RESUMO
In this study, an ultrasensitive detection platform for tobramycin (TOB) was developed, featuring a "sandwich" structure guided by AgNCs@PDANSs and Thi-AuNCs@ZnONSs. To address the issue of large background current peak signals in tagless sensors, Thi-AuNCs@ZnONSs composites were synthesized as signal tags. Zinc oxide nanosheets (ZnONSs) served as the loading agent, and AuNCs with the electroactive molecule Thi acted as carriers. Furthermore, AgNPs@PDANSs nanocomposites, possessing excellent electrical conductivity and large specific surface areas, were prepared as substrate materials for the modified electrodes. A "sandwich" structure strategy was also introduced to enhance the accuracy of the electrochemical aptasensor. This strategy, utilizing a dual sequence for target labeling and capture, yielded higher sensitivity and simplified the sensor construction compared to methods employing a single sequence. Under optimal conditions, the detection limit for TOB was established at 1.41 pM, with a detection range of 0.05-5000 nM. The aptasensor was effectively applied in the detection of TOB in tap and lake water, demonstrating outstanding reproducibility, selectivity, and stability. These results may serve as a reference for environmental TOB detection.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Eletroquímicas , Nanopartículas Metálicas , Prata , Tobramicina , Poluentes Químicos da Água , Tobramicina/análise , Tobramicina/química , Aptâmeros de Nucleotídeos/química , Técnicas Eletroquímicas/métodos , Prata/química , Nanopartículas Metálicas/química , Poluentes Químicos da Água/análise , Técnicas Biossensoriais/métodos , Limite de Detecção , Antibacterianos/análise , Antibacterianos/química , Reprodutibilidade dos Testes , Óxido de Zinco/química , Lagos/análise , Lagos/químicaRESUMO
Using a chemiluminescence reaction between luminol and H2O2 in basic solution, an ultrasensitive electrochemiluminescence (ECL) aptasensor was developed for the determination of tobramycin (TOB), as an aminoglycoside antibiotic. Ti3C2/Ni/Sm-LDH-based nanocomposite effectively catalyzes the oxidation of luminol and decomposition of H2O2, leading to the formation of different reactive oxygen species (ROSs), thus amplifying the ECL signal intensity of luminol, which can be used for the determination of TOB concentration. To evaluate the performance of the electrochemiluminescence aptasensor and synthesized nanocomposite, different methods such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) analyses were performed. The considerable specific area, large number of active sites, and enhanced electron transfer reaction on this nanocomposite led to the development of an ECL aptasensor with high sensitivity and electrocatalytic activity. After optimizing the preparation method and analysis conditions, the aptasensor revealed a wide linear response ranging from 1.0 pM to 1.0 µM with a detection limit of 18 pM, displaying outstanding accuracy, specificity, and response stability. The developed ECL sensor was found to be applicable to the determination of TOB in human serum samples and is anticipated to possess excellent clinical potentials for detecting other antibiotics, as well.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Eletroquímicas , Limite de Detecção , Medições Luminescentes , Nanocompostos , Tobramicina , Nanocompostos/química , Humanos , Técnicas Eletroquímicas/métodos , Aptâmeros de Nucleotídeos/química , Medições Luminescentes/métodos , Técnicas Biossensoriais/métodos , Tobramicina/sangue , Tobramicina/análise , Luminol/química , Antibacterianos/sangue , Antibacterianos/análise , Peróxido de Hidrogênio/química , Níquel/química , Titânio/químicaRESUMO
In this study, an ultrasensitive photoelectrochemical (PEC) aptasensor based on dual-sensitized heterojunction Ag2S/ZnS/NiS composites as a signal probe was proposed for the detection of tobramycin (TOB) by combining a cascaded quadratic signal amplification strategy. Specifically, compared to the limited visible light-harvesting capability of single sensitized composites, Ag2S/ZnS/NiS composites with p-n and n-n heterojunction could greatly improve the light energy utilization to tremendously strengthen the optical absorption in the entire visible-light region. Moreover, dual-sensitized heterojunction could effectively hinder the rapid recombination of photoelectrons and holes (carriers) to obtain a good photocurrent for improving the sensitivity of the aptasensor. Furthermore, a cascaded quadratic signal amplification strategy was applied to convert trace target TOB into plentiful gold nanoclusters (Au NCs) labelled double-stranded DNA for the construction of PEC aptasensor, with a broad linear detection range from 0.01 to 100 ng mL-1 and a low detection limit of 3.38 pg mL-1. Importantly, this study provided a versatile and sensitive PEC biosensing platform for TOB analysis, and demonstrated its successful application for TOB detection in milk samples. This protocol provides a novel dual-sensitized heterojunction composites to develop a highly efficient and harmfulless PEC aptasensor, which is expected to be used in food safety, environmental monitoring and other areas.