target-cell-specific delivery and release it based on the target sequence recognition for intracellular miRNA detection. In brief, the DMH is self-assembled from six single-stranded oligonucleotide strands through rational design, one of which containing AS1411 sequence for specific uptake. Two fluorescent dye labeled recognition strands are inserted into two DMH edges with quencher groups through partially complementary hybridization. We find that this DMH possesses great biocompatibility, good trans-membrane ability and are able to protect the gene cargo against enzymatic degradation and protein binding. Fluorescence restoration caused by the target-mediated competitive chain replacement reaction allows to simultaneous detection of two cancer-related intracellular miRNAs with little false-positive signal, providing a powerful tool to discriminate healthy normal cell and cancerous cell. Thus, the construct opens a new avenue to circumvent the challenges in gene delivery, specific delivery and intrinsic interferences resistance.


Assuntos
DNA de Cadeia Simples/química , MicroRNAs/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Linhagem Celular Tumoral , DNA de Cadeia Simples/genética , Portadores de Fármacos/química , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Humanos , MicroRNAs/genética , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética
7.
Anal Chim Acta ; 1078: 42-52, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358227

RESUMO

Hemoglobin A1c (HbA1c) is a standard biomarker to measure long-term average glucose concentration for diagnosis and monitoring of diabetes. Various methods have been reported for measuring HbA1c, however, portable and precise determination is still challenging. Herein, a new highly sensitive electrochemical nanobiosensor is developed for the specific determination of HbA1c. A nanocomposite of reduced graphene oxide (rGO) and gold with hierarchical architecture structure was electrochemically deposited on a cheap and flexible graphite sheet (GS) electrode. The nanocomposite increased the surface area, improved the electron transfer on the electrode surface and augmented the signal. It also provided a suitable substrate for linkage of thiolated DNA aptamer as a bioreceptor on the electrode surface by strong covalent bonding. The quantitative label free detection was carried out by differential pulse voltammetry (DPV) in a phosphate-buffered saline (PBS) solution containing redox probe Fe(CN)63-/4-. The detection is based on insulating the surface in presence of HbA1c and decreasing the current, which is directly related to the HbA1c concentration. The nanobiosensor demonstrated high sensitivity of 269.2 µA. cm-2, wide linear range of 1 nM-13.83 µM with a low detection limit of 1 nM. The biosensor was successfully used for measuring HbA1c in blood real sample. Furthermore, it is promising to use it as a part of a point of care device for low-invasive screening and management of diabetes.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Hemoglobina A Glicada/análise , Grafite/química , Nanopartículas Metálicas/química , Papel , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Técnicas Biossensoriais/instrumentação , DNA/química , DNA/genética , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Humanos , Limite de Detecção , Nanocompostos/química , Reprodutibilidade dos Testes
8.
Anal Chim Acta ; 1075: 128-136, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31196418

RESUMO

A fluorescent detection of Staphylococcus aureus (S. aureus) is established based on a finely designed functional chimera sequence, a molecular beacon (MB), and strand displacement target recycling. The chimera sequence, which consists of the aptamer sequence of S. aureus and the complementary sequence of MB, can form a hairpin structure due to the existence of intramolecular complementary regions. When S. aureus is present, it binds to the aptamer region of the chimera, opens the hairpin and unlocks the complementary sequence of MB. Subsequently, the MB is opened and intensive fluorescence signal is restored. To increase the sensitivity of the detection, signal amplification is achieved through strand displacement-based target recycling. With the catalysis of Nb. Bpu10I nicking endonuclease and Bsm DNA polymerase, the MB sequence can be cleaved and then elongated to form a complete duplex with the chimera, during which S. aureus is displaced from the chimera and proceeded to the next round of the reaction. This assay displays a linear correlation between the fluorescence intensity and the logarithm of the concentration of S. aureus within a broad concentration range from 80 CFU/mL to 8 × 106 CFU/mL. The detection limit of 39 CFU/mL can be derived. The assay was applied to detect S. aureus in different water samples, and satisfactory recovery and repeatability were achieved. Hence the designed chimera sequence and established assay have potential application in environmental pollution monitoring.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA Bacteriano/análise , Staphylococcus aureus/isolamento & purificação , Aptâmeros de Nucleotídeos/genética , Técnicas de Tipagem Bacteriana/métodos , Sequência de Bases , Técnicas Biossensoriais/métodos , DNA Bacteriano/química , DNA Bacteriano/genética , Desoxirribonuclease I/química , Água Potável/microbiologia , Monitoramento Ambiental/métodos , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Sequências Repetidas Invertidas , Lagos/microbiologia , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Tanques/microbiologia , Espectrometria de Fluorescência , Staphylococcus aureus/genética
9.
Int J Nanomedicine ; 14: 3543-3555, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31190811

