RESUMO
Developing precise tumor cell-specific mitochondrial ferroptosis-related inhibition miRNA imaging methods holds enormous potential for anticancer drug screening and cancer treatment. Nevertheless, traditional amplification methods still tolerated the limited tumor specificity because of the "off-tumor" signal leakage resulting from their "always-active" sensing mode. To overcome this limitation, we herein developed a dual (exogenous 808 nm NIR light and endogenous APE1) activated nanoladder for precise imaging of mitochondrial ferroptosis-related miRNA with tumor cell specificity and improved imaging resolution. Exogenous NIR light-activation can regulate the ferroptosis-related inhibition miRNA imaging signals within mitochondria, and endogenous enzyme-activation can confine signals to tumor cells. Based on this dual activation design, off-tumor signals were greatly reduced and tumor-to-background contrast was enhanced with an improved tumor/normal discrimination ratio, realizing tumor cell-specific precise imaging of mitochondrial ferroptosis-related inhibition miRNA.
Assuntos
Ferroptose , MicroRNAs , Mitocôndrias , Ferroptose/efeitos dos fármacos , Humanos , MicroRNAs/metabolismo , MicroRNAs/análise , Mitocôndrias/metabolismo , Animais , Camundongos , Imagem Óptica , Linhagem Celular Tumoral , Raios Infravermelhos , Nanopartículas/químicaRESUMO
The ultrasensitive DNA methyltransferase (Dam MTase) assay is of high significance for biomedical research and clinical diagnosis because of its profound effect on gene regulation. However, detection sensitivity is still limited by shortcomings, including photobleaching and weak signal intensities of conventional fluorophores at low concentrations. Plasmonic nanostructures with ultrastrong electromagnetic fields and fluorescence enhancement capability that can overcome these intrinsic defects hold great potential for ultrasensitive bioanalysis. Herein, a silica-coated gold nanostars (Au NSTs@SiO2)-based plasmon-enhanced fluorescence (PEF) probe with 20 "hot spots" was developed for ultrasensitive detection of Dam MTase. Here, the Dam Mtase assay was achieved by detecting the byproduct PPi of the rolling circle amplification reaction. It is worth noting that, benefiting from the excellent fluorescence enhancement capability of Au NSTs originating from their 20 "hot spots", the detection limit of Dam Mtase was reduced by nearly 105 times. Moreover, the proposed Au NST-based PEF probe enabled versatile evaluation of Dam MTase inhibitors as well as endogenous Dam MTase detection in GW5100 and JM110 Escherichia coli cell lysates, demonstrating its potential in biomedical analysis.
Assuntos
Técnicas Biossensoriais , DNA Metiltransferases Sítio Específica (Adenina-Específica) , DNA Metiltransferases Sítio Específica (Adenina-Específica)/análise , Dióxido de Silício , Ouro/química , Metilases de Modificação do DNA , Escherichia coli , Corantes Fluorescentes/química , DNA , Sondas de DNA/químicaRESUMO
BACKGROUND: There is currently a lack of comprehensive prevalence information on arthritis and its various classifications among adults in the U.S., particularly given the notable absence of detailed data regarding the Asian population. We examined the trends in the prevalence of arthritis, including osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis (PsA), and other types of arthritis, among U.S. adults by race between 2011 and 2018. METHODS: We analyzed data from the National Health and Nutrition Examination Survey (NHANES), spanning from 2011 to 2018. Our study focused on a nationally representative sample of U.S. adults aged 20 and older. Participants who answered "y es" to the research question "Doctors ever said you had arthritis?" were classified as having arthritis. Further classification into specific diseases was based on responses to the question "Which type of arthritis was it?" with options including "OA or degenerative arthritis, " "RA, " "PsA, " or "Other. " RESULTS: We analyzed 22,566 participants from NHANES (2011-2018), averaging 44.8 years, including 10,927 males. The overall arthritis prevalence rose significantly from 22.98% (95% CI: 21.47-24.55%) in 2011-12 to 27.95% (95% CI: 26.20-29.76%) in 2017-18 (P for trend < 0.001). OA increased from 12.02% (95% CI: 10.82-13.35%) in 2011 to 14.93% (95% CI: 13.47-16.51%) in 2018 (P for trend < 0.001). RA and PsA remained stable (P for trend = 0.220 and 0.849, respectively), while other arthritis rose from 2.03% (95% CI: 1.54-2.67%) in 2011-12 to 3.14% (95% CI: 2.56-3.86%) in 2017-18 (P for trend = 0.001). In Whites, Asians, and other races , arthritis and RA prevalence increased significantly (P for trend < 0.05). OA and other arthritis rose in Whites and other races (P for trend < 0.05), but no significant change occurred in the black population. The prevalence of PsA remained stable across all racial groups, with no statistically significant changes. CONCLUSIONS: In this nationally representative U.S. adult survey spanning 2011 to 2018, we identified a rising prevalence trend in arthritis, OA, and other arthritis, with notable variations among different racial groups.
Assuntos
Artrite , Inquéritos Nutricionais , Humanos , Masculino , Estados Unidos/epidemiologia , Adulto , Feminino , Prevalência , Pessoa de Meia-Idade , Artrite/epidemiologia , Adulto Jovem , Idoso , Grupos Raciais/estatística & dados numéricosRESUMO
BACKGROUND: Accurate classification can facilitate the selection of appropriate interventions to delay the progression of osteonecrosis of the femoral head (ONFH). This study aimed to perform the classification of ONFH through a deep learning approach. METHODS: We retrospectively sampled 1,806 midcoronal magnetic resonance images (MRIs) of 1,337 hips from 4 institutions. Of these, 1,472 midcoronal MRIs of 1,155 hips were divided into training, validation, and test datasets with a ratio of 7:1:2 to develop a convolutional neural network model (CNN). An additional 334 midcoronal MRIs of 182 hips were used to perform external validation. The predictive performance of the CNN and the review panel was also compared. RESULTS: A multiclass CNN model was successfully developed. In internal validation, the overall accuracy of the CNN for predicting the severity of ONFH based on the Japanese Investigation Committee classification was 87.8%. The macroaverage values of area under the curve (AUC), precision, recall, and F-value were 0.90, 84.8, 84.8, and 84.6%, respectively. In external validation, the overall accuracy of the CNN was 83.8%. The macroaverage values of area under the curve, precision, recall, and F-value were 0.87, 79.5, 80.5, and 79.9%, respectively. In a human-machine comparison study, the CNN outperformed or was comparable to that of the deputy chief orthopaedic surgeons. CONCLUSION: The CNN is feasible and robust for classifying ONFH and correctly locating the necrotic area. These findings suggest that classifying ONFH using deep learning with high accuracy and generalizability may aid in predicting femoral head collapse and clinical decision-making.
Assuntos
Aprendizado Profundo , Necrose da Cabeça do Fêmur , Humanos , Estudos Retrospectivos , Cabeça do Fêmur/diagnóstico por imagem , Necrose da Cabeça do Fêmur/diagnóstico por imagem , Necrose da Cabeça do Fêmur/cirurgia , Quadril/patologiaRESUMO
Developing an endogenous stimuli-responsive and ultrasensitive DNA sensing platform that contains a logic gate biocomputation for precise cell subtype identification holds great potential for disease diagnosis and prognostic estimation. Herein, a fluorescence-enhanced "OR-AND" DNA logic platform dual-driven by intracellular apurinic/apyrimidinic endonuclease 1 (APE 1) or a DNA strand anchored on membrane protein Mucin 1 (MUC 1) for sensitive and accurate cell subtype identification was rationally designed. The recognition toehold of the traditional activated probe (TP) was restrained by introducing a blocking sequence containing an APE 1 cleavable site (AP-site) that can be either cleaved by APE 1 or replaced by Mk-apt, ensuring the "OR-AND" gated molecular imaging for cell subtype identification. It is worth noting that this "OR-AND" gated design can effectively avoid the missing logical computation caused by membrane protein heterogeneous spatial distribution as a single input. In addition, a benefit from the excellent plasmon-enhanced fluorescence (PEF) ability of Au NSTs is that the detection limit can be decreased by nearly 165 times. Based on this, not only different kinds of MCF-7, HepG2, and L02 cells, but also different breast cancer cell subtypes, including malignant MCF-7, metastatic MDA-MB-231, and nontumorigenic MCF-10A cells, can be accurately identified by the proposed "OR-AND" gated DNA logic platform, indicating the prospect of this simple and universal design in accurate cancer screening.
Assuntos
DNA , DNA/genética , Fluorescência , Proteínas de Membrana , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Mucina-1 , Humanos , Linhagem Celular TumoralRESUMO
Strategies for achieving tumor-specific molecular imaging based on signal amplification hold great potential for evaluating the risk of tumor metastasis and progression. However, traditional amplification strategies are still constrained with limited tumor specificity because of the off-tumor signal leakage. Herein, an endogenous enzyme-activated autonomous-motion DNAzyme signal amplification strategy (E-DNAzyme) was rationally designed for tumor-specific molecular imaging with improved spatial specificity. The sensing function of E-DNAzyme can be specifically activated by the overexpressed apurinic/apyrimidinic endonuclease 1 (APE1) in the cytoplasm of tumor cells instead of normal cells, ensuring the tumor cell-specific molecular imaging with improved spatial specificity. Of note, benefiting from the target analogue-triggered autonomous motion of the DNAzyme signal amplification strategy, the detection limit can be decreased by approx. â¼7.8 times. Moreover, the discrimination ratio of tumor/normal cells of the proposed E-DNAzyme was â¼3.44-fold higher than the traditional amplification strategy, indicating the prospect of this universal design for tumor-specific molecular imaging.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Imagem Molecular , Técnicas Biossensoriais/métodosRESUMO
Osteoradionecrosis (ORN) of the femoral head is an important issue for orthopedists and radiologists in clinical practice. With the rapid development of technological advances in radiation therapy and the improvement in cancer survival rates, the incidence of ORN is rising, and there is an unmet need for basic and clinical research. The pathogenesis of ORN is complex, and includes vascular injury, mesenchymal stem cell injury, bone loss, reactive oxygen species, radiation-induced fibrosis, and cell senescence. The diagnosis of ORN is challenging and requires multiple considerations, including exposure to ionizing radiation, clinical manifestations, and findings on physical examination and imaging. Differential diagnosis is essential, as clinical symptoms of ORN of the femoral head can resemble many other hip conditions. Hyperbaric oxygen therapy, total hip arthroplasty, and Girdlestone resection arthroplasty are effective treatments, each with their own advantages and disadvantages. The literature on ORN of the femoral head is incomplete and there is no criterion standard or clear consensus on management. Clinicians should gain a better and more comprehensive understanding on this disease to facilitate its early and better prevention, diagnosis, and treatment. This article aims to review the pathogenesis, diagnosis, and management of osteoradionecrosis of the femoral head.
Assuntos
Artroplastia de Quadril , Osteorradionecrose , Humanos , Osteorradionecrose/diagnóstico , Osteorradionecrose/etiologia , Osteorradionecrose/terapia , Cabeça do Fêmur , Diagnóstico Diferencial , Radiação IonizanteRESUMO
PURPOSE: The aim of this study was to develop a deep convolutional neural network (DCNN) for detecting early osteonecrosis of the femoral head (ONFH) from various hip pathologies and evaluate the feasibility of its application. METHODS: We retrospectively reviewed and annotated hip magnetic resonance imaging (MRI) of ONFH patients from four participated institutions and constructed a multi-centre dataset to develop the DCNN system. The diagnostic performance of the DCNN in the internal and external test datasets was calculated, including area under the receiver operating characteristic curve (AUROC), accuracy, precision, recall, and F1 score, and gradient-weighted class activation mapping (Grad-CAM) technique was used to visualize its decision-making process. In addition, a human-machine comparison trial was performed. RESULTS: Overall, 11,730 hip MRI segments from 794 participants were used to develop and optimize the DCNN system. The AUROC, accuracy, and precision of the DCNN in internal test dataset were 0.97 (95% CI, 0.93-1.00), 96.6% (95% CI: 93.0-100%), and 97.6% (95% CI: 94.6-100%), and in external test dataset, they were 0.95 (95% CI, 0.91- 0.99), 95.2% (95% CI, 91.1-99.4%), and 95.7% (95% CI, 91.7-99.7%). Compared with attending orthopaedic surgeons, the DCNN showed superior diagnostic performance. The Grad-CAM demonstrated that the DCNN placed focus on the necrotic region. CONCLUSION: Compared with clinician-led diagnoses, the developed DCNN system is more accurate in diagnosing early ONFH, avoiding empirical dependence and inter-reader variability. Our findings support the integration of deep learning systems into real clinical settings to assist orthopaedic surgeons in diagnosing early ONFH.
Assuntos
Cabeça do Fêmur , Osteonecrose , Humanos , Estudos Retrospectivos , Redes Neurais de Computação , Imageamento por Ressonância Magnética/métodos , Osteonecrose/diagnóstico por imagemRESUMO
Nanogap antennas with strong electromagnetic fields of the "hot spot" in the gap region of two adjacent particles that can significantly improve the optical properties of fluorophores hold great potential for ultrasensitive bioanalysis. Herein, a DNA computation-mediated self-assembly of Au NBP dimer-based plasmonic nanogap antennas was designed for imaging of intracellular correlated dual disease biomarkers. It is worth noting that with the benefit from the electromagnetic fields of the "hot spot" in the gap region and strand displacement amplification, the fluorescence intensity can be enhanced â¼14.7-fold by Au NBP dimer-based plasmonic nanogap antennas. In addition, the AND-gate sensing mechanism was confirmed through monitoring the response of three designed nAP-PH1, m-PH1, and PH1 probes, the fluorescence recovery in different cell lines (Hela and L02), and inhibitor-treated cells, respectively. Furthermore, thanks to the "dual keys" activation design, such an "AND-gate" sensing manner can be used for ultrasensitive correlated multiplexed molecular imaging, demonstrating its feasible prospect in correlated multiplexed molecular imaging.
Assuntos
Computadores Moleculares , Corantes Fluorescentes , Polímeros , Imagem MolecularRESUMO
Optical cross-reactive sensor arrays inspired by the mammalian olfactory system that can realize straightforward discrimination of plasma from cancer patients hold great potential for point-of-care diseases diagnostics. Herein, a pH programmed fluorescence sensor array based on protein-responsive patterns was designed for straightforward discrimination of different types of cancer plasma. It is worth noting that plasma discrimination can be realized only by programming one nanomaterial using different pH values, which greatly simplifies the programmable design of the sensor array, making it an important highlight of this work. In addition, the mechanism of the pH programmed fluorescence sensor array for protein responsiveness was systematically investigated through molecular docking simulation, fluorescence resonance energy transfer (FRET), and fluorescence lifetime experiments. Most importantly, not only can the differences between plasma from healthy people and and from patients with different cancer species including gastric cancer, liver cancer, breast cancer, and cervical cancer be discriminated by this pH programmed fluorescence sensor array, but also the blind test of unknown plasma samples can be well identified with 100% accuracy, indicating its promising prospect in clinical application.
Assuntos
Nanoestruturas , Neoplasias , Animais , Transferência Ressonante de Energia de Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Mamíferos , Simulação de Acoplamento Molecular , Nanoestruturas/químicaRESUMO
Signal amplification strategies with spatiotemporally high sensitivity can provide more accurate information and hold great promise for improving the accuracy of disease diagnosis. Herein, a 808 nm near-infrared (NIR) light-activated plasmon enhanced fluorescence-catalytic hairpin assembly (PEF-CHA) signal amplification strategy was proposed for spatiotemporally controllable precise imaging of miRNA in vitro and in vivo with ultrasensitivity. The proposed 808 nm NIR light-activated PEF-CHA signal amplification strategy is constructed through combining up-conversion photocontrol and PEF technologies with CHA. It is worth noting that the laser irradiation-induced overheating effect could be effectively alleviated by using Nd3+-sensitized upconversion nanoparticles (UCNPs) to convert 808 nm NIR light to ultraviolet (UV) light, which is almost nondestructive to cells or tissues. In addition, nonspecific activation as well as false positive signals can be effectively avoided. Moreover, the detection limit can be reduced by approximate 38 times thanks to the high sensitivity of the proposed strategy. Furthermore, we demonstrate that the 808 nm NIR light-activated PEF-CHA signal amplification strategy can be expanded to sensitive and activatable imaging of intratumoral miRNAs in living mice, showing feasible prospects for precise biological and medical analysis.
Assuntos
Técnicas Biossensoriais , MicroRNAs , Nanopartículas , Animais , Técnicas Biossensoriais/métodos , Catálise , Fluorescência , Camundongos , MicroRNAs/análiseRESUMO
Programming ultrasensitive and stimuli-responsive DNAzyme-based probes that contain logic gate biocomputation hold great potential for precise molecular imaging. In this work, a DNA computation-mediated DNAzyme platform that can be activated by 808 nm NIR light and target c-MYC was designed for spatiotemporally controlled ultrasensitive AND-gated molecular imaging. Particularly, the sensing and recognition function of the traditional DNAzyme platform was inhibited by introducing a blocking sequence containing a photo-cleavable linker (PC-linker) that can be indirectly cleaved by 808 nm NIR light and thus enables the AND-gated molecular imaging. According to the responses toward three designed SDz, nPC-SDz, and m-SDz DNAzyme probes, the fluorescence recovery in diverse cell lines (MCF-7, HeLa, and L02) and inhibitor-treated cells was investigated to confirm the AND-gated sensing mechanism. It is worth noting that thanks to the strand displacement amplification and the ability of gold nanopyramids (Au NBPs) to enhance fluorescence, the fluorescence intensity increased by â¼7.9 times and the detection limit decreased by nearly 40.5 times. Moreover, false positive signals can be also excluded due to such AND-gated design. Furthermore, such a designed "AND-gate" sensing manner can also be applied to spatiotemporally controlled ultrasensitive in vivo molecular imaging, indicating its promising potential in precise biological molecular imaging.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Técnicas Biossensoriais/métodos , Computadores Moleculares , DNA Catalítico/genética , Ouro , Imagem MolecularRESUMO
"On-demand" accurate imaging of multiple intracellular miRNAs will significantly improve the detection reliability and accuracy. However, the "always-active" design of traditional multicomponent detection probes enables them to passively recognize and output signals as soon as they encounter targets, which will inevitably impair the detection accuracy and, inevitably, result in false-positive signals. To address this scientific problem, in this work, we developed a near-infrared (NIR) light-activated multicomponent detection intelligent nanoprobe for spatially and temporally controlled on-demand accurate imaging of multiple intracellular miRNAs. The proposed intelligent nanoprobe is composed of a rationally designed UV light-responsive triangular DNA nano sucker (TDS) and upconversion nanoparticles (UCNPs), named UCNPs@TDS (UTDS), which can enter cells autonomously through endocytosis and enable remote regulation of on-demand accurate imaging for multiple intracellular miRNAs using NIR light illumination at a chosen time and place. It is worth noting that the most important highlight of the UTDS we designed in this work is that it can resist nonspecific activation as well as effectively avoid false-positive signals and improve the accuracy of imaging of multiple intracellular miRNAs. Moreover, distinguishing different kinds of cell lines with different miRNA expressions levels can be also achieved through this NIR light-activated intelligent UTDS, showing feasible prospects in precise imaging and disease diagnosis.
Assuntos
MicroRNAs , Nanopartículas , DNA , Raios Infravermelhos , Reprodutibilidade dos TestesRESUMO
Plasmon-enhanced fluorescence (PEF) is considered to be a powerful signal amplification technology to overcome intrinsic shortcomings of photobleaching and brightness of the traditional fluorescent dyes. Nevertheless, exploitation of PEF-based probes for bioimaging application is still at a very early stage. In this work, a simple but powerful gold nanostar (Au NST)@SiO2-based PEF probe with 20 symmetric "hot spots" was developed for highly sensitive "lighting up" in situ imaging of intracellular microRNAs (miRNAs). By regulating the thickness of the silica shell, the distance between Au NSTs and fluorescent dyes was controlled, and the optimum fluorescence enhancement (21-fold) was obtained with the silica shell thickness of approximately 22 nm. Thanks to the 20 more powerful "hot spots" that can produce stronger localized electric fields, the Au NST-based PEF probe exhibits stronger PEF effects than the traditional plasmonic nanostructures such as gold nanorods (Au NRs), gold nanobipyramids (Au NBPs), and triangular gold nanoprisms (Au NPRs), resulting in high sensitivity and improved detection limit (LOD) of 0.21 pM for miRNA-21 analysis. Moreover, not only cancer cells (MCF-7 and Hela) and normal cells (L02) with distinct miRNA-21 expression levels can be discriminated but also tumor cells in co-cultured mixtures can be recognized, indicating its promising potential in clinical diagnosis.
RESUMO
Versatile all-in-one nanoplatforms that inherently possess both diagnostic imaging and therapeutic capabilities are highly desirable for efficient tumor diagnosis and treatment. Herein, we have developed a novel core-shell multifunctional nanomaterial-based all-in-one nanoplatform composed of gold nanobipyramids@polydopamine (Au NBPs@PDA) and gold nanoclusters (Au NCs) for simultaneous in situ multilayer imaging of dual types of tumor biomarkers (using a single-wavelength excitation) with different intracellular spatial distributions and fluorescence-guided photothermal therapy. The competitive combination between target transmembrane glycoprotein mucin1 (MUC1) and its aptamer caused Au NCs (620 nm) labeled with MUC1 aptamer to detach from the surface of Au NBPs@PDA, turning on the red fluorescence. Meanwhile, the hybridization between microRNA-21 (miRNA-21) and its complementary single-stranded DNA triggered the green fluorescence of Au NCs (515 nm). Based on this, simultaneous in situ multilayer imaging of dual types of tumor biomarkers with different intracellular spatial distributions was achieved. In addition, the potential of Au NBPs@PDA/Au NCs was also confirmed by simultaneous multilayer in situ imaging within not only three cell lines (MCF-7, HepG2, and L02 cells) with different expression levels of MUC1 and miRNA-21 but also cancer cells treated with different inhibitors. Moreover, the remarkable photothermal properties of Au NBPs@PDA resulted in the more efficient killing of cancer cells, demonstrating the great promise of the all-in-one nanoplatform for accurate diagnosis and tumor therapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Imagem Molecular/métodos , Nanoestruturas/química , Fototerapia , Nanomedicina Teranóstica/métodos , Linhagem Celular Tumoral , HumanosRESUMO
Gold nanoclusters (Au NCs) coated with various peptides have been widely used as fluorescent probes, and nowadays the most commonly used are cysteine (C) and tyrosine (Y) based ones. Herein, we report the preparation and clinical application of highly efficient and stable fluorescent Au NCs protected by screened peptides with a specific amino acid sequence Cys-Met-Met-Met-Met-Met (CMMMMM). Compared with traditional C, Y based peptide (CYYYYY) protected Au NCs, the fluorescence intensity of the CMMMMM-Au NCs increased by 230%, and the photobleaching resistance or stability of the CMMMMM-Au NCs increased by about 300% (after continuous ultraviolet irradiation for 60 min, the fluorescence of the CMMMMM-Au NCs remained more than 90% of their initial intensity, while the CYYYYY-Au NCs remained less than 30%). Assaying arrays based on CMMMMM protected Au NCs with different positive or negative charges as sensing receptors were developed through regulating different pH values, and multivariate analysis on the patterns obtained by these arrays allowed effective identification of not only ten proteins separately but also complex protein mixtures with subtly diverse compositions. The docking simulation and isothermal titration confirmed that target proteins interacted with CMMMMM-Au NCs mainly through electrostatic interactions and partly hydrophobic interactions, which affected the binding energy and fluorescence lifetime of CMMMMM-Au NCs, resulting in the unique fingerprint-like recognition patterns. Furthermore, serums from breast cancer, severe osteoarthritis, and rectal cancer patients can be effectively identified with healthy people using this CMMMMM-Au NCs based sensor array.
Assuntos
Ouro , Nanopartículas Metálicas/química , Peptídeos/química , Sequência de Aminoácidos , Neoplasias da Mama/diagnóstico , Corantes Fluorescentes/química , Osteoartrite/diagnóstico , Fotodegradação , Proteômica/métodos , Neoplasias Retais/diagnóstico , Eletricidade EstáticaRESUMO
With the increase in cancer risk, early immunodiagnosis is of great significance for timely therapy. In this work, a DNA-mediated immunosensor for the highly sensitive detection of prostate specific antigen (PSA) is proposed, which is mainly based on a portable personal glucose meter (PGM). Gold nanoparticles (AuNPs) functionalized with PSA detection antibodies and DNA primers are introduced. When the target of the PSA is present, rolling circle amplification (RCA) reactions on AuNPs are triggered and numerous repeated RCA products hybridize with the DNA-conjugated invertase; thus the signal of the PGM is generated and the PSA is quantified indirectly. With the use of a portable PGM, our method realizes a linear detection range of 0.003-50 ng mL-1, with a low detection limit of 0.1 pg mL-1, which is comparable to that of the traditional methods using expensive apparatus. Besides, the analysis of clinical human serum samples is performed to investigate its good practicability. This simple, low-cost, and miniaturized immunosensor is promising for the point-of-care testing of cancer markers.
Assuntos
Anticorpos Imobilizados/química , Técnicas Biossensoriais/métodos , DNA/química , Ouro/química , Nanopartículas Metálicas/química , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/diagnóstico , Humanos , Imunoensaio , Limite de Detecção , Masculino , Neoplasias da Próstata/sangueRESUMO
A sensitive and rapid fluorometric "switch on" assay is described for the detection of microRNA-21. It is based on the use of a fluorescence resonance energy transfer pair consisting of lysozyme-modified gold nanoclusters (Lys-Au NCs) and carbon nanotubes (CNTs). The Lys-Au NCs can be synthesized by a microwave-assisted technique within 2.5 min. They were modified with the ss-DNA probe (a 22-mer) for microRNA-21. Once the ss-DNA associates with the CNTs due to π stacking, the orange-red fluorescence (with excitation/emission peaks at 500/610 nm) is quenched. Nevertheless, the quenched fluorescence can be recovered after addition of microRNA-21 because of the stronger affnity between ss-DNA and microRNA-21. On the basis of the fluorescence recovery at 610 nm caused by microRNA-21, the latter can be quantified in the 0.01 to 100 nM concentration range, with a 36 pM detection limit. The method was applied to the determination of microRNA-21 in spiked serum with recoveries ranging from 98.6% to 110.0%. It also enables normal and cancer cells to be differentiated by direct imaging of intracellular microRNA-21. Graphical abstract A sensitive "switch on" FRET-based fluorometric assay for microRNA-21 is described. It is based on the use of lysozyme-modified gold nanoclusters (Lys-Au NCs) and carbon nanotubes (CNTs) as energy donor and energy acceptor, respectively.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , MicroRNAs/sangue , Nanotubos de Carbono/química , Ouro/química , Humanos , Limite de Detecção , Microscopia de Fluorescência/métodosRESUMO
A novel fluorescence resonance energy transfer (FRET)-based platform using polydopamine nanospheres (PDANSs) as energy acceptors and dual colored Au NCs as energy donors for simultaneous detection of multiple tumor-related microRNAs with DNase-I-assisted target recycling amplification was developed for the first time. On the basis of monitoring the change of the recovered fluorescence intensity at 445 and 575 nm upon the addition of targets miRNA-21 and let-7a, these two microRNAs (miRNAs) can be simultaneously quantitatively detected, with detection limits of 4.2 and 3.6 pM (3σ) for miRNA-21 and let-7a, which was almost 20 times lower than that without DNase I. Additionally, semiquantitative determination of miRNA-21 and let-7a can also be realized through photovisualization. Most importantly, serums from normal and breast cancer patients can be visually and directly discriminated without any sample pretreatment by confocal microscope experiments, demonstrating promising potential for auxiliary clinical diagnosis.
Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Ouro/química , Indóis/química , MicroRNAs/análise , Nanosferas/química , Polímeros/química , Neoplasias da Mama/sangue , Desoxirribonuclease I/química , Feminino , Humanos , MicroRNAs/sangue , Nanosferas/ultraestruturaRESUMO
An electrochemical biosensor for the detection of microRNA was prepared via chemical grafting of a Methylene Blue labeled reporter (MB-Rep) duplex onto a nanostructured surface that was obtained by electrodeposition of cobalt oxide and poly(o-phenylenediamine). This is followed by the attachment of hyaluronic acid and gold nanoclusters. In the presence of the target (microRNA), the probe-target duplex and the MB-Rep hairpin are formed. These will displace the labeled reporter from the sensor surface, and this results in a decrease of the amperometric signal for MB at a typical working voltage of -0.28 V (vs. Ag/AgCl). The electrode modified with hyaluronic acid possesses a large electroactive surface area and an excellent antifouling property. This makes it useful for ultrasensitive quantitation of microRNA even in complex biological media. The sensor has a linear response in the 100 f. to 0.1 µM microRNA concentration range, and a 33.3 f. detection limit. It was successfully applied to the determination of microRNA in cancer cells. Graphical abstract á .