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1.
Chem Commun (Camb) ; 56(13): 1976-1979, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-31960850

RESUMO

We herein used Ag2Se quantum dots (QDs) as a target-modulated sensitizer for upconversion nanoparticles (UCNPs) and the target thrombin as the sensitizing switch to construct a biosensor, circumventing the limited luminescence resonance energy transfer (LRET) efficiency of UCNPs, with enhanced signal-to-background ratio (SBR) and assay sensitivity.


Assuntos
Técnicas Biossensoriais/métodos , Raios Infravermelhos , Pontos Quânticos/química , Trombina/análise , Transferência Ressonante de Energia de Fluorescência , Humanos , Limite de Detecção , Razão Sinal-Ruído
2.
Anal Bioanal Chem ; 412(4): 915-922, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31900531

RESUMO

A tetrahedral DNA probe can effectively overcome the steric effects of a single-stranded probe to obtain well-controlled density and minimize nonspecific adsorption. Herein, a highly sensitive electrochemical biosensor is fabricated for determination of protein using a tetrahedral DNA probe and rolling circle amplification (RCA). N- and P-co-doped graphene (NP-rGO) is prepared, and AuNPs are then electrodeposited on it for DNA probe immobilization. Benefitting from the synergistic effects of the excellent electrical conductivity of NP-rGO, the stability of the tetrahedral DNA probe and the signal amplification of RCA, the biosensor achieves a low limit of 3.53 × 10-14 M for thrombin and a wide linear range from 1 × 10-13 to 1 × 10-7 M. This study provides a sensitive and effective method for the detection of protein in peripheral biofluids, and paves the way for future clinical diagnostics and treatment of disease. Graphical abstract.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Grafite/química , Trombina/análise , Sondas de DNA/química , Técnicas Eletroquímicas/métodos , Ouro/química , Humanos , Ácidos Nucleicos Imobilizados/química , Limite de Detecção , Nanopartículas Metálicas/química , Técnicas de Amplificação de Ácido Nucleico/métodos
3.
Talanta ; 207: 120300, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31594586

RESUMO

A "signal-on" chemiluminescence biosensor was established for detecting thrombin. The thrombin aptamer1-functionalized magnetic sodium alginate (Malg-Apt1) hydrogel was synthesized by physical interaction between sodium alginate and Ca2+, and it was used in the biosensor for separating and enriching thrombin. Ethylenediamine tetraacetic acid (EDTA) was used to chelate with Ca2+ to dissolve the hydrogel and release thrombin. A metalloporphyrinic metal-organic framework nanosheet, named as Cu-TCPP(Co) MOFs, was prepared as signal amplification strategy. Cu-TCPP(Co) MOFs/Au-ssDNA (ssDNA: single-strand DNA) was synthesized for controllable further amplification of chemiluminescent signal. The thrombin aptamer2-functionalized magnetic carbon nanotubes (MCNTs-Apt2) were used as a matrix, and Cu-TCPP(Co) MOFs/Au-ssDNA was adsorbed on the MCNTs by the complementary pairing of the partial bases between ssDNA and Apt2. Compared with ssDNA, Apt2 has a stronger interaction with thrombin. Therefore, thrombin can trigger the release of Cu-TCPP(Co) MOFs/Au-ssDNA to achieve signal amplification. Under the optimal conditions, the biosensor could detect thrombin as low as 2.178 × 10-13 mol/L with the range from 8.934 × 10-13 to 5.956 × 10-10 mol/L and exhibited excellent selectively. Moreover, the "signal-on" chemiluminescence biosensor showed potential application for the detection of thrombin in body fluids.


Assuntos
Alginatos/química , Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/química , Hidrogéis/química , Estruturas Metalorgânicas/química , Nanotubos de Carbono/química , Trombina/análise , Adsorção , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , DNA de Cadeia Simples/genética , Medições Luminescentes , Imãs/química , Modelos Moleculares , Conformação Molecular , Porfirinas/química , Trombina/metabolismo
4.
Res Vet Sci ; 127: 113-121, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31693942

RESUMO

The aim of this study was to establish a thrombin generation assay (calibrated automated thrombogram, CAT) in cats by determining the precision (repeatability), reference values, and the sensitivity to anticoagulant treatment with the factor Xa inhibitor apixaban. The CAT method was performed on citrated plasma with different commercial tissue factor (TF) reagents (PPP Reagent 1 pM [LOW], PPP Reagent 5 pM, PPP Reagent 20 pM [HIGH]) according to the manufacturers` test instruction. Measurements in triplicate were performed in platelet poor plasma (PPP) of 58 healthy cats and in 6 cats at different times following the oral administration of 2.5 mg apixaban. The median CVs in healthy cats usually were < 10% with the exception of thrombin peak height measured using PPP Reagent 1 pM (14.6%). Reference values of all parameters showed marked inter-individual variability and depended largely on the TF concentration of the used activating reagent. Thrombin generation was significantly influenced by apixaban and reacted more sensitively than other tests of haemostasis including the prothrombin time, aPTT, and rotational elastometry. In conclusion, thrombin generation measured by the CAT method using commercially available reagents seems suitable for the examination of feline PPP and may be a valuable method to establish effective anticoagulant therapies for the feline patient and monitoring of such therapies in cats.


Assuntos
Testes de Coagulação Sanguínea/veterinária , Inibidores do Fator Xa/farmacologia , Pirazóis/farmacologia , Piridonas/farmacologia , Trombina/análise , Tromboplastina/análise , Animais , Testes de Coagulação Sanguínea/métodos , Gatos , Valores de Referência
5.
Chem Commun (Camb) ; 55(83): 12479-12482, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31566643

RESUMO

The electrochemiluminescence (ECL) from europium hydroxide nanorods (EHNs) was reported for the first time using K2S2O8 as an efficient cathodic co-reactant, and an ultrasensitive ECL aptasensor based on the system for the detection of thrombin has been formulated successfully.


Assuntos
Técnicas Eletroquímicas , Európio/química , Hidróxidos/química , Medições Luminescentes , Nanotubos/química , Trombina/análise , Humanos , Tamanho da Partícula , Propriedades de Superfície , Trombina/metabolismo
6.
Analyst ; 144(20): 6048-6054, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31524217

RESUMO

This proof-of-principle study demonstrates the feasibility of a leaky waveguide (LW) aptasensor, where aptamers were immobilised in a mesoporous chitosan waveguiding film for the detection of thrombin. This work has demonstrated that aptamers immobilised in hydrogels retain their affinity and selectivity towards their target and thus can be used as bioreceptors. The use of antibodies as bioreceptors for sensing thrombin is not viable because it is a serine protease, which will cleave the antibodies. Currently used assays based on clotting time and chromogenic/fluorogenic substrates have limited potential for thrombin measurement in whole blood. Using the initial binding rate over the first 5 min, the limit of detection of our LW aptasensor for thrombin was ∼22 nM. The sensor was tested with spiked serum samples, giving a reading of 46.1 ± 4.6 nM for a sample containing 50 nM thrombin. Our proposed sensor combines the robustness and low cost of aptamers as molecular recognition elements with the simple fabrication process of the chitosan-based leaky waveguide, making LW aptasensors highly attractive for applications in point-of-care diagnostics and healthcare monitoring.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Quitosana/química , Técnicas Eletroquímicas/métodos , Trombina/análise , Estudos de Viabilidade , Humanos , Limite de Detecção
7.
Surgery ; 166(6): 1122-1127, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31522748

RESUMO

BACKGROUND: Plasma thrombin generation has been used to characterize trauma-induced coagulopathy, but description of whole blood thrombin generation is lacking. This study aimed to evaluate plasma and whole blood thrombin generation in healthy volunteers and trauma patients. We hypothesized that (1) plasma and whole blood thrombin generation are distinct, (2) whole blood thrombin generation is more pronounced in trauma patients than in healthy volunteers, and (3) thrombin generation correlates with clinical coagulation assays. METHODS: Blood was collected from healthy volunteers and trauma patients at a single, level-1 trauma center. Whole blood thrombin generation was assessed with a prototype point-of-care whole blood thrombin generation device, and plasma thrombin generation was measured with a calibrated automated thrombogram analogue. Plasma and whole blood thrombin generation were compared and correlated with international normalized ratio and thrombelastography. RESULTS: Overall, 10 healthy volunteers (average age 30, 50% men) were included and 58 trauma patients (average age 34, 76% men, 55% blunt mechanism, and with a median new injury severity score of 17) were included. Plasma and whole blood thrombin generation differed with more robust thrombin generation in plasma. Trauma patients had a significantly increased whole blood thrombin generation compared with healthy volunteers]. Plasma thrombin generation correlated with international normalized ratio, whereas whole blood thrombin generation did not correlate with thrombelastography. CONCLUSION: Plasma and whole blood thrombin generation are distinct, highlighting the need to perform standardized assays to better understand their correlation and to assess how whole blood thrombin generation confers differential outcomes in trauma.


Assuntos
Transtornos da Coagulação Sanguínea/diagnóstico , Plasma/metabolismo , Trombina/metabolismo , Ferimentos e Lesões/complicações , Adulto , Transtornos da Coagulação Sanguínea/sangue , Transtornos da Coagulação Sanguínea/etiologia , Feminino , Voluntários Saudáveis , Humanos , Escala de Gravidade do Ferimento , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Tromboelastografia/métodos , Trombina/análise , Ferimentos e Lesões/sangue , Ferimentos e Lesões/diagnóstico
8.
Anal Chim Acta ; 1079: 180-191, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31387709

RESUMO

In this research, a novel Resonance Rayleigh Scattering (RRS) aptasensor was developed for thrombin monitoring using in-situ synthesized and embedded Au nanoparticles (AuNPs) into poly vinyl alcohol -borax hydrogel (PBH). Thiolated-thrombin binding aptamer (thiolated-TBA) was attached to the surface of AuNPs embedded into PBH to design the PBH-aptasensor for thrombin detection (thiolated-TBA@AuNPs-PBH). To verify the characteristic and morphology of PBH nanocomposite, energy dispersive X-ray analysis, TEM, average particle size analizer and UV-Vis spectra were performed. The difference in RRS intensities in the absence and presence of thrombin was calculated and selected as the monitoring signal. Effect of different parameters on the RRS signal was investigated at excitation wavelength of 500 nm. Under the approved conditions, the linear detection range was validated over the concentration of 0.70 pM- 0.02 µM. The limit of detection based on 3Sb was 0.10 pM. The relative standard deviation for 5.6 pM and 3.6 nM were 4.0 and 2.7% (n = 10), respectively. The proposed aptasensor was successfully applied as an experimental model for thrombin detection in serum samples of healthy volunteers with acceptable results.


Assuntos
Aptâmeros de Nucleotídeos/química , Hidrogéis/química , Trombina/análise , Técnicas Biossensoriais/métodos , Boratos/química , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Álcool de Polivinil/química , Espalhamento de Radiação , Análise Espectral/métodos , Trombina/química
9.
Anal Chim Acta ; 1081: 59-64, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446964

RESUMO

Herein, a signal-on electrochemical aptasensor for highly sensitive detection of thrombin (TB) was constructed based on the DNAzyme-driven DNA walker strategy. We developed a new dual functional hairpin DNA (HP) containing a substrate sequence of the Mg2+-dependent DNAzyme (in the loop region) and the G-quadruplex forming segment (in the stem region). The DNA walker (TBA2-DWs), containing a TB aptamer and an enzymatic sequence, was introduced onto gold electrode (GE) by aptamers-target specific recognition, and thus initiated the enzymatic sequences to hybridize with the substrate sequence. Then, the DNA walker could repeatedly bind and cleave HP in the assistance of Mg2+, unlocking many active G-quadruplex forming sequences. Finally, hemin can further bind the G-quadruplex to form G-quadruplex/hemin complexes and generate enhanced current output. The aptasensor for TB assay showed a linear detection range from 1 pM to 60000 pM with a lower detection limit of 0.58 pM. And more, the proposed detection strategy was enzyme-free and label-free.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA Catalítico/química , DNA/química , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , DNA/genética , DNA Catalítico/genética , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Quadruplex G , Ouro/química , Hemina/química , Sequências Repetidas Invertidas , Limite de Detecção , Hibridização de Ácido Nucleico , Trombina/análise
10.
Talanta ; 205: 120140, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450469

RESUMO

Herein, we synthesized a kind of conductive supramolecular polymer hydrogel (CSPH) based on polyaniline (PANI) which can not only improve the conductivity but also promote antifouling performance of the aptasensor for the specific recruitment of thrombin (TB) from complex samples. With the electrochemical copolymerization of aniline (AN) and 3-aminophenylboronic acid (ABA) on glassy carbon electrode (GCE), the electrode was then inserted into the polyvinyl alcohol (PVA) solution to obtain robust CSPH through boric acid groups incorporated onto PANI to cause gelation of PVA solution, owing to the hydrophilicity of CSPH and nearly electrical neutrality, the modified electrode is antifouling without integration of other antifouling materials. A sandwich-type electrochemical aptasensor was constructed on the CSPH based electrode interfaces. Thrombin aptamer 1 (TBA1) were modified on the CSPH through amide bond, and thrombin aptamer 2 modified magnetic nanoparticles (MNP-TBA2) are used as signal amplification probes, the aptasensor has good sensitivity with a linear range from 1 pmol/L to 10 nmol/L and has a detection limit down to 0.64 pmol/L. The strategy of utilizing eletropolymerization of CSPH films to undergo highly selective thrombin recognition is, of course, readily extended to a broad range of targets in the real samples, and the recovery was ranging from 95.2% to 106.3% and RSDs varying from 2.3% to 4.5%.


Assuntos
Compostos de Anilina/química , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Condutividade Elétrica , Hidrogéis/química , Limite de Detecção , Trombina/análise , Incrustação Biológica/prevenção & controle , Eletroquímica , Humanos , Trombina/química , Trombina/metabolismo
11.
Biosens Bioelectron ; 142: 111510, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31319327

RESUMO

Enzyme-mediated cascade reaction is applied to amplify signal and decrease the background because enzyme can catalyze inactive substrates into active substrates to generate the signal. In this work, Au nanoparticles, as signal probe, are used to load DNA probe and ALP for dual signal amplification. Based on enzyme-mediated cascade reaction, a homogeneous biosensor is constructed for bioassay by employing thrombin as target molecule. When the target is present in the solution, ALP catalyzes the PPi into Pi and then reacts with molybdate in conjunction with Pi in the DNA backbone to produce redox precipitates on the surface of the reduced graphene oxide modified electrode with the help of magnetic separation. Compared with the conventional heterogeneous biosensor, the immobilization-free strategy, proposed in this homogeneous biosensor, improves the sensitivity because of its lower steric hindrance. As a result, this biosensor displayed a great sensitivity with a wide linear range from 1 fM to 10 nM and a detection limit of 0.26 fM, providing a promise and easy operating method for various proteins detection.


Assuntos
Técnicas Biossensoriais/métodos , Sondas de DNA/química , Ouro/química , Nanopartículas Metálicas/química , Trombina/análise , Fosfatase Alcalina/química , Técnicas Eletroquímicas/métodos , Grafite/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/ultraestrutura , Molibdênio/química , Oxirredução
12.
Anal Chim Acta ; 1077: 281-287, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31307720

RESUMO

Herein, a novel signal-off photoelectrochemical (PEC) aptasensor was proposed for sensitive detection of thrombin on the basis of C60@C3N4 nanocomposites as quencher and Au nanoparticles (depAu) decorated perylene tetracarboxylic acid (PTCA) as sensing platform. Owing to the excellent membrane-forming of PTCA and superior conductivity of depAu, the PTCA between two depAu layers can simply and effectively produce an extremely high initial photocurrent to afford a precondition for sensitive biodetection. Thereafter, the assembly of C60@C3N4 nanocomposites on electrode via typical sandwich reaction enabled the generation of a significantly decreased photocurrent. Here, the C3N4 with high surface area not only provided massive binding sites for C60 immobilization, but also partly competed with PTCA in light absorption for producing a significantly smaller photocurrent in the presence of electron donor ascorbic acid (AA). Additionally, both the C3N4 and C60 have the poor conductivity, which could inhibit the electron transfer to achieve a further decreased photocurrent, effectively improving the sensitivity of proposed biosensor. As a result, the PEC biosensor in a "signal-off" mode showed an extremely low detection limit down to 1.5 fM, providing a sensitive and universal strategy for protein detection.


Assuntos
Fulerenos/química , Grafite/química , Nanopartículas Metálicas/química , Nanocompostos/química , Compostos de Nitrogênio/química , Perileno/análogos & derivados , Trombina/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Fulerenos/efeitos da radiação , Ouro/química , Grafite/efeitos da radiação , Limite de Detecção , Nanocompostos/efeitos da radiação , Compostos de Nitrogênio/efeitos da radiação , Perileno/química , Reprodutibilidade dos Testes , Raios Ultravioleta
13.
Pediatr Blood Cancer ; 66(11): e27886, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31348595

RESUMO

BACKGROUND: Emicizumab is a bispecific antibody that bridges factor IXa and factor X to restore hemostasis in patients with hemophilia A (HA). Its efficacy and safety have been proven in multicenter trials. However, real world data regarding its use in very young children are currently lacking. Ancillary test results for monitoring emicizumab's hemostatic effect and their clinical correlations are scarce. METHODS: Children with HA and inhibitors treated by emicizumab were prospectively followed at our center. Laboratory follow-up included rotational thromboelastometry (ROTEM) and thrombin generation (TG), prior to and during treatment. RESULTS: Eleven children whose median age was 26 months were treated by emicizumab and followed for a median of 36 weeks. During follow-up, none experienced hemarthrosis or any other spontaneous bleeds. For 7/11 patients, emicizumab prophylaxis was sufficient to maintain hemostasis without additional supplemental therapy. Only 4/11 patients were occasionally treated with recombinant activated FVII for trauma. Two minor surgeries were safely performed without supplemental therapy while another procedure was complicated by major bleeding. TG parameters improved for all patients, correlating with their clinical status. Interestingly, the lowest TG values were obtained for patients experiencing bleeding episodes, while ROTEM parameters in all patients were close to the normal range. CONCLUSIONS: This study confirms the safety and efficacy of emicizumab in reducing bleeds in young children with HA with inhibitors, including infants. However, surgeries warrant caution as emicizumab prophylaxis may not be sufficient for some procedures. TG may more accurately reflect the hemostasis state than ROTEM in pediatric patients treated with emicizumab.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemorragia/prevenção & controle , Perda Sanguínea Cirúrgica/prevenção & controle , Pré-Escolar , Fator IXa/imunologia , Fator X/imunologia , Seguimentos , Hemofilia A/sangue , Hemofilia A/complicações , Hemorragia/etiologia , Humanos , Lactente , Masculino , Estudos Prospectivos , Tromboelastografia , Trombina/análise , Resultado do Tratamento
14.
Biophys Chem ; 252: 106193, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31195341

RESUMO

Enzymes are among the most important drug targets in the pharmaceutical industry. The bioassays used to screen enzyme modulators can be affected by unaccounted interferences such as time-dependent inactivation and inhibition effects. Using procaspase-3, caspase-3, and α-thrombin as model enzymes, we show that some of these effects are not eliminated by merely ignoring the reaction phases that follow initial-rate measurements. We thus propose a linearization method (LM) for detecting spurious changes of enzymatic activity based on the representation of progress curves in modified coordinates. This method is highly sensitive to signal readout distortions, thereby allowing rigorous selection of valid kinetic data. The method allows the detection of assay interferences even when their occurrence is not suspected a priori. By knowing the assets and liabilities of the bioassay, enzymology results can be reported with enhanced reproducibility and accuracy. Critical analysis of full progress curves is expected to help discriminating experimental artifacts from true mechanisms of enzymatic inhibition.


Assuntos
Caspase 3/análise , Ensaios Enzimáticos , Trombina/análise , Caspase 3/biossíntese , Caspase 3/metabolismo , Humanos , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Trombina/antagonistas & inibidores , Trombina/metabolismo
15.
Anal Chim Acta ; 1074: 142-149, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159934

RESUMO

A simple proximity hybridization-induced on particle DNA walker was designed for ultrasensitive detection of proteins, for example platelet-derived growth factor (PDGF-BB) secreted by cancer cells, in which the DNA walker was activated by specific target binding and powered by an enzymatic cleavage to produce amplified signal. High-density FAM-labeled hairpin oligonucleotides (FAM-DNA1) were functionalized on AuNPs to construct three-dimensional (3D) DNA tracks. The specific binding of PDGF-BB with two aptamer probes (DNA3 and DNA4) led to the proximity hybridization-induced DNA displacement and the free of DNA walker (DNA2) to perform movement on the 3D tracks by an enzymatic cleavage, resulting in the release of massive FAM-DNA1 fragments from the AuNPs and the generation of fluorescent signal. This DNA walker based sensing strategy could detect PDGF-BB in a concentration range of 4 orders of magnitude with a detection limit down to sub-pM level. The practical applicability of the assay was demonstrated by detecting PDGF-BB secreted from MCF-7 cells with satisfactory results. The proposed DNA walker based assay could conveniently detect PDGF-BB with high sensitivity and good accuracy, along with the good extensibility of the assay, showing promise for practical diagnosis.


Assuntos
Aptâmeros de Nucleotídeos/química , Becaplermina/análise , DNA/química , Trombina/análise , Aptâmeros de Nucleotídeos/genética , Bioensaio/métodos , DNA/genética , Ouro/química , Humanos , Limite de Detecção , Células MCF-7 , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico
16.
Mikrochim Acta ; 186(6): 356, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31098714

RESUMO

The authors describe a versatile aptasensing scheme based on the use of polypyrrole nanoparticles (PPyNPs) and DNA-silver nanoclusters (DNA-AgNCs) for multiple target detection. The DNA-AgNCs consist of two functional domains, viz. (a) a nucleation domain for attaching the metal core of the nanoclusters, and (b) a recognition domain which consists of a single-stranded aptamer. In the absence of analytes, the single-strand recognition domain will be absorbed onto the surface of the PPyNPs through π stacking and hydrophobic interactions. As a result, the red fluorescence of the DNA-AgNCs (with excitation/emission peaks at 535/625 nm) is quenched by the PPyNPs. On introducing the analytes, the DNA-AgNCs will bind them. This leads to the desorption of DNA-AgNCs and the recovery of the red fluorescence. Based on the above strategy, a versatile, sensitive and selective aptasensor was established for detection of adenosine, thrombin and interferon-gamma. The method was applied to the detection of the above targets in (spiked) serum samples and gave satisfactory results, with detection limit of 0.58 nM for IFN-γ, 0.39 nM for adenosine, and 2.2 nM for thrombin. The use of PPyNPs results in uniquely low non-specific absorption and in improved analytical results in case of real-sample analysis when compared to previously reported methods. Graphical abstract Schematic illustration of DNA-silver nanoclusters and polypyrrole nanoparticles in an aptasensor for detection of multiple targets.


Assuntos
Adenosina/análise , DNA/química , Interferon gama/análise , Polímeros/química , Pirróis/química , Prata/química , Trombina/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Fluorometria , Nanoestruturas/química
17.
Anal Chim Acta ; 1070: 112-122, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31103164

RESUMO

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has proven to be an effective technique for aptamers selection. In this study, we present an online reaction based convenient single-step CE-SELEX (ssCE-SELEX) mode with human thrombin (H-Thr) as a model target. The selection progress was monitored through bulk Kd analysis, which showed more than a 1000-fold improvement over the initial library after two rounds of selection. Three selected candidate sequences presented high binding affinities against H-Thr with nanomolar (nM) Kd determined by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM, 56.4-177.1 nM) and CE based non-linear fitting (CE-NLF, 98.2-199.7 nM). They also exhibited high specificities towards H-Thr compared with bovine thrombin, IgG, lysozyme, and lactoferrin. Meanwhile, the Kd results by isothermal titration calorimetry (ITC) confirmed the effective CE in measuring the aptamer affinity. In addition, three candidates were applied as aptasensors in the AuNPs based colorimetric assay, which showed visible color change and good linear relationships (R2 > 0.93) with H-Thr concentration. Furthermore, molecular dynamics (MD) simulation was performed to validate the binding of the three candidates with H-Thr by binding sites and binding free energy. The ssCE-SELEX method avoids off-line incubation, saves time and sample, and may provide a universal and convenient method for aptamers selection.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Sistemas On-Line , Técnica de Seleção de Aptâmeros/métodos , Animais , Calorimetria , Bovinos , Eletroforese Capilar , Humanos , Imunoglobulina G/análise , Lactoferrina/análise , Ligantes , Muramidase/análise , Muramidase/metabolismo , Trombina/análise
18.
Anal Chim Acta ; 1064: 104-111, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-30982507