RESUMO

Background: In recent years, non-invasive imaging technologies for early cancer detection have drawn worldwide attention. In this study, an antinucleolin aptamer, AS1411, was successfully conjugated to BODIPY-labeled chitosan and studied on T47D and HEK-293 cell lines. Methods: After conjugation of the aptamer to chitosan nanoparticles and purification, its structure was confirmed using atomic force microscopy (AFM), electrophoretic light scattering (ELS) and dynamic light scattering (DLS). Results of AFM, DLS and ELS of both conjugation and chitosan were compared for confirmation of conjugation. Conjugates were mixed with BODIPY FL fluorescent dye, purified and lyophilized. The labeled conjugate was characterized using Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, ELS and DLS. In vitro cellular uptake and cytotoxic effects of BODIPY-labeled chitosan-AS1411 aptamer conjugates were evaluated using the XTT assay on T47D and HEK-293 cells and flow cytometry on T47D cells. Results: The data showed that uptake of BODIPY-labeled chitosan-AS1411 aptamer conjugate was satisfactory. Moreover, there was no statistically significant cytotoxicity of the conjugate on either cell line. Conclusion: The outcomes confirmed the potential application of this new targeted imaging agent as a novel cancer diagnostic agent for molecular imaging.


Assuntos
Compostos de Boro/química , Quitosana/química , Imagem Molecular/métodos , Nanopartículas/química , Oligodesoxirribonucleotídeos/química , Tamanho da Partícula , Aptâmeros de Nucleotídeos/química , Linhagem Celular Tumoral , Proliferação de Células , Endocitose , Fluorescência , Corantes Fluorescentes/química , Células HEK293 , Humanos , Microscopia de Força Atômica , Nanoconjugados/química , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática
10.
Food Chem ; 295: 36-41, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174769

RESUMO

TOB aptamer can be adsorbed on the AuNPs surface to form AuNPs-aptamer complexation to prevent AuNPs aggregation in high salt solution. When TOB was added to the AuNPs solution, the aptamer would bind with TOB and depart from the AuNPs surface. The amount of the AuNPs-aptamer complexation depends on the TOB concentration. Different concentration of AuNPs-aptamer can catalyze the reduction reaction of CuSO4 to produce different size Cu2O particle. The resonance scattering peak intensities are correlated with the Cu2O size. Large size Cu2O particle as a resonance scattering spectroscopy probe can remarkable improve the TOB detection sensitivity. We have succeeded to detect the trace TOB in aqueous solutions. The linear range and limit of detection were 0.50-17 nM and 0.19 nM, respectively. This simple and inexpensive method exhibited high sensitivity and selectivity, which was successfully used to detect TOB in milk. The results indicated the accuracy and precision were satisfied.


