RESUMO
Upcoversion nanoparticles are an emerging luminescent nanomaterial with excellent photophysical properties that have great benefits in biological sensing. In this study, a luminescent turn-on biosensor for cell-secreted protease activity assay is established based on resonance energy transfer in an upconversion nanoparticle-graphene oxide nano-assembly. The proposed biosensor consists of a blue-emitting upconversion nanoparticle covered with a quenching complex, comprising gelatin as the proteinase substrate and graphene oxide nanosheets as luminescence acceptors. After enzymatic digestion, the upconversion nanoparticles lose the gelatin cover due to the disassembly of the quenching complex, thus the upconverting luminescence in the blue region is restored (a turn-on response). The recovered upconverting luminescence is proportional to the protease concentration; the limit of detection was 12 ng ml(-1). Finally, the upconversion-graphene oxide nanocomplex was successfully applied in the detection of cell-secreted protease-metalloproteinase in MCF-7 cancer cells with high sensitivity and specificity.
Assuntos
Nanopartículas , Técnicas Biossensoriais , Sobrevivência Celular , Grafite , Humanos , Luminescência , Metaloproteinase 9 da MatrizRESUMO
In this study, we provide a facile, effective technique for a simple isolation and enrichment of low metastatic prostate tumor cell LNCaP using biocompatible, magnetic particles asissted impedimetric sensing system. Hydrophobic cell membrane anchors (BAM) were generated onto magnetic particles which diameters vary from 50 nm to 5 µm and were used to capture LNCaP cells from the suspension. Finally, magnetic particle-LNCaP complex were addressed onto the surface of the interdigitated microelectrode (IDM). Cell viability was monitored by our laboratory developed-technique Electrical Cell Substrate Impedance Sensing (ECIS). The results reavealed that 50 nm-magnetic particles showed best performance in terms of cell separation and cell viability. This technique provides a simple and efficient method for the direct addressing of LNCaP cell on the surface and enhances better understanding of cell behavior for cancer management in the near future.
Assuntos
Magnetismo , Microeletrodos , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Humanos , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de TransmissãoRESUMO
This study introduces the facile fabrication of a bimodal nanohybrid for the luminescent ON/OFF detection of glutathione. The proposed nanosensor consists of magnetic (Fe3O4) and upconversion nanoparticles (UCP) co-encapsulated in a silica matrix, and decorated with gold nanoparticle (AuNP) as a luminescent quencher. The detection mechanism is based on the Luminescent Resonance Energy Transfer (LRET) between the donor (UCP) and the acceptor (AuNP) with the help of a disulfide bond as a bridging element. In the presence of glutathione, the disulfide bridges between AuNPs and Fe3O4/UCP@SiO2 was cleaved and the amount of glutathione could be traced by the restored luminescence (ON state) of the nanohybrids after magnetic separation.
Assuntos
Ouro/química , Medições Luminescentes/métodos , Nanopartículas de Magnetita/química , Nanopartículas Metálicas/química , Dióxido de Silício/químicaRESUMO
Rare-earth doped nanoparticle (RE), termed upconversion nanoparticle (UCNP), is a new generation of phosphorescence which has recently attracted significant research interest. Due to the unique upconversion properties, UCNP has been considered to be an excellent alternative for conventional fluorescence. Since its first emergence in mid-1960s, UCNPs have been studied in a wide range of fields, including those in biological applications. Owing to its suitable size distribution and biocompatibility, UCNP could be conjugated with various kinds of biomolecules, resulting in the development of numerous biological platforms such as biodetection assays and therapeutic modalities. The unique optical properties of UCNP such as prominent luminescence, deep penetration to biological tissues without damaging the cells, low background and high resistance to photo-bleaching enhance UCNP prospects as an excellent contrast agent in both in vitro and in vivo. In this review, we discuss the recent developments of UCNP in bioassays, optical imaging, and therapy, also the prospects and challenges of UCNP-based detection in the development of biomedical science.
