Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.451
Filtrar
1.
Nat Commun ; 11(1): 3859, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737298

RESUMO

Non-enzymatic proteins including antibodies function as biomarkers and are used as biopharmaceuticals in several diseases. Protein-responsive soft materials capable of the controlled release of drugs and proteins have potential for use in next-generation diagnosis and therapies. Here, we describe a supramolecular/agarose hydrogel composite that can release a protein in response to a non-enzymatic protein. A non-enzymatic protein-responsive system is developed by hybridization of an enzyme-sensitive supramolecular hydrogel with a protein-triggered enzyme activation set. In situ imaging shows that the supramolecular/agarose hydrogel composite consists of orthogonal domains of supramolecular fibers and agarose, which play distinct roles in protein entrapment and mechanical stiffness, respectively. Integrating the enzyme activation set with the composite allows for controlled release of the embedded RNase in response to an antibody. Such composite hydrogels would be promising as a matrix embedded in a body, which can autonomously release biopharmaceuticals by sensing biomarker proteins.


Assuntos
Anidrase Carbônica II/química , Preparações de Ação Retardada/síntese química , Hidrogéis/química , Ribonucleases/química , Sefarose/química , Animais , Anticorpos/química , Avidina/química , Biotina/química , Anidrase Carbônica II/antagonistas & inibidores , Inibidores da Anidrase Carbônica/química , Bovinos , Ativação Enzimática , Transição de Fase , Reologia , Ribonucleases/antagonistas & inibidores , Sulfonamidas/química
2.
Nucleic Acids Res ; 48(15): e88, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32710620

RESUMO

DNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no single method can identify the location, direction and speed of replication forks with high resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labeling of replicating or repaired DNA. Here, we demonstrate the use of the Oxford Nanopore Technologies MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU as well as EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show that the large adduct Biotin can be distinguished from the smaller analog IdU, which opens the possibility of using analog combinations to identify the location and direction of DNA synthesis. Furthermore, we detect IdU label on single DNA molecules in the genome of mouse pluripotent stem cells and using CRISPR/Cas9-mediated enrichment, determine replication rates using newly synthesized DNA strands in human mitochondrial DNA. We conclude that this novel method, termed Replipore sequencing, has the potential for on target examination of DNA replication in a wide range of biological contexts.


Assuntos
Bromodesoxiuridina/química , Sequenciamento por Nanoporos , Timidina/genética , Animais , Biotina/química , Biotina/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Nanoporos , Timidina/química
3.
Nucleic Acids Res ; 48(10): 5254-5267, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329781

RESUMO

Guanine-rich, single-stranded DNAs and RNAs that fold to G-quadruplexes (GQs) are able to complex tightly with heme and display strongly enhanced peroxidase activity. Phenolic compounds are particularly good substrates for these oxidative DNAzymes and ribozymes; we recently showed that the use of biotin-tyramide as substrate can lead to efficient GQ self-biotinylation. Such biotinylated GQs are amenable to polymerase chain reaction amplification and should be useful for a relatively non-perturbative investigation of GQs as well as GQ-heme complexes within living cells. Here, we report that in mixed solutions of GQ and duplex DNA in vitro, GQ biotinylation is specifically >104-fold that of the duplex, even in highly concentrated DNA gels; that a three-quartet GQ is tagged by up to four biotins, whose attachment occurs more or less uniformly along the GQ but doesn't extend significantly into a duplex appended to the GQ. This self-biotinylation can be modulated or even abolished in the presence of strong GQ ligands that compete with heme. Finally, we report strong evidence for the successful use of this methodology for labeling DNA and RNA within live, freshly dissected Drosophila larval salivary glands.