Assuntos
Aptâmeros de Nucleotídeos/química , Análise de Alimentos/métodos , Leite/química , Análise Espectral/métodos , Tobramicina/análise , Adsorção , Animais , Técnicas Biossensoriais/métodos , Catálise , Sulfato de Cobre/química , Análise de Alimentos/instrumentação , Contaminação de Alimentos/análise , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Pasteurização , Sensibilidade e Especificidade , Análise Espectral/instrumentação
12.
Bioelectrochemistry ; 129: 270-277, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31254804

RESUMO

Two typical kanamycin-A (KAN-A) electrochemical aptamer-based sensors employing different signal transduction mechanisms were deliberately designed and constructed with a similar structure. One sensor (sensor-1) was based on the classical probe conformation changing mode (PCCM) with a methylene blue (MB) label used as an electrochemical tag; the other sensor (sensor-2) used the target-induced signal probe shifting (TISPS) method with a free MB label in the assay solution. The difference in signal transduction mechanisms resulted in big differences in basic electrochemical behavior and comprehensive sensing performance. The results show that both sensor types exhibit different electrochemical behavior in square wave voltammetry, cyclic voltammetry, and in sensitivity, with detection limits of 3.0 nM for sensor-1 and 60.0 pM for sensor-2 in buffer. When validated for practical and quantitative detection of tap water and milk samples, both sensing methods performed well with detection limits of <260 nM and measurement times of <40 min. In addition, accuracy was good with mean recoveries of 72.3-92.6% and precision was acceptable with a relative standard deviation (RSD) of ≤14.2%. The basis for the similarities and differences in performance is also presented.


Assuntos
Antibacterianos/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Análise de Alimentos/métodos , Canamicina/análise , Leite/química , Poluentes Químicos da Água/análise , Animais , Água Potável/análise , Técnicas Eletroquímicas/métodos , Limite de Detecção
13.
Food Chem ; 297: 124929, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253344

RESUMO

A novel signal-on portable sensing system has been developed for OTA detection using personal glucose meter (PGM) as signal transducer. In the study, we explore the potential of using a short dsDNA as template to trigger the "click" ligation of two DNA strands, further improve the stability of DNA strand on the magnetic beads (MBs) surface, and thereby reduce the background signal. Compared with no "click" ligation, the background signal decreases 7.5 times. Both the sensitivity and selectivity are greatly promoted. A high sensitivity with OTA detection down to 72 pg/mL is achieved, which is comparable with several existing detectors, such as fluorescence-based detectors and electrochemical detectors. The feasibility of the strategy in real samples is well verified and evaluated by detecting OTA in feed samples, indicating the potential application in the food safety field.


Assuntos
Química Click , DNA/química , Ocratoxinas/análise , Aptâmeros de Nucleotídeos/química , Cobre/química , Técnicas Eletroquímicas , Limite de Detecção , Ocratoxinas/química , Espectrometria de Fluorescência
14.
Talanta ; 202: 123-135, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171160

RESUMO

Polychlorinated biphenyls (PCBs) are persistent pollutants, which have expanded in foods and the environment. Detection of PCBs is considered essential due to recognized side-effects of PCBs on health and the public concerns in this regard. On the other hand, due to the trace levels of these organic chlorine compounds, reliable and sensitive assays must be developed. Recognition elements are essential parts of analytical detection assays and sensors of PCBs since these elements are involved in the selective identification of the analytes of interest. Understanding the fundamentals of the recognition elements of PCBs and the benefits of the sensor strategies result in the development of next-generation recognition devices. This review aimed to highlight the recent progress in the recognition elements as key parts of biosensors. We initially, focused on the developed antibody-based biosensors for the detection of PCBs, followed by discussing the aptamers as novel recognition elements. Furthermore, the recent advancement in the development of aptamer-based solid phase extractions has been evaluated. These findings could contribute to improving the design of commercial PCB-kits in the future.


Assuntos
Anticorpos/química , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Bifenilos Policlorados/análise , Animais , Humanos
15.
Talanta ; 202: 152-158, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171163