Assuntos
Bioensaio/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Terapia de Alvo Molecular/métodos , Nanopartículas/uso terapêutico , Fotoquimioterapia/métodos , Animais , Humanos , Nanopartículas/químicaRESUMO
This study described the prospect of silica nanotube surface modification in simultaneous detection of pathogenic bacteria by coupling cation exchange magnetic separation with quantum dot labeling. The outer surface of magnetic nanoparticles embedded long silica nanotube (capturing SNT) was modified with poly-L-lysine to serve as a cation exchange magnetic nano-complex to capture and isolate Escherichia coli (E. coli) O157:H7 and Salmonella enteritis typhimurium (S. typhimurium) in aqueous phase. Antibody conjugated quantum dot-embedded short SNT (reporting SNT) was used to simultaneously detect both bacteria, giving a high intensity and photo-stable fluorescent image that can detect single leveled bacterium binding on the capturing SNT. The fluorescent intensity generated from the capturing of both bacteria at different concentration was in the range of 10(2)-10(5) CFU/ml for both E. coli and S. typhimurium.
Assuntos
Bactérias/isolamento & purificação , Nanotubos , Dióxido de Silício/química , Bactérias/patogenicidade , Microscopia de Fluorescência , Pontos Quânticos , Propriedades de SuperfícieRESUMO
This study described a novel fabrication of fluorescence co-encapsulating silica nanotubes (F@SNT) and the further application of the as-synthesized nanostructure as a ratiometric pH sensor in buffer solution. Silica nanotubes (SNTs) embedded anodic alumina oxide (AAO) template was fabricated by sol-gel technique, tetramethyl rhodamine (TMR-the reference dye) was incorporated directly onto silica layer via hydrophobic interaction. Subsequently, fluorescent isothiocyanate (FITC-pH sensitive dye) was encapsulated inside poly-dimethylsiloxane (PDMS) matrix and the FITC-PDMS nanocomposite was doped into the hollow structure of SNT using nano-molding lithography. On removing AAO, free-standing SNTs were obtained and were subsequently applied as a ratiometric pH sensor in phosphate buffer solution. The dual dye-doped SNTs showed excellent fluorescence and a good pH sensing performance from pH 5.2-8.0. The results were distinguishable by the emission spectra and by fluorescent visualization. High photostability, sensitivity, biocompatibility with adjustable sizes make dual dye doped-SNT a promising nanostructure for bioapplications.
Assuntos
Corantes Fluorescentes/química , Nanotubos/química , Dióxido de Silício/química , Espectrometria de Fluorescência/métodos , Fluoresceína-5-Isotiocianato , Concentração de Íons de Hidrogênio , RodaminasRESUMO
Alzheimer's disease (AD) is a progressive form of dementia affecting almost 55 million people worldwide. It is characterized by the abnormal deposition of amyloid plaques and neurofibrillary tangles within the brain, leading to a pathological cascade of neuron degeneration and death as well as memory loss and cognitive decline. Amyloid beta (Aß) is an AD biomarker present in cerebrospinal fluid and blood serum and correlates with the presence of amyloid plaques and tau tangles in the brain. Measuring the levels of Aß can help with early diagnosis of AD, which is key for studying novel AD drugs and delaying the symptoms of dementia. However, this goal is difficult to achieve due to the low levels of AD biomarkers in biofluids. Here we demonstrate for the first time the use of FLOWER (frequency locked optical whispering evanescent resonator) for quantifying the levels of post-mortem cerebrospinal fluid (CSF) Aß42 in clinicopathologically classified control, mild cognitive impairment (MCI), and AD participants. FLOWER is capable of measuring CSF Aß42 (area under curve, AUC = 0.92) with higher diagnostic performance than standard ELISA (AUC = 0.82) and was also able to distinguish between control and MCI samples. Our results demonstrate the capability of FLOWER for screening CSF samples for early diagnosis of Alzheimer's pathology.
RESUMO
G-protein coupled receptors help regulate cellular function and communication, and are targets of small molecule drug discovery efforts. Conventional techniques to probe these interactions require labels and large amounts of receptor to achieve satisfactory sensitivity. Here, we use frequency-locked optical microtoroids for label-free characterization of membrane interactions in vitro at zeptomolar concentrations for the kappa opioid receptor and its native agonist dynorphin A 1-13, as well as big dynorphin (dynorphin A and dynorphin B) using a supported biomimetic membrane. The measured affinity of the agonist dynorphin A 1-13 to the κ-opioid receptor was also measured and found to be 3.1 nM. Radioligand assays revealed a dissociation constant in agreement with this value (1.1 nM). The limit of detection for the κOR/DynA 1-13 was calculated as 180 zM. The binding of Cholera Toxin B-monosialotetrahexosyl ganglioside was also monitored in real-time and an equilibrium dissociation constant of 1.53 nM was found. Our biosensing platform provides a method for highly sensitive real-time characterization of membrane embedded protein binding kinetics that is rapid and label-free, for drug discovery and toxin screening among other applications.