Assuntos
Biotina/química , Biotinilação , DNA/química , Quadruplex G , Heme/química , RNA/química , Animais , Sequência de Bases , Biotina/análogos & derivados , Drosophila melanogaster , Ligantes , Masculino , Salmão , Espermatozoides , Tiramina/análogos & derivados , Tiramina/química
4.
Nat Chem Biol ; 16(6): 702-709, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32203413

RESUMO

When the primitive translation system first emerged in the hypothetical RNA world, ribozymes could have been responsible for aminoacylation. Given that naturally occurring T-box riboswitches selectively sense the aminoacylation status of cognate tRNAs, we introduced a domain of random sequence into a T-box-tRNA conjugate and isolated ribozymes that were self-aminoacylating on the 3'-terminal hydroxyl group. One of them, named Tx2.1, recognizes the anticodon and D-loop of tRNA via interaction with its stem I domain, similarly to the parental T-box, and selectively charges N-biotinyl-L-phenylalanine (Bio-lPhe) onto the 3' end of the cognate tRNA in trans. We also demonstrated the ribosomal synthesis of a Bio-lPhe-initiated peptide in a Tx2.1-coupled in vitro translation system, in which Tx2.1 catalyzed specific tRNA aminoacylation in situ. This suggests that such ribozymes could have coevolved with a primitive translation system in the RNA world.


Assuntos
RNA Catalítico/genética , RNA Catalítico/metabolismo , Riboswitch/genética , Aminoacilação de RNA de Transferência/efeitos dos fármacos , Bacillus subtilis/enzimologia , Sequência de Bases , Biotina/química , Domínio Catalítico , Biblioteca Gênica , Modelos Genéticos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Fenilalanina/química , Ligação Proteica , Estreptavidina/metabolismo
5.
Clin Chim Acta ; 505: 130-135, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32084383

RESUMO

BACKGROUND: Biotin is an interference in many streptavidin-biotin based immunoassays, causing falsely decreased results with sandwich immunoassays and falsely increased results with competitive immunoassays. It has been discussed that premixing streptavidin coated beads and biotinylated capturing molecules may prevent biotin interference. This study was designed to test whether such modification could mitigate biotin interference in two originally susceptible sandwich immunoassays. METHODS: Roche C-peptide and human growth hormone (hGH) immunoassays utilize three reagent containers for streptavidin coated beads (M), biotinylated capturing antibody (R1) and ruthenylated antibody (R2). The reagents were modified by premixing reagent M and R1. Following incubation, the beads were placed back in the M-container and R1-supernatant back to R1-container. Patient specimens were selected, spiked with biotin to 1055 ng/mL, and measured by both the original, unmodified reagent and modified reagent on Roche cobas e411 analyzer. The biotin interference dose response curves were also compared using pooled patient specimen spiked with different concentrations of biotin. RESULTS: For the original reagent, 1055 ng/mL of biotin decreased C- peptide results by 88% and hGH results by 97%. After reagent modification, this interference effect was nearly eliminated for C- peptide but remained about 20% decreased for hGH. CONCLUSION: Premixing streptavidin beads and biotinylated capturing molecules is an effective approach to mitigate biotin interference for certain immunoassays.


Assuntos
Biotina/análise , Biotina/química , Imunoensaio/métodos , Estreptavidina/química , Biotinilação , Peptídeo C/análise , Reações Falso-Positivas , Hormônio do Crescimento Humano/análise , Humanos , Indicadores e Reagentes
6.
Int J Pharm Compd ; 24(1): 69-76, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32023218

RESUMO

Androgenetic alopecia is the most common form of hair loss. This condition affects both men and women causing significant psychological distress and a decrease in the quality of life. The objective of this study was to investigate the clinical efficacy and patient satisfaction of a topical compounded formulation (minoxidil 10%, finasteride 0.1%, biotin 0.2%, and caffeine citrate 0.05% hydroalcoholic solution) in male androgenetic alopecia patients. A total of five individual, prospective case studies were conducted in the private hair transplant practice of Dr. James C. Marotta. Patients were provided with the topical formulation and instructed to apply a measured 1-mL dose to the entire frontal, parietal, and occipital scalp, twice daily for 6 months. Patients visited the practice periodically (90 days, 120 days, and 180 days post-treatment) for clinical evaluation, photographic assessment, and measurement of their treatment satisfaction by the Men's Hair Growth Questionnaire. By the end of the study, at 180 days, the dermatologist-in-charge concluded that the topical treatment was successful for all five patients. Although moderate, the clinical improvements were visually noticeable as most patients had thicker, more voluminous hair; improved scalp coverage; and improved general hair appearance. These results were consistent with the photographic assessment, which demonstrated a global average increase of +1.05 in the patients' hair density. According to the patients' self-assessment, the topical compounded formulation was effective following 3 months and 6 months of continuous treatment. At 120 days, the patients' satisfaction was neutral or negative, which was likely due to negligible differences in the patients' hair growth and appearance in 90 days compared to 120 days. The results from this study suggest that the new hair-loss topical solution may be considered a safe and effective treatment option in male AGA patients.