RESUMO

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world, which can lead to considerably high mortality rate. It was reported that the prognosis is extremely poor and survival is often measured in months once CRC metastases become clinically evident. Therefore, the development of effective approach for metastatic CRC cells detection and imaging may potentially be significant and helpful for CRC prognosis and treatment. Therefore, we proposed a sensitive and specific approach for high-metastatic CRC LoVo cells detection and imaging by using terminal deoxynucleotidyl transferase (TdT)-initiated molecule beacons (MBs) arrayed fluorescent aptamer probes (denoted as TMAP). In this approach, the aptamer W3 targeting high-metastatic CRC LoVo cells was elongated to form W3-poly A at the 3'-hydroxyl terminus with repeated A bases in the presence of TdT and dATP. The MBs designed with poly T sequence in the loop were then hybridized with the poly A in the aptamer W3. The TMAP was easily constructed without the need of aptamer modification. It was demonstrated that this approach could specifically detect and image the high-metastatic CRC LoVo cells from the mixture of high-metastatic CRC LoVo cells and non-metastatic HCT-8 cells. Compared with 6-carboxyfluorescein (6-FAM) labeled aptamer W3, the TMAP was demonstrated to have a much stronger fluorescence signal on the target cells, realizing a 4-fold increase in signal-to-background ratio (SBR). Determination by flow cytometry allowed for detection of as low as 23 CRC LoVo cells in 200 µL cell culture medium. The high sensitivity and the capability for using in complicate biological samples imply that this approach holds considerable potential for metastatic CRC detection and therapy.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Neoplasias Colorretais/diagnóstico por imagem , DNA Nucleotidilexotransferase/química , Corantes Fluorescentes/química , Neoplasias Colorretais/metabolismo , DNA Nucleotidilexotransferase/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Espectrometria de Fluorescência , Células Tumorais Cultivadas
16.
Talanta ; 202: 452-459, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171207

RESUMO

Despite the success in long-term storage of food and dietary products using antibiotics as supplements, enormous levels of their residues have remained as a significant health concern, leading to severe toxicity issues on consumption. Herein, we report an ultrasensitive and highly selective aptasensor based on carbon nanoparticles (CNPs) through a fluorescence-based aptamer-linked immunosorbent assay (FALIA) for rapid detection of kanamycin (KAA) residue. The fabricated CNP-aptasensor exhibited superior selectivity with exceptional photoluminescence properties. Under the optimal conditions, the linear equation of standard KAA solution was Y = -0.2279LogX+1.3648 (R = -0.9893) ranged from 10-4 to 10-7 ppb with excellent relative standard deviations (RSD) between 3.12 and 5.59 % (n = 3). Moreover, the limit of detection (LOD) was lower than 5.0 × 10-8 ppb. Together, the excellent recovery and significant efficacy in the rapid detection of antibiotics at a low level in milk indicate that this fabricated CNP-aptasensor has a great potential in the establishment of an efficient antibiotic detector system in food and other nutraceutical industries.


Assuntos
Aptâmeros de Nucleotídeos/química , Carbono/química , Técnicas de Imunoadsorção , Canamicina/análise , Luminescência , Pontos Quânticos/química
17.
Chem Commun (Camb) ; 55(51): 7378-7381, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31173001

RESUMO

We propose synthetic DNA/RNA transcription circuits based on specific aptamer recognition. By mimicking transcription factor regulation, combined with specific enzyme/DNA aptamer binding, multiple biomolecules including DNA, RNA, polymerase, restriction enzymes and methylase were used as regulators. In addition, multi-level cascading networks and methylation-switch circuits were also established. This regulation strategy has the potential to expand the toolkit of in vitro synthetic biology.


Assuntos
Aptâmeros de Nucleotídeos/química , Fatores de Transcrição/química , DNA/química , Enzimas de Restrição do DNA/química , DNA Polimerase Dirigida por DNA/química , RNA Polimerases Dirigidas por DNA/química , Metilação , RNA/química , Transcrição Genética
18.
Comput Biol Chem ; 80: 452-462, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31170561

