Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 27.257
Filtrar
1.
Molecules ; 26(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201296

RESUMO

Osteosarcoma (OSA) is the most common malignant bone neoplasia in humans and dogs. In dogs, treatment consists of surgery in combination with chemotherapy (mostly carboplatin and/or doxorubicin (Dox)). Chemotherapy is often rendered ineffective by multidrug resistance. Previous studies have revealed that Dox conjugated with 4 nm glutathione-stabilized gold nanoparticles (Au-GSH-Dox) enhanced the anti-tumor activity and cytotoxicity of Dox in Dox-resistant feline fibrosarcoma cell lines exhibiting high P-glycoprotein (P-gp) activity. The present study investigated the influence of Au-GSH-Dox on the canine OSA cell line D17 and its relationship with P-gp activity. A human Dox-sensitive OSA cell line, U2OS, served as the negative control. Au-GSH-Dox, compared to free Dox, presented a greater cytotoxic effect on D17 (IC50 values for Au-GSH-Dox and Dox were 7.9 µg/mL and 15.2 µg/mL, respectively) but not on the U2OS cell line. All concentrations of Au-GSH (ranging from 10 to 1000 µg/mL) were non-toxic in both cell lines. Inhibition of the D17 cell line with 100 µM verapamil resulted in an increase in free Dox but not in intracellular Au-GSH-Dox. The results indicate that Au-GSH-Dox may act as an effective drug in canine OSA by bypassing P-gp.


Assuntos
Doxorrubicina/química , Doxorrubicina/farmacologia , Glutationa/química , Ouro/química , Nanopartículas Metálicas/química , Osteossarcoma/tratamento farmacológico , Adolescente , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Criança , Cães , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos
2.
Int J Nanomedicine ; 16: 4391-4407, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34234433

RESUMO

Background: Gold nanocages have been widely used as multifunctional platforms for drug and gene delivery, as well as photothermal agents for cancer therapy. However, their potential as gene delivery systems for cancer treatment has been reported in combination with chemotherapeutics and photothermal therapy, but not in isolation so far. The purpose of this work was to investigate whether the conjugation of gold nanocages with the cancer targeting ligand lactoferrin, polyethylene glycol and polyethylenimine could lead to enhanced transfection efficiency on prostate cancer cells in vitro, without assistance of external stimulation. Methods: Novel lactoferrin-bearing gold nanocages conjugated to polyethylenimine and polyethylene glycol have been synthesized and characterized. Their transfection efficacy and cytotoxicity were assessed on PC-3 prostate cancer cell line following complexation with a plasmid DNA. Results: Lactoferrin-bearing gold nanocages, alone or conjugated with polyethylenimine and polyethylene glycol, were able to condense DNA at conjugate:DNA weight ratios 5:1 and higher. Among all gold conjugates, the highest gene expression was obtained following treatment with gold complex conjugated with polyethylenimine and lactoferrin, at weight ratio 40:1, which was 1.71-fold higher than with polyethylenimine. This might be due to the increased DNA cellular uptake observed with this conjugate, by up to 8.65-fold in comparison with naked DNA. Conclusion: Lactoferrin-bearing gold nanocages conjugates are highly promising gene delivery systems to prostate cancer cells.


Assuntos
Portadores de Fármacos/química , Técnicas de Transferência de Genes , Ouro/química , Lactoferrina/química , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , DNA/administração & dosagem , DNA/química , DNA/genética , Terapia Genética , Humanos , Masculino , Plasmídeos/genética , Polietilenoglicóis/química , Polietilenoimina/química , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Transfecção
3.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208594

RESUMO

This article describes the synthesis and characterization of ß-cyclodextrin-based nano-sponges (NS) inclusion compounds (IC) with the anti-tumor drugs melphalan (MPH) and cytoxan (CYT), and the addition of gold nanoparticles (AuNPs) onto both systems, for the potential release of the drugs by means of laser irradiation. The NS-MPH and NS-CYT inclusion compounds were characterized using scanning electron microscopy (SEM), X-ray powder diffraction (XRPD), energy dispersive spectroscopy (EDS), thermogravimetric analysis (TGA), UV-Vis, and proton nuclear magnetic resonance (1H-NMR). Thus, the inclusion of MPH and CYT inside the cavities of NSs was confirmed. The association of AuNPs with the ICs was confirmed by SEM, EDS, TEM, and UV-Vis. Drug release studies using NSs synthesized with different molar ratios of ß-cyclodextrin and diphenylcarbonate (1:4 and 1:8) demonstrated that the ability of NSs to entrap and release the drug molecules depends on the crosslinking between the cyclodextrin monomers. Finally, irradiation assays using a continuous laser of 532 nm showed that photothermal drug release of both MPH and CYT from the cavities of NSs via plasmonic heating of AuNPs is possible.


Assuntos
Ciclodextrinas , Ciclofosfamida/administração & dosagem , Portadores de Fármacos , Ouro , Melfalan/administração & dosagem , Nanopartículas Metálicas , Técnicas de Química Sintética , Ciclodextrinas/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos/efeitos da radiação , Ouro/química , Luz , Espectroscopia de Ressonância Magnética , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Temperatura , Termogravimetria , Tocoferóis , Difração de Raios X
4.
Nat Protoc ; 16(7): 3522-3546, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34089021

RESUMO

Cellular heterogeneity is pervasive and of paramount importance in biology. Single-cell analysis techniques are indispensable for understanding the heterogeneity and functions of cells. Low-copy-number proteins (fewer than 1,000 molecules per cell) perform multiple crucial functions such as gene expression, cellular metabolism and cell signaling. The expression level of low-copy-number proteins of individual cells provides key information for the in-depth understanding of biological processes and diseases. However, the quantitative analysis of low-copy-number proteins in a single cell still remains challenging. To overcome this, we developed an approach called single-cell plasmonic immunosandwich assay (scPISA) for the quantitative measurement of low-copy-number proteins in single living cells. scPISA combines in vivo microextraction for specific enrichment of target proteins from cells and a state-of-the-art technique called plasmon-enhanced Raman scattering for ultrasensitive detection of low-copy-number proteins. Plasmon-enhanced Raman scattering detection relies on the plasmonic coupling effect (hot-spot) between silver-based plasmonic nanotags and a gold-based extraction microprobe, which dramatically enhances the signal intensity of the surface-enhanced Raman scattering of the nanotags and thereby enables sensitivity at the single-molecule level. scPISA is a straightforward and minimally invasive technique, taking only ~6-15 min (from in vivo extraction to Raman spectrum readout). It is generally applicable to all freely floating intracellular proteins provided that appropriate antibodies or alternatives (for example, molecularly imprinted polymers or aptamers) are available. The entire protocol takes ~4-7 d to complete, including material fabrication, single-cell manipulation, protein labeling, signal acquisition and data analysis.


Assuntos
Dosagem de Genes , Imunoensaio/métodos , Proteínas/metabolismo , Análise de Célula Única , Anticorpos/metabolismo , Calibragem , Linhagem Celular Tumoral , Sobrevivência Celular , Análise de Dados , Ouro/química , Humanos , Proteínas Imobilizadas/metabolismo , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Coloração e Rotulagem
5.
Methods Mol Biol ; 2268: 85-95, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085263

RESUMO

Mammalian olfactory receptors (ORs) constitute the largest family of G-protein-coupled receptors, with up to about 1000 different genes per species, each having specific odorant ligands. ORs could be used as sensing elements of highly specific and sensitive bioelectronic hybrid devices such as bioelectronic noses. After optimized immobilization onto the device, natural ORs provide molecular recognition of various odors with their intrinsic sensitivity, discrimination, and detection properties. However, the main difficulties are related to the low expression level of recombinant ORs, their stability and potential loss of activity. Such drawbacks can be successfully overcome in bioelectronic noses integrating nanosomes (nanometric membrane vesicles carrying ORs) that are stably immobilized through a specific antibody. The advantages of such a platform rely on the fact that ORs stay in the natural membrane environment, and thus preserve their full activity. Thanks to their small sizes, nanosomes offer potential for micro- and nano-scale sensor development. In this paper, we summarize the key elements regarding nanosomes production and manipulation and provide an example of their immobilization onto a gold sensor surface. Rat ORI7 is used as a representative OR that can be functionally expressed in Saccharomyces cerevisiae. The receptor was not purified but only nanosomes were prepared. Nanosomes were immobilized onto functionalized gold surface using the anti-I7 antibody. Utilization of the antibody provides enrichment of ORI7 on the sensor surface but also uniform and appropriate orientation of the receptors. These features are crucial in optimization of bioelectronic nose' analytical performances.


Assuntos
Técnicas Biossensoriais/métodos , Ouro/química , Proteínas Imobilizadas/metabolismo , Proteínas de Membrana/metabolismo , Odorantes/análise , Receptores Odorantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Ratos , Receptores Odorantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética
6.
Carbohydr Polym ; 268: 118259, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34127229

RESUMO

Nitrocellulose (NC) membrane can have value-added applications for lateral flow assay (LFA)-based diagnostic tools, which has great potential for the detection of pathogens, such as COVID-19, in different environments. However, poor sensitivity of the NC membrane based LFA limits its further application in many cases. Herein, we developed a facile method for LFA sensitivity enhancement, by incorporating two-sugar barrier into LFAs: one between the conjugation pad and the test line, and the other between the test line and the control line. ORF1ab nucleic acid of COVID-19 was used as the model target to demonstrate the concept on the HF120 membrane. Results show that at optimum conditions, the two sugar barrier LFAs have a detection limit of 0.5 nM, which is compared to that of 2.5 nM for the control LFA, achieving a 5-fold sensitivity increase. This low cost, easy-to-fabricate and easy-to-integrate LFA method may have potential applications in other cellulose paper-based platforms.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Colódio/química , RNA Mensageiro/análise , Açúcares/química , Proteínas Virais/genética , Teste de Ácido Nucleico para COVID-19/instrumentação , DNA/química , Sondas de DNA/química , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Poliproteínas/genética , SARS-CoV-2/química , Sensibilidade e Especificidade
7.
Carbohydr Polym ; 268: 118259, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: covidwho-1242891

RESUMO

Nitrocellulose (NC) membrane can have value-added applications for lateral flow assay (LFA)-based diagnostic tools, which has great potential for the detection of pathogens, such as COVID-19, in different environments. However, poor sensitivity of the NC membrane based LFA limits its further application in many cases. Herein, we developed a facile method for LFA sensitivity enhancement, by incorporating two-sugar barrier into LFAs: one between the conjugation pad and the test line, and the other between the test line and the control line. ORF1ab nucleic acid of COVID-19 was used as the model target to demonstrate the concept on the HF120 membrane. Results show that at optimum conditions, the two sugar barrier LFAs have a detection limit of 0.5 nM, which is compared to that of 2.5 nM for the control LFA, achieving a 5-fold sensitivity increase. This low cost, easy-to-fabricate and easy-to-integrate LFA method may have potential applications in other cellulose paper-based platforms.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Colódio/química , RNA Mensageiro/análise , Açúcares/química , Proteínas Virais/genética , Teste de Ácido Nucleico para COVID-19/instrumentação , DNA/química , Sondas de DNA/química , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Poliproteínas/genética , SARS-CoV-2/química , Sensibilidade e Especificidade
8.
Food Chem ; 362: 130261, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34111691

RESUMO

In this study, a novel surface enhanced Raman spectroscopy (SERS) sensor was developed for the ultrasensitive determination of kanamycin in foods. The sensor used two distinct signal amplification strategies, namely the surface plasmon resonance of gold nanorods and a Zn-doped carbon quantum dots catalytic cascade oxidation-reduction reaction switch controlled by a nucleic acid aptamer. Under optimized experimental conditions, the SERS sensor demonstrated a linear range of 10-12 to 10-5 g mL-1 for the detection of kanamycin, with a limit of detection of 3.03 × 10-13 g mL-1. Experiments with antibiotics structurally similar to kanamycin and interferrants revealed that the sensor had excellent selectivity. Milkpowder and honey samples spiked with kanamycin were assayed, with recoveries ranging from 84.1% to 107.2% and a relative standard deviation of 0.74% to 2.81% being obtained. Quantification of kanamycin in milk samples revealed no significant difference between the results obtained with the sensor and by HPLC.


Assuntos
Aptâmeros de Nucleotídeos/química , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Canamicina/análise , Nanotubos/química , Pontos Quânticos/química , Zinco/química , Antibacterianos/análise , Técnicas Biossensoriais/instrumentação , Carbono/química , Catálise , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Análise Espectral Raman/métodos , Ressonância de Plasmônio de Superfície
9.
J Phys Chem Lett ; 12(23): 5616-5622, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34110174

RESUMO

Ligand shell-protected gold nanoparticles can form nanoreceptors that recognize and bind to specific molecules in solution, with numerous potential innovative applications in science and industry. At this stage, the challenge is to rationally design such nanoreceptors to optimize their performance and boost their further development. Toward this aim, we have developed a new computational tool, Nanotron. This allows the analysis of molecular dynamics simulations of ligand shell-protected nanoparticles to define their exact surface morphology and pocket fingerprints of binding cavities in the coating monolayer. Importantly, from dissecting the well-characterized pairing formed by the guest salicylate molecule and specific host nanoreceptors, our work reveals that guest binding at such nanoreceptors occurs via preformed deep pockets in the host. Upon the interaction with the guest, such pockets undergo an induced-fit-like structural optimization for best host-guest fitting. Our findings and methodological advancement will accelerate the rational design of new-generation nanoreceptors.


Assuntos
Ouro/análise , Nanopartículas Metálicas/análise , Simulação de Dinâmica Molecular , Mapeamento de Peptídeos/métodos , Biologia Computacional/métodos , Ouro/química , Nanopartículas Metálicas/química , Propriedades de Superfície
10.
Food Chem ; 362: 130118, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34082296

RESUMO

Organophosphate pesticides (OPs) are often used as insecticides and acaricides in agriculture, thus improving yields. OP residues may pose a serious threat, duetoinhibitionof the enzymeacetylcholinesterase(AChE). Therefore, a competitive bio-barcode immunoassay was designed for simultaneous quantification of organophosphate pesticide residues using AuNP signal amplification technology and Au@Pt catalysis. The AuNP probes were labelled with antibodies and corresponding bio-barcodes (ssDNAs), MNP probes coated with ovalbumin pesticide haptens and Au@Pt probes functionalized with the complementary ssDNAs were then prepared. Subsequently, pesticides competed with MNP probes to bind the AuNP probes. The recoveries of the developed assay were ranged from 71.26 to 117.47% with RSDs from 2.52 to 14.52%. The LODs were 9.88, 3.91, and 1.47 ng·kg-1, for parathion, triazophos, and chlorpyrifos, respectively. The assay was closely correlated with the data obtained from LC-MS/MS. Therefore, the developed method has the potential to be used as an alternative approach for detection of multiple pesticides.


Assuntos
Contaminação de Alimentos/análise , Imunoensaio/métodos , Nanopartículas Metálicas/química , Resíduos de Praguicidas/análise , Catálise , Clorpirifos/análise , Cromatografia Líquida , Análise de Alimentos/métodos , Ouro/química , Imunoensaio/instrumentação , Limite de Detecção , Compostos Organofosforados/análise , Organotiofosfatos/análise , Oxazinas/química , Paration/análise , Platina/química , Espectrometria de Massas em Tandem , Triazóis/análise
11.
Int J Nanomedicine ; 16: 3695-3705, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34113098

RESUMO

Purpose: Detection of single-base mutations is important for real-time monitoring of tumor progression, therapeutic effects, and drug resistance. However, the specific detection of single-base mutations from excessive wild-type background sequences with routine PCR technology remains challenging. Our objective is to develop a simple and highly specific qPCR-based single-base mutation detection method. Methods: Using EGRF T790M as a model, gold nanoparticles at different concentrations were separately added into the Taqman-MGB qPCR system to test specificity improvement, leading to the development of the optimal Taqman-MGB nanoPCR system. Then, these optimal conditions were used to test the range of improvement in the specificity of mutant-type and wild-type templates and the detection limit of mutation abundances in a spiked sample. Results: The Taqman-MGB nanoPCR was established based on the traditional qPCR, with significantly suppressed background noise and improved specificity for single-base mutation detection. With EGFR T790M as a template, we demonstrated that our Taqman-MGB nanoPCR system could improve specificity across a wide concentration range from 10-9 µM to 10 µM and detect as low as 0.95% mutation abundance in spiked samples, which is lower than what the traditional Taqman-MGB qPCR and existing PCR methods can detect. Moreover, we also proposed an experimentally validated barrier hypothesis for the mechanism of improved specificity. Conclusion: The developed Taqman-MGB nanoPCR system could be a powerful tool for clinical single-base mutation detection.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Mutação , Neoplasias/genética , Neoplasias/patologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptores ErbB/genética , Humanos
12.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064888

RESUMO

Advances in nanomedicine have seen the adaptation of nanoparticles (NPs) for subcellular delivery for enhanced therapeutic impact and reduced side effects. The pivotal role of the mitochondria in apoptosis and their potential as a target in cancers enables selective induction of cancer cell death. In this study, we examined the mitochondrial targeted delivery of betulinic acid (BA) by the mitochondriotropic TPP+-functionalized epigallocatechin gallate (EGCG)-capped gold NPs (AuNPs), comparing the impact of polyethylene glycol (PEG) and poly-L-lysine-graft-polyethylene glycol (PLL-g-PEG) copolymer on delivery efficacy. This included the assessment of their cellular uptake, mitochondrial localization and efficacy as therapeutic delivery platforms for BA in the human Caco-2, HeLa and MCF-7 cancer cell lines. These mitochondrial-targeted nanocomplexes demonstrated significant inhibition of cancer cell growth, with targeted nanocomplexes recording IC50 values in the range of 3.12-13.2 µM compared to that of the free BA (9.74-36.31 µM) in vitro, demonstrating the merit of mitochondrial targeting. Their mechanisms of action implicated high amplitude mitochondrial depolarization, caspases 3/7 activation, with an associated arrest at the G0/G1 phase of the cell cycle. This nano-delivery system is a potentially viable platform for mitochondrial-targeted delivery of BA and highlights mitochondrial targeting as an option in cancer therapy.


Assuntos
Sistemas de Liberação de Medicamentos , Ouro/química , Nanopartículas Metálicas/administração & dosagem , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Triterpenos Pentacíclicos/farmacologia , Apoptose , Células CACO-2 , Ciclo Celular , Proliferação de Células , Células HeLa , Humanos , Células MCF-7 , Nanopartículas Metálicas/química , Neoplasias/patologia , Triterpenos Pentacíclicos/química
13.
Food Chem ; 361: 129901, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34082384

RESUMO

A simple electrochemical immunosensor based on nitrogen-doped graphene and polyamide-amine (GN-PAM) composites was proposed for the detection of the CP4-EPSPS protein in genetically modified (GM) crops. In this immunosensor, the amplification of the detection signal was realized through antibodies labeled with gold nanoparticles (AuNPs). The electrochemical responses of the immunosensor were linear (R2 = 0.9935 and 0.9912) when the GM soybean RRS and maize NK603 content ranged from 0.025% to 1.0% and 0.05% to 1.5%, respectively. The limits of detection for the GM soybean RRS and maize NK603 were as low as 0.01% and 0.03%, respectively. The immunosensor also exhibited high specificity, and satisfactory stability, reproducibility, and accuracy. Our findings indicated that the constructed immunosensor provides a new approach for the sensitive detection of the CP4-EPSPS protein. Notably, the sensor may be applied to other proteins or pathogenic bacteria by simply changing the antibodies, and may also be used for multi-component analysis.


Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Produtos Agrícolas/genética , Imunoensaio/métodos , Plantas Geneticamente Modificadas/genética , Anticorpos Monoclonais/química , Produtos Agrícolas/química , Técnicas Eletroquímicas , Ouro/química , Grafite/química , Limite de Detecção , Nanopartículas Metálicas/química , Plantas Geneticamente Modificadas/química , Poliaminas/química , Reprodutibilidade dos Testes , Soja/química , Soja/genética , Zea mays/química , Zea mays/genética
14.
J Phys Chem Lett ; 12(23): 5564-5570, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34105967

RESUMO

Multiplexing capabilities and sensitivity of surface-enhanced Raman spectroscopy (SERS) nanoparticles (NPs) are strongly dependent on the selected Raman reporter. These Raman-active molecules are responsible for giving each batch of SERS NPs its unique spectral fingerprint. Herein, we studied four types of SERS NPs, namely, AuNPs labeled with trans-1,2-bis(4-pyridyl)ethylene (BPE), 4,4'-bis(mercaptomethyl)biphenyl (BMMBP), 5-(4-pyridyl)-1,3,4-oxadiazole-2-thiol (PODT), and 5-(4-pyridyl)-1H-1,2,4-triazole-3-thiol (PTT), and demonstrated that the best level of theory could be chosen based on inner products of DFT-calculated and experimental Raman spectra. We also calculated the theoretical spectra of these Raman reporters bound to Au20 clusters to interrogate how SERS enhancement would affect their spectral fingerprint. Importantly, we found a correlation between B3LYP-D3 calculated and experimental enhancement factors, which opens up an avenue toward predicting which Raman reporters could offer improved sensitivity. We observed 0.5 and 3 fM limits of detection for BMMBP- and PTT-labeled 60 nm AuNPs, respectively.


Assuntos
Ouro/análise , Ouro/química , Nanopartículas Metálicas/análise , Nanopartículas Metálicas/química , Análise Espectral Raman/métodos , Propriedades de Superfície
15.
Mikrochim Acta ; 188(6): 199, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: covidwho-1245646

RESUMO

Since the COVID-19 disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) was declared a pandemic, it has spread rapidly, causing one of the most serious outbreaks in the last century. Reliable and rapid diagnostic tests for COVID-19 are crucial to control and manage the outbreak. Here, a label-free square wave voltammetry-based biosensing platform for the detection of SARS-CoV-2 in nasopharyngeal samples is reported. The sensor was constructed on screen-printed carbon electrodes coated with gold nanoparticles. The electrodes were functionalized using 11-mercaptoundecanoic acid (MUA) which was used for the immobilization of an antibody against SARS-CoV-2 nucleocapsid protein (N protein). The binding of the immunosensor with the N protein caused a change in the electrochemical signal. The detection was realised by measuring the change in reduction peak current of a redox couple using square wave voltammetry at 0.04 V versus Ag ref. electrode on the immunosensor upon binding with the N protein. The electrochemical immunosensor showed high sensitivity with a linear range from 1.0 pg.mL-1 to 100 ng.mL-1 and a limit of detection of 0.4 pg.mL-1 for the N protein in PBS buffer pH 7.4. Moreover, the immunosensor did not exhibit significant response with other viruses such as HCoV, MERS-CoV, Flu A and Flu B, indicating the high selectivity of the sensor for SARS-CoV-2. However, cross reactivity of the biosensor with SARS-CoV is indicated, which gives ability of the sensor to detect both SARS-CoV and SARS-CoV-2. The biosensor was successfully applied to detect the SARS-CoV-2 virus in clinical samples showing good correlation between the biosensor response and the RT-PCR cycle threshold values. We believe that the capability of miniaturization, low-cost and fast response of the proposed label-free electrochemical immunosensor will facilitate the point-of-care diagnosis of COVID 19 and help prevent further spread of infection.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/análise , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , SARS-CoV-2/química , Anticorpos Imobilizados/imunologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Teste para COVID-19/instrumentação , Carbono/química , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ácidos Graxos/química , Ouro/química , Humanos , Imunoensaio/instrumentação , Limite de Detecção , Nanopartículas Metálicas/química , Nasofaringe/virologia , Fosfoproteínas/análise , Fosfoproteínas/imunologia , Compostos de Sulfidrila/química
16.
Nat Protoc ; 16(6): 3141-3162, 2021 06.
Artigo em Inglês | MEDLINE | ID: covidwho-1209962

RESUMO

The global pandemic of coronavirus disease 2019 (COVID-19) highlights the shortcomings of the current testing paradigm for viral disease diagnostics. Here, we report a stepwise protocol for an RNA-extraction-free nano-amplified colorimetric test for rapid and naked-eye molecular diagnosis of COVID-19. The test employs a unique dual-prong approach that integrates nucleic acid (NA) amplification and plasmonic sensing for point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with a sample-to-assay response time of <1 h. The RNA-extraction-free nano-amplified colorimetric test utilizes plasmonic gold nanoparticles capped with antisense oligonucleotides (ASOs) as a colorimetric reporter to detect the amplified nucleic acid from the COVID-19 causative virus, SARS-CoV-2. The ASOs are specific for the SARS-CoV-2 N-gene, and binding of the ASOs to their target sequence results in the aggregation of the plasmonic gold nanoparticles. This highly specific agglomeration step leads to a change in the plasmonic response of the nanoparticles. Furthermore, when tested using clinical samples, the accuracy, sensitivity and specificity of the test were found to be >98.4%, >96.6% and 100%, respectively, with a detection limit of 10 copies/µL. The test can easily be adapted to diagnose other viral infections with a simple modification of the ASOs and primer sequences. It also provides a low-cost, rapid approach requiring minimal instrumentation that can be used as a screening tool for the diagnosis of COVID-19 at point-of-care settings in resource-poor situations. The colorimetric readout of the test can even be monitored using a handheld optical reader to obtain a quantitative response. Therefore, we anticipate that this protocol will be widely useful for the development of biosensors for the molecular diagnostics of COVID-19 and other infectious diseases.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Ouro/química , Nanopartículas Metálicas/química , Oligonucleotídeos Antissenso/química , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/instrumentação , Colorimetria/instrumentação , Colorimetria/métodos , Humanos , Limite de Detecção , Oligonucleotídeos Antissenso/genética , Testes Imediatos , RNA Viral/genética , SARS-CoV-2/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-34022759

RESUMO

Low-molecular-weight thiols are widely present in human fluids, and are regarded as a kind of potential broad-spectrum evaluation indicators for some clinical diseases. In this work, gold nanoparticles capped with Tween 20 were used for purification and microextraction of the main free thiols (cysteine, homocysteine, glutathione and methionine) in saliva based on Au-S bond formation. Ultrasound further sped up the releasing of the target analytes, and the releasing time needed was only 10 min, and the required sample volume was only 40 µL. The desorption solution could be directly injected for electrophoretic analysis without derivatization, and field-amplified sample stacking of electrophoretic online enrichment technology further improved the detection sensitivity. The synergistic enrichment effect made the enrichment factors of four analytes reach 1119-2067 times. This developed method was applied for the analyses of saliva samples of healthy volunteers. Acceptable sensitivity (LODs: 0.15-1.5 ng mL-1) and recoveries (97.6-116%) were obtained in the saliva sample matrix. This proposed method provides an alternative for the sensitive detection of low-molecular-weight thiols in noninvasive body fluids, which has potential application prospect in the preliminary noninvasive diagnosis of diabetes, cardiovascular diseases, etc.


Assuntos
Eletroforese Capilar/métodos , Nanopartículas Metálicas/química , Polissorbatos/química , Saliva/química , Compostos de Sulfidrila , Adulto , Ouro/química , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Compostos de Sulfidrila/análise , Compostos de Sulfidrila/isolamento & purificação , Adulto Jovem
18.
Mikrochim Acta ; 188(6): 199, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34041585

RESUMO

Since the COVID-19 disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) was declared a pandemic, it has spread rapidly, causing one of the most serious outbreaks in the last century. Reliable and rapid diagnostic tests for COVID-19 are crucial to control and manage the outbreak. Here, a label-free square wave voltammetry-based biosensing platform for the detection of SARS-CoV-2 in nasopharyngeal samples is reported. The sensor was constructed on screen-printed carbon electrodes coated with gold nanoparticles. The electrodes were functionalized using 11-mercaptoundecanoic acid (MUA) which was used for the immobilization of an antibody against SARS-CoV-2 nucleocapsid protein (N protein). The binding of the immunosensor with the N protein caused a change in the electrochemical signal. The detection was realised by measuring the change in reduction peak current of a redox couple using square wave voltammetry at 0.04 V versus Ag ref. electrode on the immunosensor upon binding with the N protein. The electrochemical immunosensor showed high sensitivity with a linear range from 1.0 pg.mL-1 to 100 ng.mL-1 and a limit of detection of 0.4 pg.mL-1 for the N protein in PBS buffer pH 7.4. Moreover, the immunosensor did not exhibit significant response with other viruses such as HCoV, MERS-CoV, Flu A and Flu B, indicating the high selectivity of the sensor for SARS-CoV-2. However, cross reactivity of the biosensor with SARS-CoV is indicated, which gives ability of the sensor to detect both SARS-CoV and SARS-CoV-2. The biosensor was successfully applied to detect the SARS-CoV-2 virus in clinical samples showing good correlation between the biosensor response and the RT-PCR cycle threshold values. We believe that the capability of miniaturization, low-cost and fast response of the proposed label-free electrochemical immunosensor will facilitate the point-of-care diagnosis of COVID 19 and help prevent further spread of infection.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/análise , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , SARS-CoV-2/química , Anticorpos Imobilizados/imunologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Teste para COVID-19/instrumentação , Carbono/química , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ácidos Graxos/química , Ouro/química , Humanos , Imunoensaio/instrumentação , Limite de Detecção , Nanopartículas Metálicas/química , Nasofaringe/virologia , Fosfoproteínas/análise , Fosfoproteínas/imunologia , Compostos de Sulfidrila/química
19.
Food Chem ; 358: 129898, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33933961

RESUMO

The sensitive detection of pesticides in complex environment is important but still challenging in presence of organic-rich water sample and food matrix. Herein, we reported a nitrile-mediated SERS immunosensor for sensitive and optical anti-interference determination of imidacloprid. Raman tag contained CN bond could provide a sharp characteristic peak in the Raman-silent spectral window (1800 ~ 2800 cm-1), which could resist the optical noises from the fingerprint region (<1800 cm-1). Aucore-Agshell bimetallic nanocuboid (AuNR@Ag) connected with antigen and Raman tag was used as Raman probe, while Fe3O4 magnetic nanoparticle functionalized with anti-imidacloprid antibody was applied as signal enhancer. Owing to the specific recognition ability between antigen and antibody, the competitive system with imidacloprid was formed. Under the optimal condition, the linear relationship was developed in the range of 10-400 nM. Finally, the SERS immunosensor was successfully applied to determine imidacloprid in real samples with recoveries from 96.8% to 100.5%.


Assuntos
Análise de Alimentos/métodos , Imunoensaio/métodos , Neonicotinoides/análise , Nitrocompostos/análise , Análise Espectral Raman/métodos , Contaminação de Alimentos/análise , Ouro/química , Imunoensaio/instrumentação , Nanopartículas Magnéticas de Óxido de Ferro/química , Neonicotinoides/imunologia , Nitrocompostos/imunologia , Praguicidas/análise , Praguicidas/imunologia , Sensibilidade e Especificidade , Análise Espectral Raman/instrumentação
20.
Int J Nanomedicine ; 16: 3407-3427, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34040371

RESUMO

Purpose: Plasmonic photothermal cancer therapy by gold nanorods (GNRs) emerges as a promising tool for cancer treatment. The goal of this study was to design cationic oligoethylene glycol (OEG) compounds varying in hydrophobicity and molecular electrostatic potential as ligand shells of GNRs. Three series of ligands with different length of OEG chain (ethylene glycol units = 3, 4, 5) and variants of quaternary ammonium salts (QAS) as terminal functional group were synthesized and compared to a prototypical quaternary ammonium ligand with alkyl chain - (16-mercaptohexadecyl)trimethylammonium bromide (MTAB). Methods: Step-by-step research approach starting with the preparation of compounds characterized by NMR and HRMS spectra, GNRs ligand exchange evaluation through characterization of cytotoxicity and GNRs cellular uptake was used. A method quantifying the reshaping of GNRs was applied to determine the effect of ligand structure on the heat transport from GNRs under fs-laser irradiation. Results: Fourteen out of 18 synthesized OEG compounds successfully stabilized GNRs in the water. The colloidal stability of prepared GNRs in the cell culture medium decreased with the number of OEG units. In contrast, the cellular uptake of OEG+GNRs by HeLa cells increased with the length of OEG chain while the structure of the QAS group showed a minor role. Compared to MTAB, more hydrophilic OEG compounds exhibited nearly two order of magnitude lower cytotoxicity in free state and provided efficient cellular uptake of GNRs close to the level of MTAB. Regarding photothermal properties, OEG compounds evoked the photothermal reshaping of GNRs at lower peak fluence (14.8 mJ/cm2) of femtosecond laser irradiation than the alkanethiol MTAB. Conclusion: OEG+GNRs appear to be optimal for clinical applications with systemic administration of NPs not-requiring irradiation at high laser intensity such as drug delivery and photothermal therapy inducing apoptosis.


Assuntos
Ouro/química , Ouro/metabolismo , Nanotubos/química , Polietilenoglicóis/química , Compostos de Amônio Quaternário/química , Temperatura , Transporte Biológico , Coloides , Estabilidade de Medicamentos , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...