Assuntos
Dinorfinas , Receptores Opioides kappa , Receptores Opioides kappa/metabolismo , Receptores Opioides kappa/agonistas , Dinorfinas/metabolismo , Toxina da Cólera/metabolismo , Técnicas Biossensoriais/métodos , Ligação Proteica , Humanos , Ensaio Radioligante/métodosRESUMO
As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus mutates, finding effective drugs becomes more challenging. In this study, we use ultrasensitive frequency locked microtoroid optical resonators in combination with in silico screening to search for COVID-19 drugs that can stop the virus from attaching to the human angiotensin-converting enzyme 2 (hACE2) receptor in the lungs. We found 29 promising candidates that could block the binding site and selected four of them that were likely to bind very strongly. We tested three of these candidates using frequency locked optical whispering evanescent resonator (FLOWER), a label-free sensing method based on microtoroid resonators. FLOWER has previously been used for sensing single macromolecules. Here we show, for the first time, that FLOWER can provide accurate binding affinities and sense the inhibition effect of small molecule drug candidates without labels, which can be prohibitive in drug discovery. One of the candidates, methotrexate, showed binding to the spike protein 1.8 million times greater than that to the receptor binding domain (RBD) binding to hACE2, making it difficult for the virus to enter cells. We tested methotrexate against different variants of the SARS-CoV-2 virus and found that it is effective against all four of the tested variants. People taking methotrexate for other conditions have also shown protection against the original SARS-CoV-2 virus. Normally, it is assumed that methotrexate inhibits the replication and release of the virus. However, our findings suggest that it may also block the virus from entering cells. These studies additionally demonstrate the possibility of extracting candidate ligands from large databases, followed by direct receptor-ligand binding experiments on the best candidates using microtoroid resonators, thus creating a workflow that enables the rapid discovery of new drug candidates for a variety of applications.
RESUMO
Binding events to elements of the cell membrane act as receptors which regulate cellular function and communication and are the targets of many small molecule drug discovery efforts for agonists and antagonists. Conventional techniques to probe these interactions generally require labels and large amounts of receptor to achieve satisfactory sensitivity. Whispering gallery mode microtoroid optical resonators have demonstrated sensitivity to detect single-molecule binding events. Here, we demonstrate the use of frequency-locked optical microtoroids for characterization of membrane interactions in vitro at zeptomolar concentrations using a supported biomimetic membrane. Arrays of microtoroids were produced using photolithography and subsequently modified with a biomimetic membrane, providing high quality (Q) factors (>106) in aqueous environments. Fluorescent recovery after photobleaching (FRAP) experiments confirmed the retained fluidity of the microtoroid supported-lipid membrane with a diffusion coefficient of 3.38±0.26 µm2â s-1. Utilizing this frequency-locked membrane-on-a-chip model combined with auto-balanced detection and non-linear post-processing techniques, we demonstrate zeptomolar detection levels The binding of Cholera Toxin B- monosialotetrahexosyl ganglioside (GM1) was monitored in real-time, with an apparent equilibrium dissociation constant kd=1.53 nM. The measured affiny of the agonist dynorphin A 1-13 to the κ-opioid receptor revealed a kd=3.1 nM using the same approach. Radioligand binding competition with dynorphin A 1-13 revealed a kd in agreement (1.1 nM) with the unlabeled method. The biosensing platform reported herein provides a highly sensitive real-time characterization of membrane embedded protein binding kinetics, that is rapid and label-free, for toxin screening and drug discovery, among other applications.