RESUMO

Poisoning by organophosphates (OPs) takes one of the leading places in the total number of exotoxicoses. Detoxication of OPs at the first stage of the poison entering the body could be achieved with the help of DNA- or RNA-aptamers, which are able to bind poisons in the bloodstream. The aim of the research was to develop an approach to rational in silico design of aptamers for OPs based on the example of paraoxon. From the published sequence of an aptamer binding organophosphorus pesticides, its three-dimensional model has been constructed. The most probable binding site for paraoxon was determined by molecular docking and molecular dynamics (MD) methods. Then the nucleotides of the binding site were mutated consequently and the values of free binding energy have been calculated using MD trajectories and MM-PBSA approach. On the basis of the energy values, two sequences that bind paraoxon most efficiently have been selected. The value of free binding energy of paraoxon with peripheral anionic site of acetylcholinesterase (AChE) has been calculated as well. It has been revealed that the aptamers found bind paraoxon more effectively than AChE. The peculiarities of paraoxon interaction with the aptamers nucleotides have been analyzed. The possibility of improving in silico approach for aptamer selection is discussed.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Reativadores da Colinesterase/metabolismo , Paraoxon/metabolismo , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sítios de Ligação , Reativadores da Colinesterase/química , Desenho de Drogas , Humanos , Ligações de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Paraoxon/química , Ligação Proteica , Eletricidade Estática
19.
Chemistry ; 25(44): 10505-10510, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31173420

RESUMO

Precision cell-selective surface glycan remodeling is of vital importance for modulation of cell surface dynamics, tissue-specific imaging, and immunotherapy, but remains an unsolved challenge. Herein, we report a switchable enzymatic accessibility (SEA) strategy for highly specific editing of carbohydrate moieties of interest on the target cell surface. We demonstrate the blocking of enzyme in the inaccessible state with a metal-organic framework (MOF) cage and instantaneous switching to the accessible state through disassembly of MOF. We further show that this level of SEA regulation enables initial guided enzyme delivery to the target cell surface for subsequent cell-specific glycan remodeling, thus providing a temporally and spatially controlled tool for tuning the glycosylation architectures. Terminal galactose/N-acetylgalactosamine (Gal/GalNAc) remodeling and terminal sialic acid (Sia) desialylation have been precisely achieved on target cells even with other cell lines in close spatial proximity. The SEA protocol features a modular and generically adaptable design, a very short protocol duration (ca. 30 min or shorter), and a very high spatial resolving power (ability to differentiate immediately neighboring cell lines).


Assuntos
Membrana Celular/enzimologia , Polissacarídeos/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Aptâmeros de Nucleotídeos/química , Biocatálise , Membrana Celular/química , Ativação Enzimática , Galactose/química , Galactose/metabolismo , Galactose Oxidase/antagonistas & inibidores , Galactose Oxidase/metabolismo , Glicosilação , Células Hep G2 , Humanos , Células MCF-7 , Estruturas Metalorgânicas/química , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Imagem Óptica/métodos , Polissacarídeos/química , Propriedades de Superfície
20.
Anal Chim Acta ; 1074: 142-149, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159934

RESUMO

A simple proximity hybridization-induced on particle DNA walker was designed for ultrasensitive detection of proteins, for example platelet-derived growth factor (PDGF-BB) secreted by cancer cells, in which the DNA walker was activated by specific target binding and powered by an enzymatic cleavage to produce amplified signal. High-density FAM-labeled hairpin oligonucleotides (FAM-DNA1) were functionalized on AuNPs to construct three-dimensional (3D) DNA tracks. The specific binding of PDGF-BB with two aptamer probes (DNA3 and DNA4) led to the proximity hybridization-induced DNA displacement and the free of DNA walker (DNA2) to perform movement on the 3D tracks by an enzymatic cleavage, resulting in the release of massive FAM-DNA1 fragments from the AuNPs and the generation of fluorescent signal. This DNA walker based sensing strategy could detect PDGF-BB in a concentration range of 4 orders of magnitude with a detection limit down to sub-pM level. The practical applicability of the assay was demonstrated by detecting PDGF-BB secreted from MCF-7 cells with satisfactory results. The proposed DNA walker based assay could conveniently detect PDGF-BB with high sensitivity and good accuracy, along with the good extensibility of the assay, showing promise for practical diagnosis.


Assuntos
Aptâmeros de Nucleotídeos/química , Becaplermina/análise , DNA/química , Trombina/análise , Aptâmeros de Nucleotídeos/genética , Bioensaio/métodos , DNA/genética , Ouro/química , Humanos , Limite de Detecção , Células MCF-7 , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico
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