Assuntos
Alopecia/fisiopatologia , Biotina/química , Cafeína/química , Citratos/química , Finasterida , Minoxidil , Feminino , Humanos , Masculino , Estudos Prospectivos , Qualidade de Vida , Resultado do Tratamento
7.
Chem Commun (Camb) ; 56(18): 2787-2790, 2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32025667

RESUMO

Expanding the catalytic repertoire of ribozymes to include vitamin synthesis requires efficient labelling of RNA with the substrate of interest, prior to in vitro selection. For this purpose, we rationally designed and synthesized six GMP-conjugates carrying a synthetic pre-thiamine or biotin precursor and investigated their transcription incorporation properties by T7 RNA polymerase.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Guanosina Monofosfato/biossíntese , Proteínas Virais/metabolismo , Vitaminas/biossíntese , Biocatálise , Biotina/química , Biotina/metabolismo , Guanosina Monofosfato/química , Estrutura Molecular , Tiamina/química , Tiamina/metabolismo , Vitaminas/química
8.
Food Chem ; 316: 126356, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32045810

RESUMO

Fumonisins (FBs) exist widely in crops, foods and feeds. Consumption of FBs contaminated corn can cause oesophageal cancer. So it is necessary to develop sensitivity methods for its detection. Here, we report an enhanced assay for rapid and ultra-sensitive detection of FBs based on nanomagnetic beads (NMBs) and antibody-biotin-streptavidin-HRP. Because antibody-BNHS can bind with several number of streptavidin-HRP, the signal amplification of the catalytical oxidation of TMB was enhanced. The detection limit of sensor was down to 0.21 ng mL-1 with a linear range from 0.31 to 162.42 ng mL-1. Since NMBs provide a nearly "in solution" reaction, they lead to a shortened reaction time (22 min) than that of flat solid-phase based traditional assay. Furthermore, the recoveries obtained by standard FBs spiked to maize samples were from 100.6 to 107.3%. This enhanced assay supplied a rapid, sensitive and reliable method for detection of FBs in maize.


Assuntos
Anticorpos/imunologia , Armoracia/enzimologia , Biotina/química , Fumonisinas/análise , Peroxidase do Rábano Silvestre/metabolismo , Estreptavidina/química , Zea mays/química , Ensaio de Imunoadsorção Enzimática/métodos , Limite de Detecção
9.
Chemistry ; 26(22): 5085-5092, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32096262

RESUMO

We report a method to detect proteins via suppression of rolling circle amplification (RCA) by using an appropriate aptamer as the linear primer (denoted as an aptaprimer) to initiate RCA. In the absence of a protein target, the aptaprimer is free to initiate RCA, which can produce long DNA products that are detected via binding of a fluorescent intercalating dye. Introduction of a target causes the primer region within the aptamer to become unavailable for binding to the circular template, inhibiting RCA. Using SYBR Gold or QuantiFluor dyes as fluorescent probes to bind to the RCA reaction product, it is possible to produce a generic protein-modulated RCA assay system that does not require fluorophore- or biotin-modified DNA species, substantially reducing complexity and cost of reagents. Based on this modulation of RCA, we demonstrate the ability to produce both solution and paper-based assays for rapid and quantitative detection of proteins including platelet derived growth factor and thrombin.


