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1.
Sci Rep ; 11(1): 7792, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33833254

RESUMO

SARS-CoV-2 infection increases the risk of thrombosis by different mechanisms not fully characterized. Although still debated, an increase in D-dimer has been proposed as a first-line hemostasis test associated with thromboembolic risk and unfavorable prognosis. We aim to systematically and comprehensively evaluate the association between thrombin generation parameters and the inflammatory and hypercoagulable state, as well as their prognostic value in COVID-19 patients. A total of 127 hospitalized patients with confirmed COVID-19, 24 hospitalized patients with SARS-CoV-2-negative pneumonia and 12 healthy subjects were included. Clinical characteristics, thrombin generation triggered by tissue factor with and without soluble thrombomodulin, and also by silica, as well as other biochemical parameters were assessed. Despite the frequent use of heparin, COVID-19 patients had similar thrombin generation to healthy controls. In COVID-19 patients, the thrombin generation lag-time positively correlated with markers of cell lysis (LDH), inflammation (CRP, IL-6) and coagulation (D-dimer), while the endogenous thrombin potential (ETP) inversely correlated with D-dimer and LDH, and positively correlated with fibrinogen levels. Patients with more prolonged lag-time and decreased ETP had higher peak ISTH-DIC scores, and had more severe disease (vascular events and death). The ROC curve and Kaplan Meier estimate indicated that the D-dimer/ETP ratio was associated with in-hospital mortality (HR 2.5; p = 0.006), and with the occurrence of major adverse events (composite end-point of vascular events and death) (HR 2.38; p = 0.004). The thrombin generation ETP and lag-time variables correlate with thromboinflammatory markers, and the D-dimer/ETP ratio can predict major adverse events in COVID-19.


Assuntos
/diagnóstico , Trombina/análise , Adulto , Idoso , Testes de Coagulação Sanguínea , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/diagnóstico , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Trombose/sangue , Trombose/diagnóstico
2.
Nucleic Acids Res ; 48(12): 6431-6444, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32442276

RESUMO

While many methods are available to measure the concentrations of proteins in solution, the development of a method to quantitatively report both increases and decreases in different protein concentrations in real-time using changes in the concentrations of other molecules, such as DNA outputs, has remained a challenge. Here, we present a biomolecular reaction process that reports the concentration of an input protein in situ as the concentration of an output DNA oligonucleotide strand. This method uses DNA oligonucleotide aptamers that bind either to a specific protein selectively or to a complementary DNA oligonucleotide reversibly using toehold-mediated DNA strand-displacement. It is possible to choose the sequence of output strand almost independent of the sensing protein. Using this strategy, we implemented four different exchange processes to report the concentrations of clinically relevant human α-thrombin and vascular endothelial growth factor using changes in concentrations of DNA oligonucleotide outputs. These exchange processes can operate in tandem such that the same or different output signals can indicate changes in concentration of distinct or identical input proteins. The simplicity of our approach suggests a pathway to build devices that can direct diverse output responses in response to changes in concentrations of specific proteins.


Assuntos
Aptâmeros de Nucleotídeos/química , Trombina/química , Fator A de Crescimento do Endotélio Vascular/química , Técnicas Biossensoriais/métodos , Humanos , Ligação Proteica , Trombina/análise , Fator A de Crescimento do Endotélio Vascular/análise
3.
Mikrochim Acta ; 187(5): 295, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32347383

RESUMO

A fluorescence method based on functionalized magnetic nanoparticles (FMNPs) and hybridization chain reaction (HCR) is developed for the enzyme-free amplified determination of thrombin. In the proposed design, aptamer against thrombin was hybridized with the capture DNA-modified magnetic nanoparticles to yield the FMNPs. In the presence of thrombin, aptamers are released due to the specific and high-affinity binding between thrombin and its aptamer. The exposed capture DNA subsequently hybridized with the partial sequence of helper DNA, and the vacant sequence of helper DNA further hybridized with HCR products which is pre-formed by the alternate hybridization of single-stranded DNAs (H1 and H2). The immobilized HCR products were then labeled with YOYO-1 for fluorescence measurement. Fluorescence signal intensity of labeled YOYO-1 was measured at an emission wavelength of 519 nm (excitation under 488 nm) and used for calibration. By taking advantage of HCR amplification, this direct assay strategy showed a linear response in the 20- to 200-pM concentration range, and the limit of detection is 9.2 pM which is about 3-orders of magnitude lower than the serum thrombin concentration (10 nM) that triggers blood clotting. This developed method can efficiently differentiate the target protein from a protein matrix, and it is verified by determination of thrombin in spiked serum samples with recoveries in the range of 94.5-103.3%. Graphical abstract A fluorometry method for thrombin detection using magnetic nanoparticles and enzyme-free hybridization chain reaction.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Fluorometria , Nanopartículas de Magnetita/química , Hibridização de Ácido Nucleico , Trombina/análise , Humanos
4.
Transfusion ; 60(5): 1069-1077, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32315090

RESUMO

BACKGROUND: Phlebotomy is among the main determinants of anemia of prematurity. Blood sparing policies endorsed umbilical cord blood (here called placental) as an alternative source for laboratory testing. Little is known on the suitability of placental blood to evaluate neonatal hemostasis of newborn infants. We aimed to compare the hemostatic profile of paired placental and infant venous blood, by means of prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, antithrombin, protein C, thromboelastography (TEG) and thrombin generation assay (TGA). STUDY DESIGN: This was an observational single-center study. METHODS: We collected at birth venous citrated blood from both placental and infant venous source and performed PT, APTT, fibrinogen, antithrombin, protein C, TEG (reaction time-R; kinetics-K alpha angle-α, maximum amplitude-MA and lysis at 30 minutes-LY30), and TGA (endogenous thrombin potential-ETP). RESULTS: We enrolled 60 neonates with a median gestational age (range) of 37 weeks (28+1 -41) and birth-weight 2417 g (950-4170). Based on TEG and TGA, placental blood showed a procoagulant imbalance as indicated by lower median R (4.0 vs. 6.1 min; p < 0.001) and K (1.3 vs. 2.2 min; p < 0.001); higher α-angle (69.7 vs. 57.4°; p < 0.001) and ETP (1260 vs. 1078; p = 0.002) than those observed for infant venous blood. PT and APTT did not differ significantly between the two groups. CONCLUSIONS: While placental and neonatal blood samples are equally suitable to measure the standard coagulation tests PT and APTT, placental blood leads to a procoagulant imbalance when testing is performed with TEG or TGA. These effects should be considered when interpreting results stemming from investigation of neonatal hemostasis.


Assuntos
Transtornos da Coagulação Sanguínea/diagnóstico , Hemostasia/fisiologia , Doenças do Recém-Nascido/diagnóstico , Triagem Neonatal/métodos , Placenta/irrigação sanguínea , Transtornos da Coagulação Sanguínea/sangue , Testes de Coagulação Sanguínea , Feminino , Sangue Fetal/fisiologia , Fibrinogênio/análise , Idade Gestacional , Humanos , Recém-Nascido , Doenças do Recém-Nascido/sangue , Masculino , Tempo de Tromboplastina Parcial , Parto/sangue , Flebotomia/métodos , Flebotomia/normas , Gravidez , Tempo de Protrombina , Reprodutibilidade dos Testes , Trombina/análise
5.
Talanta ; 211: 120730, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32070579

RESUMO

Recently, various inorganic nanomaterials have been used as fluorescence anisotropy (FA) enhancers for biosensing successfully. However, most of them are size-uncontrollable and possess an intensive fluorescence quenching ability, which will seriously reduce the accuracy and sensitivity of FA method. Herein, we report a two-dimensional DNA nanosheet (DNS) without fluorescence quenching effect as a novel FA amplification platform. In our strategy, fluorophore-labeled probe DNA (pDNA) is linked onto the DNS surface through the hybridization with the handle DNA (hDNA) that extended from the DNS, resulting in the significantly enhanced FA value. After the addition of target, the pDNA was released from the DNS surface due to the high affinity between the hDNA and target, and the FA was decreased. Thus, target could be detected by the significantly decreased FA value. The linear range was 10-50 nM and the limit of detection was 8 nM for the single-stranded DNA detection. This new method is general and has been also successfully applied for the detection of ATP and thrombin sensitively. Our method improved the accuracy of FA assay and has great potential to detect series of biological analytes in complex biosensing systems.


Assuntos
Trifosfato de Adenosina/análise , Técnicas Biossensoriais/métodos , DNA/química , Polarização de Fluorescência/métodos , Corantes Fluorescentes/química , Nanoestruturas/química , Trombina/análise , DNA de Cadeia Simples/análise , Humanos , Limite de Detecção , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência
6.
ACS Appl Mater Interfaces ; 12(5): 5569-5577, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31933352

RESUMO

Thrombin is a marker of blood-related diseases, and its detection is of great significance in the fields of medical and biological research. Herein, a novel chemiluminescence (CL) sensor for thrombin detection was prepared based on dual-aptamer biorecognition and mesoporous silica encapsulated with iron porphyrin. Mesoporous silica encapsulated with hematin by aptamer1 (Apt1/hematin/M-SiO2) and magnetic microspheres modified with aptamer2 (Apt2/NH2-MS) were successfully prepared, and the two materials were used to construct a CL sensor to detect thrombin. Primarily, Apt2/NH2-MS is used for pretreatment separation of thrombin samples by the biorecognition effect between the aptamer (Apt2) and target (thrombin). Then, thrombin/Apt2/NH2-MS is again recognized with Apt1 on the surface of Apt1/hematin/M-SiO2 and Apt1/thrombin/Apt2/NH2-MS is formed, so dual-aptamer biorecognition is realized. Meanwhile, the generated Apt1/thrombin/Apt2/NH2-MS makes Apt1 shed off the surface of M-SiO2 and release hematin. The released hematin can catalyze the luminol-H2O2 CL reaction. Therefore, a sandwich-type CL sensor was constructed based on dual-aptamer biorecognition and hematin catalysis for the detection of thrombin. The sensor has a linear range of 7.5 × 10-15 to 2.5 × 10-10 mol·L-1 and a detection limit of 2.2 × 10-15 mol·L-1 and also exhibits excellent selectivity, reproducibility, and stability. The sensor was successfully used for the detection of thrombin in serum samples, which makes it possible to apply the sensor in the detection of thrombin in actual samples.


Assuntos
Aptâmeros de Nucleotídeos/química , Medições Luminescentes/métodos , Porfirinas/química , Dióxido de Silício/química , Trombina/análise , Hemina/química , Hemina/metabolismo , Humanos , Ferro/química , Limite de Detecção , Magnetismo , Reprodutibilidade dos Testes
7.
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
8.
Anal Chim Acta ; 1100: 240-249, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31987147

RESUMO

In this study, a novel label- and immobilization-free RNA aptamer-modified riboswitch-based biosensor was developed by using RNA aptamer modified secondary-structural scaffolds to control the identity of the ribosomal binding sequence (RBS). In the developed sensor, the duplex RNA aptamers-modified cis-repressor sequence is introduced upstream to the RBS of the indicating gene (gfp gene), leading to formatting an RNA bubble due to the none-complementary state of the RNA aptamers in the hairpin structure of the cis-repressor sequence. Without the presence of the target molecule, the ribosome cannot identify the RBS of the indicating gene as the RBS is hidden by the introduced cis-repressor, consequently, the indicating gene in the sensor would not be expressed, demonstrating the absence of the target. On the contrary, with the presence of the target molecule, the binding of aptamer with the target would induce the enlargement of the RNA bubble, leading to the separation of the cis-repressor sequence and RBS. Hence, the indicating gene would be expressed, manifesting the existence of the target. In addition, the developed sensor can quantitatively report the target concentrations by measuring the gfp gene-encoded GFP (green fluorescent protein) concentration. The approach proposed in this study can be used to construct sensors for detecting various chemicals by introducing the corresponding aptamers, therefore, this strategy can potentially provide a new set of analytical tools in the field of analytical chemistry.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Técnicas Eletroquímicas , Sondas Moleculares/química , RNA/sangue , Sequência de Aminoácidos , Calibragem , Humanos , RNA/genética , Trombina/análise , Trombina/metabolismo
9.
Anal Chim Acta ; 1100: 40-46, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31987151

RESUMO

In this work, a dual amplified signal enhancement approach based on coupling deoxyribozyme (DNAzyme)-driven bipedal DNA walkers (BDW) and terminal deoxynucleotidyl transferase (TdT)-mediated DNA elongation signal amplifications has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. In presence of thrombin, the BDW complex, which is comprised from the target thrombin and two DNAzyme-containing probes, can exhibit autonomous cleavage behavior on the surface of the substrate DNA (SD) modified electrode, and remove the cleaved DNA fragment from the electrode surface. Subsequently, the TdT can catalyze the elongation of the SD with free 3'-OH termini and formation of many G-quadruplex sequence replicates with the presence of 2'-deoxyaguanosine-5'-triphosphate (dGTP) and adenosine 5'-triphosphate (dATP) at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a "signal-on" and completely label-free fashion. Under optimized conditions, the response peak current was linear with the concentration of thrombin in the range from 0.5 pM to 100000 pM with detection limit of 0.31 pM. This research provides us a sustainable idea for the hyphenated multiple amplification strategies and a stable and effective method for the detection of protein biomarkers.


Assuntos
Técnicas Biossensoriais , DNA Nucleotidilexotransferase/química , DNA Catalítico/química , DNA/química , Técnicas Eletroquímicas , Técnicas de Amplificação de Ácido Nucleico , DNA/genética , DNA/metabolismo , DNA Nucleotidilexotransferase/metabolismo , DNA Catalítico/metabolismo , Eletrodos , Humanos , Trombina/análise , Trombina/metabolismo
10.
Anal Chim Acta ; 1098: 164-169, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31948580

RESUMO

Detecting molecular biomarkers in high sensitivity plays an important role in the diagnosis of various diseases at the early stage. Here, by combining the target-induced polymerization nicking reaction (TIPNR) with the loop-mediated isothermal amplification (LAMP), we describe an ultrasensitive and label-free aptamer-based sensing method for detecting low levels of proteins in human serum by using thrombin as the model target analyte. The target thrombin binds and causes spontaneous assembly of two distinct aptamer probes to form the templates for the polymerization nicking reaction recycling amplification to produce many forward inner primer sequences. Subsequently, downstream LAMP reactions are initiated by these sequences for the generation of tremendous DNA hairpins with various lengths via automated cyclic strand displacement reactions. The SYBR Green I organic dye further binds the many hairpins to show drastically amplified fluorescence for ultrasensitive detection of thrombin down to 3.6 fM in the linear range from 0.01 pM to 10 nM. Such a sensing method based on aptamers has high discrimination capability for the target molecules against other non-specific proteins and is applicable for diluted serum samples. With the successful demonstration of the substantial signal amplification ability and simplicity feature of this assay approach, highly sensitive and convenient detection of other disease biomarkers with this method can be envisioned in the near future.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas de Amplificação de Ácido Nucleico , Trombina/análise , Humanos , Polimerização , Trombina/metabolismo
11.
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
12.
ACS Appl Mater Interfaces ; 12(2): 2871-2877, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31849211

RESUMO

In this work, the array arrangement of cascade enzymes was implemented by alternately and equidistantly anchoring two model enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) to the vertexes of rigid DNA tetrahedron units in lattice-like nucleic acid scaffold, in which the distance between any adjacent cascade enzymes had been regulated to the optimum for obtaining high enzyme cascade catalytic efficiency. Compared to the enzyme cascade system with no-array arrangement of cascade enzymes, the proposed enzyme cascade system allowed the intermediate H2O2 produced by GOx catalyzing substrate glucose to concurrently and equidistantly diffuse toward the four adjacent HRP enzyme surfaces. In this case, the invalid diffusion effect of intermediate H2O2 between cascade enzymes could be effectively avoided, thereby promoting the enzyme cascade reaction with high catalytic efficiency. The specific catalytic efficiency (kcat/Km) of the cascade enzyme system with array arrangement had been evaluated, which exhibited catalytic efficiency about 3.6 times higher than that of the randomly arranged cascade enzyme system. As a result, this strategy provided a new avenue for constructing a highly efficient enzyme cascade system with ultimate applications in biosynthesis, bioanalysis, and biodiagnostics.


Assuntos
DNA/química , Glucose Oxidase/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Nanoestruturas/química , Conformação de Ácido Nucleico , Biocatálise , Técnicas Biossensoriais , Técnicas Eletroquímicas , Humanos , Cinética , Reprodutibilidade dos Testes , Trombina/análise , Fatores de Tempo
13.
J Vet Emerg Crit Care (San Antonio) ; 30(1): 102-106, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31845506

RESUMO

OBJECTIVE: To evaluate the utility of the chemiluminescent enzyme immunoassay (CLEIA) method for point-of-care (POC) measurement of canine plasma thrombin-antithrombin complex (TAT) concentration. ASSESSMENT AND MAIN RESULTS: Plasma TAT concentration was measured in 54 healthy dogs and in 72 dogs with various diseases. A significant correlation was found between TAT concentration measured by CLEIA and that measured by an ELISA that was previously used in dogs. The upper limit of the reference value of TAT concentrations measured by CLEIA was determined to be 0.2 ng/mL based on the TAT concentration in 54 healthy dogs. TAT concentrations exceeded the reference interval in a portion of dogs when a hypercoagulable state may be present. CONCLUSIONS: Canine plasma TAT concentrations measured using CLEIA were correlated with that measured using ELISA. Hence, a POC testing instrument may be used for early detection of activation of thrombin generation in emergency and critical care settings.


Assuntos
Coagulação Intravascular Disseminada/veterinária , Doenças do Cão/diagnóstico , Peptídeo Hidrolases/sangue , Trombina/análise , Animais , Antitrombina III , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/diagnóstico , Doenças do Cão/sangue , Cães , Ensaio de Imunoadsorção Enzimática/normas , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Medições Luminescentes/normas , Medições Luminescentes/veterinária , Masculino , Sistemas Automatizados de Assistência Junto ao Leito , Valor Preditivo dos Testes , Estudos Prospectivos
14.
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
15.
Artif Organs ; 44(3): 296-304, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31520401

RESUMO

The formation of thrombi in medical devices that come into contact with blood is a common cause of increased morbidity and mortality. Prolonged use of central venous catheters (CVCs) may cause high infection rates or compromise CVC patency due to thrombus development. In this study, we sought insights into possible changes in the hemostatic system during prolonged use of inserted CVCs for hemodialysis by assessing platelets by CD62P and CD41a expression and the potential for thrombin generation (TG). This study included patients with chronic renal failure who were undergoing hemodialysis three times a week using a CVC, and healthy subjects as controls. The participants were distributed into three groups: Group 1: clinically and laboratorially healthy individuals matched by sex and age to the patients (controls); Group II: patients who had completed 1 month of CVC insertion; and Group III: the same patients after they had completed 4 months of CVC insertion. Platelet activation analysis and TG evaluation were performed using blood samples obtained through two different accesses, that is, through a peripheral vein and directly from the CVC lumen. The data showed platelet activation and an increase in the generation of thrombin, particularly after 4 months of CVC use. The results also indicated that insertion of the catheter into the blood stream stimulated the intrinsic rather than the extrinsic pathway. Taken together, the data showed a direct relationship between the use of CVCs in hemodialysis patients and a state of hypercoagulability, most likely associated with endothelial damage and the contact of the medical device with blood components such as platelets and coagulation factors.


Assuntos
Cateteres Venosos Centrais/efeitos adversos , Selectina-P/análise , Diálise Renal/efeitos adversos , Trombina/análise , Trombose/etiologia , Adulto , Idoso , Coagulação Sanguínea , Cateterismo Venoso Central/efeitos adversos , Cateterismo Venoso Central/instrumentação , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária , Diálise Renal/instrumentação , Trombose/sangue
16.
Mikrochim Acta ; 187(1): 53, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848726

RESUMO

A fluorometric method is described for the determination of thrombin. Polymer nanoparticles containing the luminol-terbium(III) complex (luminol-Tb) were prepared where luminol acts as the bridging ligand, and Tb(III) acts as the central metal ion. Thrombin possesses a large number of electrons donating groups that coordinate with luminol-Tb. Following coordination, the rigidity of the linker is increased, and this decreases the non-radiative decay rate and induces an increase in fluorescence intensity at 430 nm. Hence, thrombin can be fluorometrically determined. The detection limit of thrombin is as low as 3.5 pM (at an SNR of 3). This is about 10 times lower than assays using an aptamer. The method was applied in the determination of thrombin in human serum via the standard addition method and gave satisfying results. Graphical abstractSchematic representation of the preparation of the luminol-Tb(III) complex in a nanoparticle host by the self-assembly of luminol and Tb(III) ions. Thrombin readily coordinates with the luminol-Tb(III) system, and this results in particle aggregation. The blue fluorescence of luminol increases strongly, and this effect provides the basis for fluorometric determination of thrombin.


Assuntos
Complexos de Coordenação/química , Fluorescência , Nanopartículas/química , Polímeros/química , Térbio/química , Trombina/análise , Complexos de Coordenação/síntese química , Luminol/química , Estrutura Molecular , Tamanho da Partícula , Propriedades de Superfície
17.
Mikrochim Acta ; 187(1): 25, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31811449

RESUMO

The detection of thrombin by using CdS nanocrystals (CdS NCs), gold nanoparticles (AuNPs) and luminol is investigated in this work. Thrombin is detected by three methods. One is called the quenching method. It is based on the quenching effect of AuNPs on the yellow fluorescence of CdS NCs (with excitation/emission wavelengths of 355/550 nm) when placed adjacent to CdS NCs. The second method (called amplification method) is based on an amplification mechanism in which the plasmonics on the AuNPs enhance the emission of CdS NCs through distance related Förster resonance energy transfer (FRET). The third method is ratiometric and based on the emission by two luminophores, viz. CdS NCs and luminol. In this method, by increasing the concentration of thrombin, the intensity of CdS NCs decreases, while that of luminol increases. The results showed that ratiometric method was most sensitive (with an LOD of 500 fg.mL-1), followed by the amplification method (6.5 pg.mL-1) and the quenching method (92 pg.mL-1). Hence, the latter is less useful. Graphical abstract Schematic representation of three different methods (quenching, amplification and ratiometric) were applied for detection of thrombin via aptasensor. The CdS nanocrystals, streptavidin (Str) coated AuNPs and also Str-luminol coated AuNPs were used for the construction steps of the electrochemiluminescence (ECL)-based biosensor.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Compostos de Cádmio/química , Pontos Quânticos/química , Sulfetos/química , Trombina/análise , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Eletroquímica , Eletrodos , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Medições Luminescentes , Trombina/metabolismo
18.
World J Emerg Surg ; 14: 57, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31857822

RESUMO

Background: An acute traumatic coagulopathy (ATC) is observed in about one third of severely traumatized patients. This early, specific, and endogenous disorder is triggered by the association of trauma and hemorrhage. The early phase of this condition is characterized by the expression of a bleeding phenotype leading to hemorrhagic shock and the late phase by a prothrombotic profile leading to multiple organ failure. The physiopathology of this phenomenon is still poorly understood. Hypotheses of disseminated intravascular coagulation, activated protein C-mediated fibrinolysis, fibrinogen consumption, and platelet functional impairment were developed by previous authors and continue to be debated. The objective of this study was to observe general hemostasis disorders in case of ATC to confront these hypotheses. Method: Four groups of 15 rats were compared: C, control; T, trauma; H, hemorrhage; and TH, trauma and hemorrhage. Blood samples were drawn at baseline and 90 min. Thrombin generation tests, platelet aggregometry, and standard hemostasis tests were performed. Results: Significant differences were observed between the baseline and TH groups for aPTT (17.9 ± 0.8 s vs 24.3 ± 1.4 s, p < 0.001, mean ± SEM), MAP (79.7 ± 1.3 mmHg vs 43.8 ± 1.3 mmHg, p < 0.001, mean ± SEM), and hemoglobin (16.5 ± 0.1 g/dL vs 14.1 ± 0.3 g/dL, p < 0.001, mean ± SEM), indicating the presence of an hemorrhagic shock due to ATC. Compared to all other groups, coagulation factor activities were decreased in the TH group, but endogenous thrombin potential was (paradoxically) higher than in group C (312 ± 17 nM/min vs. 228 ± 23 nM/min; p = 0.016; mean ± SEM). We also observed a subtle decrease in platelet count and function in case of ATC and retrieved an inversed linear relationship between fibrinogen concentration and aPTT (intercept, 26.53 ± 3.16; coefficient, - 3.40 ± 1.26; adjusted R 2: 0.1878; p = 0.0123). Conclusions: The clinical-biological profile that we observed, combining normal thrombin generation, fibrinogen depletion, and a hemorrhagic phenotype, reinforced the hypothesis of activated protein C mediated-fibrinolysis. The key role of fibrinogen, but not of the platelets, was confirmed in this study. The paradoxical preservation of thrombin generation suggests a protective mechanism mediated by rhabdomyolysis in case of major trauma. Based on these results, we propose a new conception concerning the pathophysiology of ATC.


Assuntos
Coagulação Intravascular Disseminada/fisiopatologia , Coagulação Intravascular Disseminada/terapia , Animais , Pressão Arterial/fisiologia , Modelos Animais de Doenças , Fibrinogênio/análise , Ácido Láctico/análise , Ácido Láctico/sangue , Potássio/análise , Potássio/sangue , Protrombina/análise , Tempo de Protrombina/métodos , Ratos , Ratos Sprague-Dawley/sangue , Trombina/análise , Ferimentos e Lesões/sangue , Ferimentos e Lesões/complicações
19.
Sci Rep ; 9(1): 18740, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822733

RESUMO

The development of optical biosensors for the rapid and costless determination of clinical biomarkers is of paramount importance in medicine. Here we report a fast and low-cost biosensor based on a plasmonic D-shaped plastic optical fibre (POF) sensor derivatized with an aptamer specific for the recognition of thrombin, the target marker of blood homeostasis and coagulation cascade. In particular, we designed a functional interface based on a Self Assembled Monolayer (SAM) composed of short Poly Ethylene Glycol (PEG) chains and biotin-modified PEG thiol in ratio 8:2 mol:mol, these latter serving as baits for the binding of the aptamer through streptavidin-chemistry. The SAM was studied by X-ray Photoelectron Spectroscopy (XPS) analysis, static contact angle (CA), Surface Plasmon Resonance (SPR) in POFs, and fluorescence microscopy on gold surface. The optimized SAM composition enabled the immobilization of about 112 ng/cm2 of aptamer. The thrombin detection exploiting POF-Aptasensor occurred in short times (5-10 minutes), the reached Limit of Detection (LOD) was about 1 nM, and the detection range was 1.6-60 nM, indicating the POF-Aptasensor well addresses the needs for a low-cost, simple to use and to realize, rapid, small size and portable diagnostic platform.


Assuntos
Técnicas Biossensoriais/instrumentação , Fibras Ópticas , Plásticos/química , Ressonância de Plasmônio de Superfície/instrumentação , Trombina/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/economia , Biotina/química , Limite de Detecção , Polietilenoglicóis/química , Trombina/genética
20.
Anal Chem ; 91(23): 15317-15324, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31710462

RESUMO

As an important biomarker, thrombin (TB) is a major player in thrombosis and hemostasis and has attracted increasing attention involving its determination. Herein a universal and ultrasensitive fluorescence biosensor based on a binding-induced 3D-bipedal DNA walker and catalytic hairpin assembly (CHA) strategy has been proposed for cascade signal amplification detection of thrombin. In this study, we designed two proximity probes (foot 1 and foot 2) which include a specific affinity ligand for TB binding and a Pb2+-dependent DNAzyme tail sequence. In the presence of TB, the simultaneous binding of TB to foot 1 (F1) and foot 2 (F2) via TB aptamer (TBA) brings the tail sequences into close proximity and the melting temperature for tail sequences and track DNA is increased, allowing the Pb2+-dependent DNAzyme to cleave the track DNA into two short fragments which have lower affinities for the DNAzyme and, finally, leading to the release of trigger DNA (T-DNA) for subsequent CHA reaction. In the meantime, the dissociated DNA walkers (F1 and F2) explore adjacent unwound track DNA, and the walking procedure is conducted. Unlike the conventional unipedal DNA walkers that anchor foot DNA and track DNA on the same sensing surface, the proposed 3D-bipedal DNA walking machine can not only increase the local concentration of track DNA but can also improve the walking efficiency and expand the range of the walkers to some extent due to the two free feet. Moreover, with the advantages of superior sensitivity and excellent specificity, this biosensing platform exhibits a huge potential in practical application in biomedical research and clinical diagnosis.


Assuntos
Técnicas Biossensoriais , Sondas de DNA/química , DNA Catalítico/química , DNA/química , Técnicas de Amplificação de Ácido Nucleico , Trombina/análise , Sítios de Ligação , Biocatálise , Biomarcadores/análise , Biomarcadores/metabolismo , DNA/metabolismo , Sondas de DNA/metabolismo , DNA Catalítico/metabolismo , Fluorescência , Humanos , Ligantes , Técnicas de Sonda Molecular , Trombina/metabolismo
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