RESUMO
Rapid detection of toxic and hazardous gases at trace concentrations plays a vital role in industrial, battlefield, and laboratory scenarios. Of interest are both sensitive as well as highly selective sensors. Whispering-gallery mode (WGM) microresonator-based biochemical sensors are among the most sensitive sensors in existence due to their long photon confinement times. One main concern with these devices, however, is their selectivity toward specific classes of target analytes. Here, we employ frequency locked WGM microtoroid optical resonators covalently modified with various polymer coatings to selectively detect the chemical warfare agent surrogate diisopropyl methylphosphonate (DIMP) as well as the toxic industrial chemicals formaldehyde and ammonia at parts-per-trillion concentrations (304, 434, and 117 ppt, respectively). This is 1-2 orders of magnitude better than previously reported, depending on the target, except for pristine graphene and pristine carbon nanotube sensors, which demonstrate similar detection levels but in vacuum and without selectivity. Selective polymer coatings include polyethylene glycol for DIMP sensing, accessed by the modification of commercially available materials, and 3-(triethoxysilyl) propyl-terminated polyvinyl acetate (PVAc) for ammonia sensing. Notably, we developed for the first time an efficient one-pot procedure to access 3-(triethoxysilyl) propyl-terminated PVAc that utilizes cobalt-mediated living radical polymerization and a nitroxyl polymer-terminating agent. Alkaline hydrolysis of PVAc coatings to form polyvinyl alcohol coatings directly bound to the microtoroid proved to be reliable and reproducible, leading to WGM sensors capable of the rapid and selective detection of formaldehyde vapors. The selectivity of these three polymer coatings as sensing media was predicted, in part, based on their functional group content and known reactivity patterns with the target analytes. Furthermore, we demonstrate that microtoroids coated with a mixture of polymers can serve as an all-in-one sensor that can detect multiple agents. We anticipate that our results will facilitate rapid early detection of chemical agents, as well as their surrogates and precursors.
RESUMO
Sensitive and rapid biosensors are of critical importance for a variety of applications including infectious disease detection and monitoring as well as medical diagnostics and drug discovery. Whispering gallery mode microtoroid optical resonators are among the most sensitive biochemical sensors in existence. When combined with frequency-locking and data-processing techniques, these sensors have been shown to be capable of single-molecule detection in under 30 s. The sensitivity of these sensors is affected by how a concentration of analyte molecules is transported to the surface of the sensors and the average time it takes the molecules to bind at that concentration. Currently, one question in the field is that at these low concentrations, how these microsensors achieve such rapid response times. Here, we reconcile theory and experiment and demonstrate through flow visualization experiments and finite-element simulations that the total analyte arrival and binding time can be on the order of seconds. This fast response time provides an advantage over nanoscale sensors such as nanowires or nanorods. We anticipate that these results can help us to control, with confidence, when and how many molecules bind to these sensors, thus enabling the building of faster and more sensitive sensors.
Assuntos
Técnicas Biossensoriais , NanotubosRESUMO
Phospholipid nanoshells, for example, liposomes, provide a versatile enabling platform for the development of nanometer-sized biosensors and molecular delivery systems. Utilization of phospholipid nanoshells is limited by the inherent instability in complex biological environments, where the phospholipid nanoshell may disassemble and degrade, thus releasing the contents and destroying sensor function. Polymer scaffold stabilization (PSS), wherein the phospholipid nanoshells are prepared by partitioning reactive monomers into the lipid bilayer lamella followed by radical polymerization, has emerged to increase phospholipid nanoshell stability. In this work, we investigated the effects of three different radical initiator conditions to fabricate stable PSS-phospholipid nanoshells yet retain the activity of encapsulated model fluorescent sensor proteins. To identify nondestructive initiation conditions, UV photoinitiation, neutral redox initiation, and thermal initiation were investigated as a function of PSS-phospholipid nanoshell stabilization and fluorescence emission intensity of enhanced green fluorescent protein (eGFP) and tandem dimer Tomato (td-Tomato). All three initiator approaches yielded comparably stable PSS-phospholipid nanoshells, although slight variations in PSS-phospholipid nanoshell size were observed, ranging from ca. 140 nm for unstabilized phospholipid nanoshells to 300-500 nm for PSS-phospholipid nanoshells. Fluorescence emission intensity of encapsulated eGFP was completely attenuated under thermal initiation (0% vs control), moderately attenuated under UV photoinitiation (40 ± 4% vs control), and unaffected by neutral redox initiation (97 ± 3% vs control). Fluorescence emission intensity of encapsulated td-Tomato was significantly attenuated under thermal initiation (13 ± 3% vs control), moderately attenuated UV photoinitiation (64 ± 5% vs control), and unaffected by neutral redox initiation (98% ± 4% vs control). Therefore, the neutral redox initiation method provides a significant advancement toward the preparation of protein-functionalized PSS-phospholipid nanoshells. These results should help to guide future applications and designs of biosensor platforms using PSS-phospholipid nanoshells and other polymer systems employing protein transducers.