Assuntos
Biotina/química , DNA/metabolismo , Proteínas/metabolismo , Trombina/química , Biotina/metabolismo , Primers do DNA , Corantes Fluorescentes , Proteínas/química , Trombina/metabolismo
10.
Nat Commun ; 11(1): 32, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896744

RESUMO

Many intracellular pathogens, such as mammalian reovirus, mimic extracellular matrix motifs to specifically interact with the host membrane. Whether and how cell-matrix interactions influence virus particle uptake is unknown, as it is usually studied from the dorsal side. Here we show that the forces exerted at the ventral side of adherent cells during reovirus uptake exceed the binding strength of biotin-neutravidin anchoring viruses to a biofunctionalized substrate. Analysis of virus dissociation kinetics using the Bell model revealed mean forces higher than 30 pN per virus, preferentially applied in the cell periphery where close matrix contacts form. Utilizing 100 nm-sized nanoparticles decorated with integrin adhesion motifs, we demonstrate that the uptake forces scale with the adhesion energy, while actin/myosin inhibitions strongly reduce the uptake frequency, but not uptake kinetics. We hypothesize that particle adhesion and the push by the substrate provide the main driving forces for uptake.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Orthoreovirus Mamífero 3/fisiologia , Nanopartículas Metálicas/química , Actinas/metabolismo , Animais , Avidina/química , Biotina/química , Capsídeo/química , Células Cultivadas , Fibroblastos/virologia , Ouro , Células HeLa , Humanos , Integrinas/metabolismo , Cinética , Orthoreovirus Mamífero 3/química , Orthoreovirus Mamífero 3/patogenicidade , Nanopartículas Metálicas/virologia , Modelos Teóricos , Miosinas/metabolismo , Ratos , Vírion/patogenicidade , Vírion/fisiologia
11.
Inorg Chem ; 59(1): 913-924, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31825210

RESUMO

The ruthenium(II) complexes [RuCl(L1)(L3)]Cl (1), [RuCl(L1)(L4)]Cl (2), [RuCl(L2)(L4)]Cl (3), [RuCl(L1)(L5)]Cl (4), and [RuCl(L2)(L5)]Cl (5) of NNN-donor dipicolylamine (dpa) bases (L4, L5) having BODIPY (boron-dipyrromethene) moieties, NN-donor phenanthroline derivatives (L1, L2), and benzyldipicolylamine (bzdpa, L3) were prepared and characterized by spectroscopic techniques and their cellular localization/uptake and photocytotoxicity studied. Complex 1, as its PF6 salt (1a), has been structurally characterized with help of a single-crystal X-ray diffraction technique. It has a RuN5Cl core with the Cl bonded trans to the amine nitrogen atom of bzdpa. The complexes showed intense absorption spectral bands near 500 nm (ε ≈ 58000 M-1 cm-1) in 2 and 3 and 654 nm (ε ≈ 80000 M-1 cm-1) in 4 and 5 in 1/1 DMSO/DPBS (v/v). Complex 5 having biotin and PEGylated-disteryl BODIPY gave a singlet oxygen quantum yield (ΦΔ) of ∼0.65 in DMSO. Complex 5 exhibited remarkable PDT (photodynamic therapy) activity (IC50 ≈ 0.02 µM) with a photocytotoxicity index (PI) value of >5000 in red light of 600-720 nm in A549 cancer cells. The biotin-conjugated complexes showed better photocytotoxicity in comparison to nonbiotinylated analogues in A549 cells. The complexes displayed less toxicity in HPL1D normal cells in comparison to A549 cancer cells. The emissive BODIPY complexes 3 and 5 (ΦF ≈ 0.07 in DMSO) showed significant mitochondrial localization.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Luz , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Células A549 , Antineoplásicos/síntese química , Antineoplásicos/química , Biotina/química , Biotina/farmacologia , Boro/química , Boro/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Clivagem do DNA/efeitos dos fármacos , Teoria da Densidade Funcional , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Imagem Óptica , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/química , Porfobilinogênio/análogos & derivados , Porfobilinogênio/química , Porfobilinogênio/farmacologia , Rutênio/química , Rutênio/farmacologia
12.
Eur J Med Chem ; 186: 111831, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31740052