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Eletroquímicas , Luz , Limite de Detecção , Leite , Compostos de Prata , Sulfetos , Tobramicina , Compostos de Zinco , Tobramicina/análise , Tobramicina/química , Técnicas Eletroquímicas/métodos , Aptâmeros de Nucleotídeos/química , Compostos de Prata/química , Compostos de Zinco/química , Sulfetos/química , Leite/química , Animais , Nanopartículas Metálicas/química , Antibacterianos/análise , Ouro/química , Contaminação de Alimentos/análiseRESUMO
Electrochemical aptamer-based sensors support the high-frequency, real-time monitoring of molecules-of-interest in vivo. Achieving this requires methods for correcting the sensor drift seen during in vivo placements. While this correction ensures EAB sensor measurements remain accurate, as drift progresses it reduces the signal-to-noise ratio and precision. Here, we show that enzymatic cleavage of the sensor's target-recognizing DNA aptamer is a major source of this signal loss. To demonstrate this, we deployed a tobramycin-detecting EAB sensor analog fabricated with the DNase-resistant "xenonucleic acid" 2'O-methyl-RNA in a live rat. In contrast to the sensor employing the equivalent DNA aptamer, the 2'O-methyl-RNA aptamer sensor lost very little signal and had improved signal-to-noise. We further characterized the EAB sensor drift using unstructured DNA or 2'O-methyl-RNA oligonucleotides. While the two devices drift similarly in vitro in whole blood, the in vivo drift of the 2'O-methyl-RNA-employing device is less compared to the DNA-employing device. Studies of the electron transfer kinetics suggested that the greater drift of the latter sensor arises due to enzymatic DNA degradation. These findings, coupled with advances in the selection of aptamers employing XNA, suggest a means of improving EAB sensor stability when they are used to perform molecular monitoring in the living body.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Eletroquímicas , Aptâmeros de Nucleotídeos/química , Técnicas Eletroquímicas/métodos , Técnicas Biossensoriais/métodos , Animais , Ratos , Tobramicina/análiseRESUMO
The diversity of microbial species in the gut has a strong influence on health and development of the host. Further, there are indications that the variation in expression of gut bacterial metabolic enzymes is less diverse than the taxonomic profile, underlying the importance of microbiome functionality, particularly from a toxicological perspective. To address these relationships, the gut bacterial composition of Wistar rats was altered by a 28 day oral treatment with the antibiotics tobramycin or colistin sulfate. On the basis of 16S marker gene sequencing data, tobramycin was found to cause a strong reduction in the diversity and relative abundance of the microbiome, whereas colistin sulfate had only a marginal impact. Associated plasma and fecal metabolomes were characterized by targeted mass spectrometry-based profiling. The fecal metabolome of tobramycin-treated animals had a high number of significant alterations in metabolite levels compared to controls, particularly in amino acids, lipids, bile acids (BAs), carbohydrates, and energy metabolites. The accumulation of primary BAs and significant reduction of secondary BAs in the feces indicated that the microbial alterations induced by tobramycin inhibit bacterial deconjugation reactions. The plasma metabolome showed less, but still many alterations in the same metabolite groups, including reductions in indole derivatives and hippuric acid, and furthermore, despite marginal effects of colistin sulfate treatment, there were nonetheless systemic alterations also in BAs. Aside from these treatment-based differences, we also uncovered interindividual differences particularly centering on the loss of Verrucomicrobiaceae in the microbiome, but with no apparent associated metabolite alterations. Finally, by comparing the data set from this study with metabolome alterations in the MetaMapTox database, key metabolite alterations were identified as plasma biomarkers indicative of altered gut microbiomes resulting from a wide activity spectrum of antibiotics.