RESUMO
This study introduces the double-ligands stabilizing gold nanoclusters and the fabrication of gold nanocluster/graphene nanocomplex as a "turn-on" fluorescent probe for the detection of cancer-related enzyme matrix metalloproteinase-9. A facile, one-step approach was developed for the synthesis of fluorescent gold nanoclusters using peptides and mercaptoundecanoic acid as co-templating ligands. The peptide was designed to possess a metalloproteinase-9 cleavage site and to act not only as a stabilizer but also as a targeting ligand for the enzyme detection. The prepared gold nanoclusters show an intense red fluorescence with a broad adsorption spectrum. In the presence of the enzyme, due to the excellent quenching properties and the negligible background of graphene oxide, the developed peptide-gold nanocluster/graphene nanocomplex yielded an intense "turn-on" fluorescent response, which strongly correlated with the enzyme concentration. The limit of detection of the nanocomplex was 0.15nM. The sensor was successfully applied for "turn-on" detection of metalloproteinase-9 secreted from human breast adenocarcinoma MCF-7 cells with high sensitivity, selectivity, significant improvement in terms of detection time and simplicity.
Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Ouro/química , Grafite/química , Metaloproteinase 9 da Matriz/análise , Nanoestruturas/química , Humanos , Ligantes , Limite de Detecção , Células MCF-7 , Nanoestruturas/ultraestrutura , Óxidos/química , Peptídeos/química , Espectrometria de Fluorescência/métodosRESUMO
MMP-9 (92 kDa gelatinease), which is member of matrix metalloproteinases (MMPs) family, plays a crucial role in the breakdown of extracellular matrix (ECM) by degrading the major components of ECM that lead to tumor cell invasion and metastasis through the basement membrane. Our study presents the on-chip dual-sensing device for rapid detection of cell-secreted MMP-9 and corresponding cell morphology changes in real-time domain. The device consists of 2 sensing platforms (both are interdigitated array microelectrodes - IDAMs) within 1 common fluidic chamber: one detects the cell morphology responses via Electric Cell-substrate Impedance Sensing (ECIS) technique, meanwhile the other records the cleavage effect between cell-secreted MMP-9 and the surface immobilized peptide via the capacitance-based sensing method. Thanks to the selectivity of designed peptide, this approach allows the rapid and specific detection of MMP-9. In comparison with gold standard ELISA assay, the detection time was significantly reduced from over 4h to within 30 min with the wide detection range from 10 pM to 10nM. Finally, this study provides the novel model for MMP-9 protease direct detection from living cell and new insights in multi-purpose detection of cancer associated enzyme and cell migration behavior.
Assuntos
Técnicas de Cultura de Células/instrumentação , Movimento Celular/fisiologia , Tamanho Celular , Condutometria/instrumentação , Dispositivos Lab-On-A-Chip , Metaloproteinase 9 da Matriz/metabolismo , Sistemas Computacionais , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Células MCF-7 , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
An electrical measurement known as Electric Cell-substrate Impedance Sensing (ECIS) has become increasingly applied to the study of cellular viability, proliferation and cytotoxicity with the advantages of label-free, non-invasion and real-time monitoring capability in comparison with other conventional methods (MTS, MTT). With this technique, cells are grown on the micro-sized gold electrodes where the micro-ampere alternative current is applied to measure the impedance changes due to the physiological changes caused by internal or external stimuli. In another field, Silica Nanotubes (SNTs) are a novel class of inorganic structures with promising potentials in bio-separation, drug delivery, imaging and other biomedical applications. In this study, by using ECIS-based self-fabricated cell chip, Cells were cultured on the working electrodes and separately exposure to the 0, 2 microm, 2 microm and 10 microm long at the varying concentrations of SNTs to evaluate the cellular responses such as viability, multiplication time and cytotoxicity. Final results were additionally compared with the MTS method as a reference to review the reliability