RESUMO

Heparanase is regarded as a promising target for anticancer drugs and Ronepastat is one of the most promising heparanase inhibitors insert in clinical study for Multiple Myeloma Therapy. To improve its pharmacokinetic/pharmacodynamic profile, as well to have an antidote able to neutralize its activity in case of over dosages or intolerance, a new class of its derivatives was obtained inserting non-carbohydrate moieties of different length between the polysaccharide chain and biotin or its derivatives. In vitro these novel derivatives maintain the anti-heparanase activity without induced toxicity. The newly synthesized compounds retained the ability to attenuate the growth of CAG myeloma tumors in mice with potency similar, or in one case even higher than that of the reference compound Roneparstat as well as inhibited metastatic dissemination (lung colonization) of murine B16-F10 melanoma cells in vivo.


Assuntos
Antineoplásicos/farmacologia , Biotina/química , Glucuronidase/antagonistas & inibidores , Glicóis/farmacologia , Inibidores de Glicosídeo Hidrolases/farmacologia , Heparina/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Glucuronidase/metabolismo , Glicóis/síntese química , Glicóis/química , Inibidores de Glicosídeo Hidrolases/síntese química , Inibidores de Glicosídeo Hidrolases/química , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Imagem Óptica , Relação Estrutura-Atividade
13.
Biochem Cell Biol ; 98(1): 31-41, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-30931575

RESUMO

RNA is involved in all domains of life, playing critical roles in a host of gene expression processes, host-defense mechanisms, cell proliferation, and diseases. A critical component in many of these events is the ability for RNA to interact with proteins. Over the past few decades, our understanding of such RNA-protein interactions and their importance has driven the search and development of new techniques for the identification of RNA-binding proteins. In determining which proteins bind to the RNA of interest, it is often useful to use the approach where the RNA molecule is the "bait" and allow it to capture proteins from a lysate or other relevant solution. Here, we review a collection of methods for modifying RNA to capture RNA-binding proteins. These include small-molecule modification, the addition of aptamers, DNA-anchoring, and nucleotide substitution. With each, we provide examples of their application, as well as highlight their advantages and potential challenges.


Assuntos
Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/química , RNA/análise , RNA/química , Biotina/química , Humanos , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química
14.
Talanta ; 206: 120228, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514892

RESUMO

The asymmetric flow field-flow fractionation (AF4) coupled on-line with elemental (inductively coupled plasma-mass spectrometry, ICP-MS) and molecular (fluorescence and UV) detection has been investigated as a powerful tool for the characterization of bioinorganic nano-conjugates. In this study, we described methods for the characterization of biotin-antibody complexes bioconjugated with streptavidin quantum dots (QDs-SA-b-Ab). Operating parameters of AF4 separation technique were optimized and two procedures are proposed using a channel thickness of 350 µm and 500 µm. The use of a 500 µm spacer allowed to achieve an efficient AF4 separation of the QDs-SA-b-Ab complexes from the excess of individual species used in the bioconjugation that was required for a proper characterization of the bioconjugates. Optimization of the AF4 allowed a separation resolution good enough to isolate the QDs-SA-b-Ab bioconjugates from the free excess of b-Ab and QD-SA. The efficiency of the bioconjugation process could be then calculated, obtaining a value of 86% for a 1 QDs-SA: 5 b-Ab bioconjugation ratio. In addition, sample recovery around 90% was achieved.


Assuntos
Pontos Quânticos/análise , Água/química , Anticorpos/química , Biotina/química , Compostos de Cádmio/análise , Compostos de Cádmio/química , Fluorescência , Fracionamento por Campo e Fluxo/métodos , Limite de Detecção , Espectrometria de Massas/métodos , Pontos Quânticos/química , Espalhamento de Radiação , Compostos de Selênio/análise , Compostos de Selênio/química , Estreptavidina/química , Sulfetos/análise , Sulfetos/química , Compostos de Zinco/análise , Compostos de Zinco/química
16.
Anal Chim Acta ; 1095: 138-145, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31864614