RESUMO

A proximity ligation assay (PLA) induced hairpin to DNAzyme structure switching strategy has been described for entropy-driven amplified detection of thrombin. The enzyme-strand (E-DNA) and substrate-strand (S-DNA) of DNAzyme are locked in hairpins structure, and the catalytic activity of DNAzyme is inhibited simultaneously. However, in the presence of thrombin, the PLA can induce the unlocking of hairpin, and then the forming of active DNAzyme. Subsequently, the cleavage of DNAzyme can release DNA fragment to induce the entropy-driven amplification reaction, resulting significant recovery of fluorescent intensity by the separation of FAM from quencher. There was a good linear relationship in the range of 5 pM - 1 nM. This method provides high reliability and sensitivity under enzyme- and hairpin-free conditions.


Assuntos
Técnicas Biossensoriais , DNA Catalítico/química , Entropia , Técnicas de Amplificação de Ácido Nucleico , Trombina/análise , DNA Catalítico/metabolismo , Espectrometria de Fluorescência , Trombina/metabolismo
19.
Thromb Haemost ; 119(6): 899-905, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31005063

RESUMO

BACKGROUND: During liver transplantation (LT), thrombin generation (TG) is altered. The most frequently used assay for TG is the Calibrated Automated Thrombogram (CAT). It is designed for series of plasmas and is semi-automated. Complete automation has led to a new device, the ST-Genesia, enabling quantitative standardized TG evaluation. OBJECTIVE: The aim of this observational study was to compare the TG results of the CAT and the ST-Genesia on frozen-thawed plasma samples prepared from the blood of LT patients. PATIENTS AND METHODS: Poor platelet plasma aliquots were prepared from blood samples from six LT patients selected to get the whole range of TG and were assessed with CAT (recombinant human tissue factor [TF] concentration 5 pm) and with ST-Genesia Bleedscreen assay (BS, using 'low' recombinant human TF concentration) and Thromboscreen assay (TS, using 'medium' recombinant human TF concentration). The TG parameters studied were: lag time, peak, time to peak, endogenous thrombin potential, velocity index and start tail. RESULTS: BS and TS did not differ significantly from each other whatever the parameter studied, whereas most of the CAT parameters were significantly different from those obtained with BS and TS. Hierarchical clustering analysis of the different parameters of TG showed three homogeneous groups. One cluster gathered TG quantitative parameters from ST-Genesia. A second cluster gathered all the kinetic parameters. The last cluster isolated the quantitative parameters of CAT. CONCLUSION: In patients undergoing LT, TG performed with CAT and with ST-Genesia provided different results, for unknown reasons.


Assuntos
Transplante de Fígado , Plasma/metabolismo , Trombina/metabolismo , Automação Laboratorial , Testes de Coagulação Sanguínea/métodos , Calibragem , Equipamentos e Provisões , Humanos , Plasma/química , Reprodutibilidade dos Testes , Trombina/análise , Tromboplastina
20.
Mater Sci Eng C Mater Biol Appl ; 99: 1399-1406, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30889674

RESUMO

The highly catalytic Ca-doped carbon dots (CDCa) were prepared by microwave procedure, that exhibit strong catalytic effect on HAuCl4-glucose (GLC) reaction to form gold nanoparticles (AuNPs) with high SERS activity, using Victoria blue B (VBB) as a molecular probe. The SERS intensity at 1615 cm-1 increased linearly with CDCa increasing, due to formation of more AuNPs nanosol substrate as indicator. When thrombin aptamer (Apt) was added in this system, Apt adsorbed on the CDCa surface to inhibit theirs catalytic activity, and the SERS intensity decreased. However, when thrombin (TB) was present, it can bind to Apt to form stable G-duplex of Apt-TB and free CDCa catalyst in the system, and the SERS signal increased linearly. Thus a free-label Apt-SERS quantitative analysis method was developed for ultratrace TB, with a linear range of 0.0058-0.115 nmol/L and a detection limit of 0.0018 nmol/L TB.


Assuntos
Aptâmeros de Nucleotídeos/química , Cálcio/química , Carbono/química , Análise Espectral Raman/métodos , Trombina/análise , Catálise , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Tamanho da Partícula , Corantes de Rosanilina/química , Espectrofotometria Infravermelho , Difração de Raios X
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