Assuntos
Antibacterianos , Microbioma Gastrointestinal , Ratos , Animais , Antibacterianos/farmacologia , Colistina/farmacologia , Colistina/análise , Tobramicina/farmacologia , Tobramicina/análise , Ácidos e Sais Biliares/análise , Ratos Wistar , Metaboloma , Fezes/química , RNA Ribossômico 16S/genéticaRESUMO
Despite advances in the development of photoelectrochemical (PEC) sensor, modulating the PEC response of assembled heterostructure interface is still a great challenge. Here, an ultrasensitive PEC aptasensor for tobramycin (TOB) assay was conducted based on one-dimensional/two-dimensional CdS nanorods@ZnIn2S4 nanosheets (1D/2D CdS NRs@ZnIn2S4 NSs) heterojunctions by tailoring enzymatic loading capacity. Firstly, alkaline phosphatase modified TOB aptamer (ALP-Apt) was linked via specific base complementary pairing, and insoluble precipitations were then produced through the ALP-triggered catalytic reaction with the aid of Ag+, which prevented the charge transfer and resulted in the decrement of photocurrent. In the presence of TOB, partial ALP-Apt detached from the electrode surface due to the strong affinity between TOB and its aptamer, leading to a reduction in the amount of ALP and insoluble precipitate, in turn the PEC response partially recovered. The photocurrents exhibited a wider linear range towards the TOB concentration of 1.0-5.0 × 104 pg mL-1, with a low detection limit of 0.96 pg mL-1. The constructed PEC aptasensor gained satisfactory results for TOB assay in milk samples as well, which also offered significant promise for other pollutants in environmental analysis.
Assuntos
Técnicas Biossensoriais , Nanotubos , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Limite de Detecção , Nanotubos/química , Tobramicina/análise , NanotecnologiaRESUMO
In this study, a novel colorimetric bioassay method was developed for the sensitive determination of tobramycin (TOB). To detect TOB, silver nanoparticles (AgNPs) were decorated with TOB-specific aptamers (apt), and positively charged poly diallyl dimethyl ammonium chloride (PDDA) was used. As long as tobramycin is not present in the assay system, PDDA can coalesce with the aptamer, and AgNPs would remain stable (λmax = 400 nm) in the dispersed system against PDDA-induced aggregation. When TOB is added, aptamer can bind to the compound, which leads to release of PDDA and subsequent aggregation of AgNPs (λmax = 540 nm). This remarkable change, as a colorimetric analytics signal, can be used for quantitative analysis of TOB. TOB can be detected by this highly sensitive colorimetric aptasensor with a limit of detection (LOD) of 70 pM. Furthermore, TOB can be detected with the naked eye at concentrations above 1 nM.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Nanopartículas Metálicas , Animais , Técnicas Biossensoriais/métodos , Colorimetria/métodos , Ouro/análise , Limite de Detecção , Leite/química , Poli A , Polímeros , Prata/análise , Tobramicina/análiseRESUMO
The rational design and preparation of available fullerene@metal-organic matrix hybrid materials are of profound significance in electrochemical biosensing applications due to their unique photoelectric properties. In this work, C60@UiO-66-NH2 nanocomposites serve as greatly promising materials to modify electrodes and fix aptamers, resulting in a remarkable electrochemical aptasensor for impedimetric sensing of tobramycin (TOB). Nanoscale composites have preferable electroactivity and small particle size with more exposed functional sites, such as Zr(IV) and -NH2, to immobilize aptamers for enhanced detection performance. As we know, most of the electrochemical impedance aptasensors require a long time to complete the detection process, but this prepared biosensor shows the rapid quantitative identification of target TOB within 4 min. This work expands the synthesis of functional fullerene@metal-organic matrix hybrid materials in electrochemical biosensing applications.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Fulerenos/química , Estruturas Metalorgânicas/química , Nanocompostos/química , Tobramicina/análise , Aminas/química , Espectroscopia Dielétrica , Humanos , Tobramicina/sangue , Tobramicina/urina , Zircônio/químicaRESUMO
A facile and versatile competitive electrochemical aptasensor for tobramycin (TOB) detection is described using electrochemical-deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework (AuNPs/P-MOF) as signal-amplification platform and a DNA probe labeled with methylene blue (MB) at the 3'-end (MB-Probe) as a signal producer. First, F-Probe (short complementary DNA strands of both the aptamer and the MB-Probe label with a sulfhydryl group at the 5'-end) was immobilized on the AuNPs/P-MOF modified electrode as detection probes, which competed with TOB in binding to the aptamer. TOB-aptamer binding resulted in F-Probe remaining unhybridized on the electrode surface, so that a significant current response was generated by hybridizing with MB-Probe instead. The developed strategy showed favorable repeatability, with a relative standard deviation (RSD) of 4.3% computed over five independent assays, and high stability, with only 6.8% degradation after 15 days of storage. Under optimal conditions, the proposed aptamer strategy exhibited a linear detection range from 100 pM to 500 nM with a limit of detection (LOD) of 56 pM (S/N = 3). The electrochemical aptasensor demonstrated remarkable selectivity, and its feasibility for accurate and quantitative detection of TOB in milk samples was confirmed (RSD < 4.5%). Due to its simple design, easy operation, and high sensitivity and selectivity, the proposed method could expect to detect other antibiotics by replacing the aptamers. In summary, this study provides a simple and effective new strategy for electrochemical aptasening based on MOF-based sensing interface. Scheme illustration of label-free competitive electrochemical aptamer-based detection of tobramycin based on electrochemically deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework as signal-amplification platform.
Assuntos
Antibacterianos/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Nanopartículas Metálicas/química , Estruturas Metalorgânicas/química , Tobramicina/análise , Animais , Antibacterianos/química , DNA/química , Técnicas Eletroquímicas/métodos , Contaminação de Alimentos/análise , Ouro/química , Ácidos Nucleicos Imobilizados/química , Ferro/química , Limite de Detecção , Azul de Metileno/química , Leite/química , Oxirredução , Polietilenoimina/química , Reprodutibilidade dos Testes , Tobramicina/químicaRESUMO
The complexity of Tobradex® ointment formulation (dexamethasone 0.1 wt% and tobramycin 0.3 wt%) and the high cost of pharmacokinetic (PK) studies in human aqueous humor may prevent generic drug companies from moving forward with a Tobradex®-equivalent product development. The in vitro drug release test would be an alternative approach for differentiating the generic formulations containing both dexamethasone (DEX) and tobramycin (TOB), and the results should be correlated with the in vivo ocular PK studies for further evaluation. To facilitate the in vivo ocular PK studies, a sensitive, rapid and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that can simultaneously quantify both DEX and TOB in rabbit ocular matrices including tear, aqueous humor and cornea was established and validated. The lower limit of quantification (LLOQ) was 1.5 ng/ml for DEX and 3 ng/ml for TOB with good precision and accuracy. Both intra- and inter-batch precisions were within ±15%, and the accuracy for all QCs was within the range of 85-115%. This new method was successfully applied for a pilot pharmacokinetic analysis of DEX and TOB in rabbit tears after topical administration of Tobradex® ointment.