RESUMO

Glycosylation on the cell surface contains abundant biological information, and detecting the glycan on cell surfaces can offer critical insight into biology and diseases. Here, a signal amplification strategy for the sensitive detection of glycan expression on the cell surface was proposed. In this approach, glycans on the cell surface were detected with poly(glycidyl methacrylate)-grafted silica nanosphere labeled with quantum dots (QDs) and biotin through the specific affinity reaction of avidin-biotin on the cancer cells. Glycans on the cell surface were first labeled via selective oxidization of sialyl groups into aldehydes by periodate. Aniline-catalyzed hydrazone ligation with biotin hydrazide was then used for the specific recognition to avidin. The nanoprobe was fabricated with "living" SiO2 nanoparticles with alkyl bromide groups on their surfaces. They were then subsequently grafted with poly(glycidyl methacrylate) (PGMA) brushes via the successive surface-initiated atom transfer radical polymerization. The CdTe QDs and biotin were immobilized through a ring-open reaction with epoxy groups in the PGMA brushes to obtain QDs/biotin-polymer brush-functionalized silica nanosphere (SiO2-PGMA-QDs/biotin). Enhanced sensitivity could be achieved by an increase in CdTe QDs loading per assay event, because of the large number of surface functional epoxy groups offered by the PGMA. As a result, fluorescence signal increased versus the unamplified method. This method successfully demonstrates a simple, specific, and potent method to detect glycans on the cell surface.


Assuntos
Corantes Fluorescentes/química , Nanosferas/química , Ácidos Polimetacrílicos/química , Polissacarídeos/análise , Pontos Quânticos/química , Dióxido de Silício/química , Biotina/análogos & derivados , Biotina/química , Compostos de Cádmio/química , Células HeLa , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Polissacarídeos/química , Dióxido de Silício/síntese química , Estreptavidina/química , Telúrio/química
17.
Anal Chim Acta ; 1095: 212-218, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31864625

RESUMO

Sensitive and selective detection of miRNA is of great significance for the early diagnosis of human diseases, especially for cancers. Quartz crystal microbalance (QCM) is an effective tool for detecting biological molecules; however, the application of QCM for miRNA detection is still very limited. One of the great needs for QCM detection is to further improve the QCM signal. Herein, for the first time, we promote a new signal enhancement strategy for the detection of miRNA by QCM. First, a hairpin biotin-modified DNA was used as a probe DNA, which exposes the biotin site when interacting with target miRNA. Then, a streptavidin@metal-organic framework (SA@MOF) complex formed by electrostatic attractions between SA and a MOF was introduced into the QCM detection system. The SA@MOF complexes serve as both a signal amplifier and a specific recognition element via specific biotin-SA interactions. The strategy was applied to the detection of a colorectal cancer marker, miR-221, by using a stable Zr(IV)-MOF, UiO-66-NH2. The detection linear range was 10 fM-1 nM, the detection limit was 6.9 fM, and the relative standard deviation (RSD) (n = 5) was lower than 10% in both simulated conditions and the real serum environment. Furthermore, the detection limit reached 0.79 aM when coupled with the isothermal exponential amplification reaction (EXPAR).


Assuntos
Estruturas Metalorgânicas/química , MicroRNAs/análise , Estreptavidina/química , Animais , Técnicas Biossensoriais/métodos , Biotina/química , Bovinos , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Técnicas de Microbalança de Cristal de Quartzo/métodos
18.
Proc Jpn Acad Ser B Phys Biol Sci ; 95(10): 602-611, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31827018

RESUMO

In advanced cancer patients, malignant cells invade and disseminate within normal cells and develop resistance to therapy with additional genetic mutations, which makes radical cure very difficult. Precision medicine against advanced cancer is hampered by the lack of systems aimed at multiple target molecules within multiple loci. Here, we report the development of a versatile diagnostic and therapeutic system for advanced cancer, named the Cupid and Psyche system. Based on the strong non-covalent interaction of streptavidin and biotin, a low immunogenic mutated streptavidin, Cupid, and a modified artificial biotin, Psyche, have been designed. Cupid can be fused with various single-chain variable fragment antibodies and forms tetramer to recognize cancer cells precisely. Psyche can be conjugated to a wide range of diagnostic and therapeutic agents against malignant cells. The Cupid and Psyche system can be used in pre-targeting therapy as well as photo-immunotherapy effectively in animal models supporting the concept of a system for precision medicine for multiple targets within multiple loci.