Assuntos
Humor Aquoso/química , Cromatografia Líquida/métodos , Dexametasona/análise , Espectrometria de Massas em Tandem/métodos , Tobramicina/análise , Animais , Antibacterianos/análise , Antibacterianos/farmacocinética , Córnea/química , Dexametasona/farmacocinética , Feminino , Modelos Lineares , Masculino , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Lágrimas/química , Tobramicina/farmacocinéticaRESUMO
Abuse of tobramycin (TOB) causes a series of diseases. Therefore, the development of rapid and sensitive method for analyzing TOB in food products is necessary. In this work, aptamer modified SnO2/Bi2S3-based photoelectrochemical (PEC) sensor was developed for the determination of TOB in milk. Under optimal condition, a wide linear response for TOB from 5 to 50 nmol/L with a limit of detection of 4.28 nmol/L is reached. The possible detection mechanism is that TOB molecules are specifically captured by aptamer, increasing electron transfer resistance and declining the photocurrent. Thanks to the favorably matched energy level of SnO2, and Bi2S3, the PEC aptasensor exhibits high sensitivity, and with the aid of oxalate, the sensitivity of the sensor is further improved. Importantly, the stability of the PEC aptasensor is also satisfactory due to the calcination of SnO2/Bi2S3 at 450 °C.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Bismuto/química , Limite de Detecção , Leite/química , Sulfetos/química , Compostos de Estanho/química , Tobramicina/análise , Animais , Técnicas Eletroquímicas/métodos , Eletroquímica , Análise de Alimentos , Processos Fotoquímicos , Tobramicina/metabolismoRESUMO
Electrochemical aptamer-based (E-AB) biosensors suffer from sensor-to-sensor signal variations due to the variation of the total number and the heterogeneity of probes immobilized on the electrode surface, with the former attracting more attention. As such, a calibration process to correct for such variations is required for this type of sensor, causing inconvenience and inaccessibility in harsh sensing environments such as blood samples, which has dramatically limited the widespread clinical use of biosensors. In response, here, we have adopted E-AB sensors to achieve calibration-free measurements of small biological/drug molecules. Specifically, we employ one probe-attached redox reporter and a second intercalated redox reporter to generate two signals, achieving good sensor-to-sensor reproducibility and thus obviating the need for calibration. We first demonstrated the capability of E-AB sensors for the accurate measurement of kanamycin, tobramycin, and adenosine triphosphate (ATP) in phosphate-buffered saline (PBS) buffer, achieving concentration ranges of approximately 4.7 × 103-, 2.0 × 103-, and 12.7-fold, respectively. Then, we applied this calibration-free approach to the measurement of these three target molecules directly in undiluted serum, achieving a concentration precision of a few micromolars.
Assuntos
Trifosfato de Adenosina/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Técnicas Eletroquímicas , Canamicina/análise , Tobramicina/análise , Calibragem , OxirreduçãoRESUMO
Background The aim of our work was to develop and validate a hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) methods for the quantification of tobramycin (TMC) and lincomycin (LMC)in plasma, microdialysis fluid and urine. Methods Protein precipitation was used to extract TMC and LMC from plasma, while microdialysis fluid and urine sample were diluted prior to instrumental analysis. Mobile phase A consisted of 2 mM ammonium acetate in 10% acetonitrile with 0.2% formic acid (v/v) and mobile phase B consisted of 2 mM ammonium acetate in 90% acetonitrile with 0.2% formic acid (v/v). Gradient separation (80%-10% of mobile phase B) for TMC was done using a SeQuant zic-HILIC analytical guard column. While separation of LMC was performed using gradient elution (100%-40% of mobile phase B) on a SeQuant zic-HILIC analytical column equipped with a SeQuant zic-HILIC guard column. Vancomycin (VCM) was used as an internal standard. A quadratic calibration was obtained over the concentration range for plasma of 0.1-20 mg/L for TMC and 0.05-20 mg/L for LMC, for microdialysis fluid of 0.1-20 mg/L for both TMC and LMC, and 1-100 mg/L for urine for both TMC and LMC. Results For TMS and LMC, validation testing for matrix effects, precision and accuracy, specificity and stability were all within acceptance criteria of ±15%. Conclusions The methods described here meet validation acceptance criteria and were suitable for application in a pilot pharmacokinetic research study performed in a sheep model.
Assuntos
Lincomicina/análise , Espectrometria de Massas em Tandem/métodos , Tobramicina/análise , Calibragem , Cromatografia Líquida de Alta Pressão/normas , Meia-Vida , Humanos , Limite de Detecção , Lincomicina/sangue , Lincomicina/normas , Lincomicina/urina , Microdiálise , Projetos Piloto , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/normas , Tobramicina/sangue , Tobramicina/normas , Tobramicina/urinaRESUMO
Tobramycin (TOB) is an aminoglycoside antibiotic. The residue of TOB in animal-derived foods and environment will be harmful to human health, and therefore the specific detection of TOB residue in food and water is of great importance. Herein, through magnetic beads-based SELEX, overall 37 ssDNA aptamers specific for TOB were screened after ten rounds of selection. The affinity and specificity of these aptamers were evaluated, among which No. 32 aptamer (Ap 32) exhibits excellent performance. Then a post-SELEX optimization of Ap 32 was carried out based on rational design, through which a truncated aptamer with the length of 34 nucleotides (Ap 32-2) was identified as the best aptamer for TOB. Finally, the application of the screened aptamer was explored. A colorimetric assay of TOB was established based on the aptamer-modified gold nanoparticles (AuNPs). In the range from 100 to 1400 nM, the absorbance of AuNPs solution at 520 nm was linearly decreased with the increased concentration of TOB. The detection limit was estimated to be 37.9 nM. The assay was applied to detect TOB residue in honey samples.
Assuntos
Aptâmeros de Nucleotídeos , Análise de Alimentos , Contaminação de Alimentos/análise , Mel/análise , Técnica de Seleção de Aptâmeros , Tobramicina , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/química , Colorimetria , Ouro/química , Nanopartículas Metálicas/química , Tobramicina/análise , Tobramicina/químicaRESUMO
A digital fluorescence detector (DFD), a handheld fluorescence detection device, can convert the fluorescence signal of samples into the corresponding fluorescer concentration. Herein, by adopting a DFD as the readout, a novel intelligent platform was developed based on a ratiometric paper-based device (RPD) for multiple aminoglycoside detection. There are five layers and four parallel channels contained in the designed RPD, functioning as reagent storage, fluidic path control and signal processing, respectively. The rationale of this design lies in the fact that aptamer/graphitic carbon nitride nanosheet (Apt/g-C3N4 NS) modified layers can catalyze o-phenylenediamine to fluorescent 2,3-diaminophenazine (DAP) in the presence of H2O2. When Apt was removed from nanosheets via the Apt-target reaction, the peroxidase-like activity would be decreased, thus decreasing the production of DAP. All the changes of the fluorescence DAP signal can be read out using a portable DFD. Based on the DFD signal change related to the concentration of the target, a quantitative reaction platform was established. Furthermore, the sample flow and Apt-target reaction time can be reasonably regulated using the H2O2-cleavable hydrophobic compound modified layer placed between the target recognition region and detection region. Then, the practicality of this platform was verified through realizing sensitive analysis of streptomycin, tobramycin, and kanamycin simultaneously. Overall, with merits including portability and ease of operation, the platform shows great potential in on-site simultaneous detection of multiple targets, especially in resource-limited settings.
Assuntos
Antibacterianos/análise , Canamicina/análise , Papel , Espectrometria de Fluorescência/métodos , Estreptomicina/análise , Tobramicina/análise , Animais , Antibacterianos/química , Aptâmeros de Nucleotídeos/química , Fluorescência , Corantes Fluorescentes/química , Contaminação de Alimentos/análise , Grafite/química , Peróxido de Hidrogênio/química , Canamicina/química , Limite de Detecção , Leite/química , Nanoestruturas/química , Compostos de Nitrogênio/química , Fenazinas/química , Fenilenodiaminas/química , Reprodutibilidade dos Testes , Rios/química , Espectrometria de Fluorescência/instrumentação , Estreptomicina/química , Tobramicina/química , Poluentes Químicos da Água/análiseRESUMO
The influence of the biofilm matrix on molecular diffusion is commonly hypothesized to be responsible for emergent characteristics of biofilms such as nutrient trapping, signal accumulation and antibiotic tolerance. Hence quantifying the molecular diffusion coefficient is important to determine whether there is an influence of biofilm microenvironment on the mobility of molecules. Here, we use single plane illumination microscopy fluorescence correlation spectroscopy (SPIM-FCS) to obtain 3D diffusion coefficient maps with micrometre spatial and millisecond temporal resolution of entire Pseudomonas aeruginosa microcolonies. We probed how molecular properties such as size and charge as well as biofilm properties such as microcolony size and depth influence diffusion of fluorescently labelled dextrans inside biofilms. The 2 MDa dextran showed uneven penetration and a reduction in diffusion coefficient suggesting that the biofilm acts as a molecular sieve. Its diffusion coefficient was negatively correlated with the size of the microcolony. Positively charged dextran molecules and positively charged antibiotic tobramycin preferentially partitioned into the biofilm and remained mobile inside the microcolony, albeit with a reduced diffusion coefficient. Lastly, we measured changes of diffusion upon induction of dispersal and detected an increase in diffusion coefficient inside the biofilm before any loss of biomass. Thus, the change in diffusion is a proxy to detect early stages of dispersal. Our work shows that 3D diffusion maps are very sensitive to physiological changes in biofilms, viz. dispersal. However, this study also shows that diffusion, as mediated by the biofilm matrix, does not account for the high level of antibiotic tolerance associated with biofilms.
Assuntos
Antibacterianos/análise , Biofilmes/crescimento & desenvolvimento , Dextranos/análise , Difusão , Matriz Extracelular de Substâncias Poliméricas/química , Pseudomonas aeruginosa/crescimento & desenvolvimento , Tobramicina/análise , Imageamento Tridimensional , Análise Espaço-Temporal , Espectrometria de FluorescênciaRESUMO
In this paper, we adapt the electrochemical, aptamer-based (E-AB) sensor platform to develop colorimetric aptamer-based sensors using a closed-bipolar electrode (C-BPE) system. The C-BPE E-AB sensors provide quantitative detection of target molecules based on the rate of color change of an electrochromic Prussian blue (PB) thin-film indicator electrode. The C-BPE cathode, or sensing electrode, is modified with a redox-labeled aptamer that binds to a specific target. More specifically, we employed sequences specific for adenosine triphosphate (ATP) and tobramycin as test-bed targets because these sequences are well vetted. The C-BPE anode, or indicator electrode, was coated with an electrochromic thin film comprising Prussian white (PW) that, when reduced to PB, is accompanied by a corresponding color change used for analytical detection. The rate of color change from PW to PB is facilitated by a potassium ferricyanide-catalyzed oxidation of leucomethylene blue (LB) to methylene blue (MB), the redox label conjugated to the aptamer on the sensing electrode. We demonstrate that the rate of color change is quantitatively related to the concentration of target analyte, which provides a means for naked eye determination. When combined with smartphone-based colorimetric detection, these C-BPE E-AB sensors present a user-friendly alternative to traditional E-AB sensors that rely on voltammetric analysis and a potentiostat, opening up the possibility of point-of-use applications.
Assuntos
Aptâmeros de Nucleotídeos/química , Colorimetria/métodos , Técnicas Eletroquímicas/métodos , Trifosfato de Adenosina/análise , Técnicas Biossensoriais , Eletrodos , Oxirredução , Smartphone , Tobramicina/análiseRESUMO
TOB aptamer can be adsorbed on the AuNPs surface to form AuNPs-aptamer complexation to prevent AuNPs aggregation in high salt solution. When TOB was added to the AuNPs solution, the aptamer would bind with TOB and depart from the AuNPs surface. The amount of the AuNPs-aptamer complexation depends on the TOB concentration. Different concentration of AuNPs-aptamer can catalyze the reduction reaction of CuSO4 to produce different size Cu2O particle. The resonance scattering peak intensities are correlated with the Cu2O size. Large size Cu2O particle as a resonance scattering spectroscopy probe can remarkable improve the TOB detection sensitivity. We have succeeded to detect the trace TOB in aqueous solutions. The linear range and limit of detection were 0.50-17â¯nM and 0.19â¯nM, respectively. This simple and inexpensive method exhibited high sensitivity and selectivity, which was successfully used to detect TOB in milk. The results indicated the accuracy and precision were satisfied.
Assuntos
Aptâmeros de Nucleotídeos/química , Análise de Alimentos/métodos , Leite/química , Análise Espectral/métodos , Tobramicina/análise , Adsorção , Animais , Técnicas Biossensoriais/métodos , Catálise , Sulfato de Cobre/química , Análise de Alimentos/instrumentação , Contaminação de Alimentos/análise , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Pasteurização , Sensibilidade e Especificidade , Análise Espectral/instrumentaçãoRESUMO
The design and construction of high-performance photoelectrode with efficient visible-light absorption and fast charge transport rate are crucial to developing photoelectrochemical (PEC) sensor. Herein, a well-aligned amorphous MoSx (a-MoSx)@ZnO core-shell nanorod arrays was uniformly grown on indium tin oxide (ITO) via a facile all-electrochemical strategy. Simultaneous boosted PEC performance and stability in the visible region were obtained. Those could be attributed to the collaboration effect of fast electron transfer path within the oriented ZnO nanorod and extended visible-light absorption upon combining a-MoSx, as well as favorable energy-band alignment between the two PEC materials. By employing tobramycin-binding aptamer as recognition element, a self-powered PEC aptasensor for tobramycin was successfully fabricated for the first time. The sensor exhibited a rapid response in a wide linear range of 0.010-50â¯ng/mL with good stability and reproducibility. The detection limit was as low as 5.7â¯pg/mL. Ultrasensitive and high-affinitive determination of tobramycin in serum samples was realized at 0â¯V (vs.SCE) with desired accuracy and satisfactory recovery. This work reveals the promising application of all-electrodeposited a-MoSx@ZnO NR arrays-based photoanode for self-powered PEC bioassay in the tobramycin-related disease diagnosis.
Assuntos
Técnicas Biossensoriais/métodos , Dissulfetos/química , Molibdênio/química , Nanotubos/química , Tobramicina/análise , Óxido de Zinco/química , Luz , Limite de DetecçãoRESUMO
An aptamer with the length of only 15 nucleotides specific for tobramycin was obtained through rationally designed truncation from a previously reported long sequence. The structural and binding properties of the aptamer were characterized. The dissociation constant (Kd) was determined to be 42.12â¯nM, indicating high affinity of the aptamer for tobramycin. Then an electrochemical sensor based on this aptamer was developed, which employed an enzymes-assisted dual signal amplification cycle through target recycling and strand-displacement DNA polymerization. A hairpin probe containing the aptamer sequence was designed and used to start the production cycle of a short ssDNA fragment in the presence of tobramycin, with the help of phi29 DNA polymerase and nicking endonuclease Nt.AlwI. The ssDNA fragment was captured by a signal transduction probe modified on gold electrode to form a triple-helix structure. With the help of [Ru(NH3)6]3+, a significant electrochemical signal was observed in differential pulse voltammetry (DPV). Under the optimal conditions, the current in DPV is linearly related with the concentration of tobramycin in the range of 10-200â¯nM, and the detection limit is 5.13â¯nM. The electrochemical sensor showed high specificity for tobramycin when it was challenged by other antibiotics. In addition, the constructed sensor was used to detect tobramycin in milk and water samples, and showed satisfactory performance. Therefore, the screened aptamer as well as the developed sensor has great application prospects in the fields of food safety control, medical test and environment monitoring.