Assuntos
Antineoplásicos/química , Biotina/química , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Estreptavidina/química , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Imunoterapia , Medicina de Precisão , Anticorpos de Cadeia Única/química
19.
Int J Nanomedicine ; 14: 9337-9349, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31819435

RESUMO

Background: Enzyme-linked immunosorbent assay (ELISA) is a common method for diagnosing swine influenza. However, the production of classical antibodies is both costly and time-consuming. As a promising alternative diagnostic tool, single-domain antibodies (sdAbs) offer the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity. Methods: Phage display technology was used to isolate sdAbs against the SIV-NP protein from a camel VHH library. The sdAb5 was fused to the biotin acceptor peptide (BAP) and a His-Tag for its expression as monomeric and site-specific biotinylation in E.coli to develop an sdAb-based blocking ELISA (sdAb-ELISA). In the sdAb-ELISA, the anti-SIV antibodies from swine samples were used to block the binding between the biotinylated sdAb5 and SIV-NP protein coated on the ELISA plate. The specificity, sensitivity, and reproducibility of sdAb-ELISA were determined. In addition, consistency among sdAb-ELISA, commercial ELISA kit, and Western blot was evaluated. Results: Six SIV-NP-specific sdAbs were isolated, among which sdAb5 was identified as a dominant sdAb with higher reactivity. The cut-off value of biotinylated sdAb5-based bELISA was determined to be 29.8%. Compared with the positive reference serum against five different types of swine viruses, the developed sdAb-ELISA showed 100% specificity. The detection limit of sdAb-ELISA was 1:160 in an anti-SIV positive reference serum, which is lower than that of the commercial ELISA kit (1:20). In 78 diluted anti-SIV positive serum (1:80), 21 and 42 samples were confirmed as positive by the commercial ELISA kit and sdAb-ELISA, respectively. The coefficients of variation of intra- and inter-assay were 1.79-4.57% and 5.54-9.98%, respectively. The sdAb-ELISA and commercial ELISA kit showed a consistency of 94.17% in clinical swine serum samples. Furthermore, the coincidence rate was 96.67% between the results detected by sdAb-ELISA and Western blot. Conclusion: A specific, sensitive, and reproducible sdAb-ELISA was successfully developed, which offers a new, promising method to detect anti-SIV antibodies in swine serum.


Assuntos
Anticorpos Antivirais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/imunologia , Anticorpos de Domínio Único/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Biotina/química , Biotinilação , Camelus , Nucleoproteínas/isolamento & purificação , Nucleoproteínas/metabolismo , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/virologia , Biblioteca de Peptídeos , Reprodutibilidade dos Testes , Anticorpos de Domínio Único/química , Suínos
20.
Opt Express ; 27(23): 34405-34415, 2019 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-31878488

RESUMO

Biosensing based on whispering-gallery mode (WGM) resonators has been continuously studied with great attention due to its excellent sensitivity guaranteeing the label-free detection. However, its practical impact is insignificant to date despite notable achievements in academic research. Here, we demonstrate a novel practical platform of on-chip WGM sensors integrated with microfluidic channels. By placing silicon nanoclusters as a stable active compound in micro-resonators, the sensor chip can be operated with a remote pump and readout, which simplifies the chip integration and connection to the external setup. In addition, silicon nanoclusters having large absorption cross-section over broad wavelength range allow active sensing for the first time with an LED pump in a top-illumination scheme which significantly reduces the complexity and cost of the measurement setup. The nano-slot structure of 25 nm gap width is embedded in the resonator where the target bio-molecules are selectively detected with the sensitivity enhanced by strongly confined mode-field. The sensitivity confirmed by real-time measurements for the streptavidin-biotin complex is 0.012 nm/nM, improved over 20 times larger than the previously reported WGM sensors with remote readout.


Assuntos
Algoritmos , Técnicas Biossensoriais , Luz , Proteínas de Bactérias/química , Biotina/análogos & derivados , Biotina/química , Processamento de Imagem Assistida por Computador
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA