Accurate Thrombin Monitoring Based on Proximity Ligation Assay-Assisted Rolling Circle Amplification (RCA).

Due to the fact that the expression level of thrombin affects the coagulation function of the injured tissue after trauma, it is considered as a very promising biomarker for the diagnosis and treatment of trauma. Nonetheless, sensitive, simple, and accurate thrombin detection continue to be extremely difficult. Here, using the two domains of thrombin as detection targets, we build a unique, accurate, isothermal thrombin analysis method. The method is constructed based on the integration of proximity ligation and rolling circle amplification (RCA). This approach specifically binds with the two functional domains of thrombin by using two intricately constructed probes. The technique has great accuracy thanks to proximity ligation, and the coupled RCA ensures acceptable sensitivity. With a limit of detection (LOD) of 0.23 pM, the method has demonstrated favorable detection persistence. Furthermore, the technique has a high selectivity for thrombin. Integrating merits including high sensitivity, low cost, and good portability, this method may enrich the arsenal for thrombin related applications.

Nucleic acid aptamer-based biosensors and their application in thrombin analysis.

Thrombin is a multifunctional serine protease that plays an important role in coagulation and anticoagulation processes. Aptamers have been widely applied in biosensors due to their high specificity, low cost and good biocompatibility. This review summarizes recent advances in thrombin quantification using aptamer-based biosensors. The primary focus is optical sensors and electrochemical sensors, along with their applications in thrombin analysis and disease diagnosis.

In-Situ Fabrication of Electroactive Cu2+-Trithiocyanate Complex and Its Application for Label-Free Electrochemical Aptasensing of Thrombin.

The preparation of an electroactive matrix for the immobilization of the bioprobe shows great promise to construct the label-free biosensors. Herein, the electroactive metal-organic coordination polymer has been in-situ prepared by pre-assembly of a layer of trithiocynate (TCY) on a gold electrode (AuE) through Au-S bond, followed by repetitive soaking in Cu(NO3)2 solution and TCY solutions. Then the gold nanoparticles (AuNPs) and the thiolated thrombin aptamers were successively assembled on the electrode surface, and thus the electrochemical electroactive aptasensing layer for thrombin was achieved. The preparation process of the biosensor was characterized by an atomic force microscope (AFM), attenuated total reflection-Fourier transform infrared (ATR-FTIR), and electrochemical methods. Electrochemical sensing assays showed that the formation of the aptamer-thrombin complex changed the microenvironment and the electro-conductivity of the electrode interface, causing the electrochemical signal suppression of the TCY-Cu2+ polymer. Additionally, the target thrombin can be label-free analyzed. Under optimal conditions, the aptasensor can detect thrombin in the concentration range from 1.0 fM to 1.0 µM, with a detection limit of 0.26 fM. The spiked recovery assay showed that the recovery of the thrombin in human serum samples was 97.2-103%, showing that the biosensor is feasible for biomolecule analysis in a complex sample.

PEC thrombin aptasensor based on Ag-Ag2S decorated hematite photoanode with signal-down effect of precipitation catalyzed by G-quadruplexes/hemin.

A photoelectrochemical (PEC) aptasensor for thrombin detection was rationally designed based on the photoanode of one-dimensional hematite nanorods (α-Fe2O3 NRs) with several steps of modifications. Uniform α-Fe2O3 NRs were grown vertically on the surface of fluorine-doped tin oxide (FTO) conductive glass through a one-step hydrothermal method; then Ag was grown on the surface of α-Fe2O3 NRs through a photoreduction method followed by a partial in-situ transformation into Ag2S, conferring an improvement on the initial photocurrent. Two main critical factors, namely, the steric hindrance of thrombin, benzoquinone (BQ) precipitation oxidized by H2O2 under the catalysis of G-quadruplexes/hemin, contributed to the sensitive signal-down response toward the target. Photocurrent signals related with thrombin concentration was established for thrombin analysis due to the non-conductive complex as well as their competitive consumption of electron donors and irradiation light. The excellent initial photocurrent was combined with the signal-down amplification in the design of the biosensor, conferring a limit of detection (LOD) as low as 40.2 fM and a wide linear range from 0.0001 nM to 50 nM for the detection of thrombin. The proposed biosensor was also assessed in terms of selectivity, stability, and applicability in human serum analyses, which provided an appealing maneuver for the specific analysis of thrombin in trace amount.

Electrochemical biomimetic enzyme cascade amplification combined with target-induced DNA walker for detection of thrombin.

Fe-N-doped carbon nanomaterials (Fe-N/CMs) were designed as a novel biomimetic enzyme with excellent peroxidase-like activity to achieve high-efficient enzyme cascade catalytic amplification with the aid of glucose oxidase (GOx), which was further combined with target-induced DNA walker amplification to develop a sensitive electrochemical biosensor for thrombin detection. Impressively, massive output DNA was transformed from small amounts of target thrombin by highly effective DNA walker amplification as protein-converting strategy, which could then induce the immobilization of functionalized nanozyme on the electrode surface to achieve the high-efficient electrochemical biomimetic enzyme cascade amplification. As a result, an amplified enzyme cascade catalytic signal was measured for thrombin detection ranging from 0.01 pM to 1 nM with a low detection limit of 3 fM. Importantly, the new biomimetic enzyme cascade reaction coupled the advantages of natural enzyme and nanozyme, which paved an avenue to construct varied artificial multienzymes amplification systems for biosensing, bioanalysis, and disease diagnosis applications.

Hemostatic In Vitro Properties of Novel Plasma Supernatants Produced from Late-storage Low-titer Type O Whole Blood.

BACKGROUND: The use of low-titer group O whole blood is increasing. To reduce wastage, unused units can be converted to packed red blood cells. Supernatant is currently discarded post-conversion; however, it could be a valuable transfusable product. The aim of this study was to evaluate supernatant prepared from late-storage low-titer group O whole blood being converted to red blood cells, hypothesizing it will have higher hemostatic activity compared to fresh never-frozen liquid plasma. METHODS: Low-titer group O whole blood supernatant (n = 12) prepared on storage day 15 was tested on days 15, 21, and 26 and liquid plasma (n = 12) on 3, 15, 21, and 26. Same-day assays included cell counts, rotational thromboelastometry, and thrombin generation. Centrifuged plasma from units was banked for microparticle characterization, conventional coagulation, clot structure, hemoglobin, and additional thrombin generation assays. RESULTS: Low-titer group O whole blood supernatant contained more residual platelets and microparticles compared to liquid plasma. At day 15, low-titer group O whole blood supernatant elicited a faster intrinsic clotting time compared to liquid plasma (257 ± 41 vs. 299 ± 36 s, P = 0.044), and increased clot firmness (49 ± 9 vs. 28 ± 5 mm, P < 0.0001). Low-titer group O whole blood supernatant showed more significant thrombin generation compared to liquid plasma (day 15 endogenous thrombin potential 1,071 ± 315 vs. 285 ± 221 nM·min, P < 0.0001). Flow cytometry demonstrated low-titer group O whole blood supernatant contained significantly more phosphatidylserine and CD41+ microparticles. However, thrombin generation in isolated plasma suggested residual platelets in low-titer group O whole blood supernatant were a greater contributor than microparticles. Additionally, low-titer group O whole blood supernatant and liquid plasma showed no difference in clot structure, despite higher CD61+ microparticle presence. CONCLUSIONS: Plasma supernatant produced from late-storage low-titer group O whole blood shows comparable, if not enhanced, in vitro hemostatic efficacy to liquid plasma.

Protein labeling and crosslinking by covalent aptamers.

In this chapter, a new approach to the selective modification of native proteins is discussed, using electrophilic covalent aptamers. These biochemical tools are generated through the site-specific incorporation of a label-transferring or crosslinking electrophile into a DNA aptamer. Covalent aptamers provide the ability to transfer a variety of functional handles to a protein of interest or to irreversibly crosslink to the target. Methods for the aptamer-mediated labeling and crosslinking of thrombin are described. Thrombin labeling is fast and selective, in both simple buffer and in human plasma and outcompetes nuclease-mediated degradation. This approach provides facile, sensitive detection of labeled protein by western blot, SDS-PAGE, and mass spectrometry.

Plasma thrombin generation kinetics vary by injury pattern and resuscitation characteristics in pediatric and young adult trauma patients.

BACKGROUND: Thrombin generation kinetics are not well studied in children. This study aimed to assess how thrombin generation kinetics vary in pediatric and young adult (YA) trauma patients by clinical characteristics and injury pattern. METHODS: Prospective cohort study where plasma samples were obtained from pediatric (ages 0-17 years) and YA (ages 18-21 years) trauma patients upon emergency department arrival. Thrombin generation (calibrated automated thrombogram [CAT]) was quantified as lag time (LT, minutes), peak height (PH, nM), time to peak (ttPeak, minutes), and endogenous thrombin potential (ETP, nM × minute). Results are expressed as median and quartiles [Q1, Q3] and compared using Wilcoxon rank sum testing with p < 0.05 considered significant. RESULTS: We enrolled 47 pediatric (median age, 15 [14, 17] years, 78% male, 87% blunt, median Injury Severity Score, 12) and 49 YA (median age 20 [18, 21] years, 67% male, 84% blunt, median Injury Severity Score, 12) patients. Pediatric and YA patients had similar rates of operative intervention (51% vs. 57%), transfusion (25% vs. 20%), and traumatic brain injury (TBI) (53% vs. 49%). Pediatric patients who required an operation had accelerated initiation of thrombin generation, with shorter LT than those who did not (2.58 [2.33, 2.67]; 2.92 [2.54, 3.00], p = 0.034). Shorter LT (2.41 [2.22, 2.67]; 2.67 [2.53, 3.00]) and ttPeak (4.50 [4.23, 4.73]; 5.22 [4.69, 5.75], both p < 0.01) were noted in pediatric patients who required transfusion as compared with those who did not. The YA patients requiring transfusion had shorter LT (2.33 [2.19, 2.74]; 2.83 [2.67, 3.27]) and ttPeak (4.48 [4.33, 5.65]; 5.33 [4.85, 6.28] both p < 0.04) than those who were not transfused. Young adults with TBI had greater ETP than those without (1509 [1356, 1671]; 1284 [1154, 1471], p = 0.032). CONCLUSION: Thrombin generation kinetics in pediatric trauma patients prior to intervention vary with need for operation and transfusion, while thrombin generation kinetics in young adult patients are influenced by TBI and need for operation or transfusion. This is a promising tool for assessing coagulopathy in young trauma patients. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

Switch to pdVWF:pdFVIII concentrate for prophylaxis in a paediatric patient with Type 3 von Willebrand disease: a case report.

OBJECTIVES: To report the long-term prophylaxis management of a child with type 3 von Willebrand disease by switching to Wilate (Octapharma AG), a plasma-derived, double virus-inactivated concentrate of freeze-dried of a 1 to 1 ratio of active Von Willebrand Factor and Factor VIII (pdVWF:pdFVIII) recently marketed as Eqwilate in France. METHODS: This is a case report of 12.6-year-old boy with congenital Type 3 VWD who had a history of frequent bleeds. Prophylaxis started at the age of 38 months with FVIII-poor pdVWF concentrate (Wilfactin, LFB) and FVIII (Wilstart, LFB). Pharmacokinetics and thrombin generation assay were performed. Annualized bleeding rate was derived from the bleeding episodes documented in the medical record during a 24-month period before and after starting pdVWF:pdFVIII concentrate. RESULTS: Both product injections promptly raised the endogenous thrombin potential (ETP). However, the maximal concentration of formed thrombin was higher following pdVWF:pdFVIII injection. Due to a high bleeds frequency and better results regarding FVIII levels and thrombin generation, the prophylaxis regimen was changed to the same dose and frequency of pdVWF:pdFVIII concentrate (42 IU/kg per day, three times a week). During the last 24 months, annualized total, trauma, and spontaneous bleeding rates were 7.5, 4.5, and 3, respectively. These rates decreased to 2, 1.5, and 0.5 respectively during the next two years. The mother reported a marked improvement in the quality of life of his son and hers. CONCLUSION: Switch to pdVWF:pdFVIII concentrate for long-term prophylaxis in a young type 3 VWD patient was safe and effective in reducing bleeds.

Comparison of Optical and Electrical Sensor Characteristics for Efficient Analysis of Attachment and Detachment of Aptamer.

Nucleic acid aptamer-based research has focused on achieving the highest performance for bioassays. However, there are limitations in evaluating the affinity for the target analytes in these nucleic acid aptamer-based bioassays. In this study, we mainly propose graphene oxide (GO)-based electrical and optical analyses to efficiently evaluate the affinity between an aptamer and its target. We found that an aptamer-coupled GO-based chip with an electrical resistance induced by a field-effect transistor, with aptamers as low as 100 pM, can detect the target, thrombin, at yields as low as 250 pM within five minutes. In the optical approach, the fluorescent dye-linked aptamer, as low as 100 nM, was efficiently used with GO, enabling the sensitive detection of thrombin at yields as low as 5 nM. The cantilever type of mechanical analysis also demonstrated the intuitive aptamer-thrombin reaction in the signal using dBm units. Finally, a comparison of electrical and optical sensors' characteristics was introduced in the attachment and detachment of aptamer to propose an efficient analysis that can be utilized for various aptamer-based research fields.

An instrument-free visual quantitative detection method based on clock reaction: the detection of thrombin as an example.

Instrument-free visual quantitative detection in chemical and biochemical analysis is of great significance in practical applications especially in point-of-care testing and in places where resources are limited. In this paper, we report the development of a time-based instrument-free visual quantitative detection method by employing a clock reaction, a type of chemical reaction displaying characteristic clocking behavior. The feasibility of the method was illustrated by the quantitative detection of thrombin in buffer solution using the lapse of time as the readout signal. The linear range of detection was from 1.3 to 43 nM (r2 = 0.990, n = 3) with a LOD of 0.9 nM, which is lower than the physiological concentrations of thrombin in the resting and activated blood, which range from low nanomolar to low micromolar, respectively. This method was also validated by detecting thrombin in the serum and a good recovery of nearly 100 ± 8.0% was obtained. To the best of our knowledge, this work is the first report that uses the characteristic time of a clock reaction as the readout signal in instrument-free colorimetry for quantitative bioanalysis.

Recent Progresses in Development of Biosensors for Thrombin Detection.

Thrombin is a serine protease with an essential role in homeostasis and blood coagulation. During vascular injuries, thrombin is generated from prothrombin, a plasma protein, to polymerize fibrinogen molecules into fibrin filaments. Moreover, thrombin is a potent stimulant for platelet activation, which causes blood clots to prevent bleeding. The rapid and sensitive detection of thrombin is important in biological analysis and clinical diagnosis. Hence, various biosensors for thrombin measurement have been developed. Biosensors are devices that produce a quantifiable signal from biological interactions in proportion to the concentration of a target analyte. An aptasensor is a biosensor in which a DNA or RNA aptamer has been used as a biological recognition element and can identify target molecules with a high degree of sensitivity and affinity. Designed biosensors could provide effective methods for the highly selective and specific detection of thrombin. This review has attempted to provide an update of the various biosensors proposed in the literature, which have been designed for thrombin detection. According to their various transducers, the constructions and compositions, the performance, benefits, and restrictions of each are summarized and compared.

A point-of-care microfluidic channel-based device for rapid and direct detection of fibrinogen in whole blood.

Hemorrhage is the leading cause of preventable death in civilian and battlefield traumatic injuries. Patients with severe traumatic hemorrhagic shock are more likely to be deficient in fibrinogen than those with other coagulation factors, and hypofibrinogenemia is an independent risk factor for mortality. Thus, rapid detection of fibrinogen levels is of great importance in these patients during damage control resuscitation. Plasma is used as an analyte in fibrinogen detection, which restricts the use of existing devices in emergencies. To meet the needs of on-site detection, we developed a point-of-care microfluidic channel-based device for direct measurement of fibrinogen concentration in whole blood. In our method, thrombin is dispersed on a reaction strip to initiate conversion of fibrinogen to fibrin. The permeability of the resulting blood clots depends on the fibrinogen level. A hydrophobic plastic protection flake between the reaction strip and a wicking strip is then removed to allow flow of unclotted blood. The rate of blood flow along the wicking strip was inversely related to the fibrinogen concentration. The whole process could be completed in as fast as 5 minutes for a whole blood sample size of 150 µL, and yielded accurate results ranging from 0 to 4 g L-1, which were unaffected by Ca2+, blood lipids, hematocrit, warfarin and tissue plasminogen activators (tPAs). Results using clinical whole blood samples were also highly consistent with those using an automatic coagulation analyzer, yielding a Pearson correlation coefficient of up to 0.919. This approach has potential for allowing rapid diagnosis of fibrinogen concentration in critically ill bleeding patients in different settings, thus helping to judge the suitability of fibrinogen replacement therapy (FRT) in cases of emergency bleeding and in patients at risk of thrombosis due to hyperfibrinogenemia.

Ultrasensitive disposable apatasensor for reagentless electrocatalytic detection of thrombin: An O2-Dependent hemin-G4-aptamer assay on gold screen-printed electrodes.

Serine protease thrombin is a strong neurotoxin produced by the injured brain and an Alzheimer's disease biomarker. To address its point-of-care testing (POCT), we adapted the O2-dependent aptamer assay for thrombin to gold screen-printed electrodes (Au SPE). The assay exploits reagentless (with no mediators) electrocatalytic activity of hemin-G4 DNAzyme in O2 reduction. Au SPEs modified with thiolated hemin-conjugated aptamer and PEG showed enhanced electrocatalytic activity in O2 reduction upon thrombin binding to the aptamer, then folding into the electroactive hemin-G4 DNAzyme structure. 0.5 fM thrombin were detected in aerated PBS and artificial cerebrospinal fluid, correspondingly, with the logarithmic linear range extending to 100 fM; dopamine, and uric and ascorbic acids did not interfere with electroanalysis. The disposable aptasensor met basic POCT requirements, with a minimal shelf life of 3 days. However, the reactivity and suitability of the Au SPE surface for thiol binding and electrocatalysis required special surface pre-treatment and modification protocols, and the fundamental problem of a long-term stability of thiol modification on gold should be addressed for practical applications of Au SPE-based apatasensors in POCT.

Colorimetric detection of thrombin based on signal amplification by transcription-reverse transcription concerted reaction using non-crosslinking aggregation of gold nanoparticles.

Gold nanoparticles (AuNPs) have been used as colorimetric biosensors by utilizing the difference in color between the dispersed (red) and aggregated (blue) states. We previously developed a biosensor that converts sandwich-type thrombin recognition to RNA amplification and color difference of AuNPs. But the sensitivity was insufficient because of the linear signal amplification mechanism. In this study, we designed an exponential signal amplification biosensor based on transcription-reverse transcription concerted (TRC) reaction.

Field-Effect Transistor with a Plasmonic Fiber Optic Gate Electrode as a Multivariable Biosensor Device.

A novel multivariable system, combining a transistor with fiber optic-based surface plasmon resonance spectroscopy with the gate electrode simultaneously acting as the fiber optic sensor surface, is reported. The dual-mode sensor allows for discrimination of mass and charge contributions for binding assays on the same sensor surface. Furthermore, we optimize the sensor geometry by investigating the influence of the fiber area to transistor channel area ratio and distance. We show that larger fiber optic tip diameters are favorable for electronic and optical signals and demonstrate the reversibility of plasmon resonance wavelength shifts after electric field application. As a proof of principle, a layer-by-layer assembly of polyelectrolytes is performed to benchmark the system against multivariable sensing platforms with planar surface plasmon resonance configurations. Furthermore, the biosensing performance is assessed using a thrombin binding assay with surface-immobilized aptamers as receptors, allowing for the detection of medically relevant thrombin concentrations.

Thrombin Generation in Cardiac Versus Noncardiac Surgical Cohorts.

BACKGROUND: Bleeding can be a significant problem after cardiac surgery. As a result, venous thromboembolism (VTE) or anticoagulation or both following mechanical valve implantation are often delayed in these patients. The calibrated automated thrombin (CAT) generation assay has become the gold standard to evaluate thrombin generation, a critical step in clot formation independent of other hemostatic processes (eg, platelet activation, fibrin cross-linking, and fibrinolysis), and is increasingly used to examine thrombotic and hemorrhagic outcomes. No study has currently used this assay to compare the thrombin generation profiles of cardiac surgical patients to noncardiac surgical patients. We hypothesize that noncardiac patients may be less prone to postoperative changes in thrombin generation. METHODS: A prospective, observational, cohort study was undertaken using blood samples from 50 cardiac and 50 noncardiac surgical patients preoperatively, immediately postoperatively, and on postoperative days 1 to 4. Platelet-poor plasma samples were obtained from patients preoperatively, on arrival to the postanesthesia care unit (PACU) or intensive care unit (ICU), and daily on postoperative days 1 to 4 if patients remained inpatient. Samples were evaluated for CAT measurements. Patient and surgical procedure characteristics were obtained from the electronic medical record. RESULTS: The primary outcome variable, median endogenous thrombin potential (ETP), measured in nanomolar × minutes (nM × min), was decreased 100% in cardiac surgical versus 2% in noncardiac patients (P < .001). All parameters of thrombin generation were similarly depressed. Cardiac (versus noncardiac) surgical type was associated with -76.5% difference of percent change in ETP on multivariable regression analysis (95% confidence interval [CI], -87.4 to -65.5; P value <.001). CONCLUSIONS: Cardiac surgical patients exhibit a profound decrease in thrombin generation postoperatively compared with noncardiac surgical patients evaluated by this study. Hemodilution and coagulation factor depletion likely contribute to this decreased thrombin generation after cardiac surgery.

Persistent hypercoagulability in dogs envenomated by the European adder (Vipera berus berus).

BACKGROUND: Envenomation by the European adder, Vipera berus berus (Vbb), is a medical emergency. The overall in vivo haemostatic effects of pro- and anticoagulant components in Vbb venom, and the downstream effects of cellular injury and systemic inflammation, are unclear. OBJECTIVES: To longitudinally describe the global coagulation status of dogs after Vbb envenomation and compare to healthy controls. A secondary aim was to investigate differences between dogs treated with and without antivenom. METHODS: Citrated plasma was collected at presentation, 12 hours (h), 24 h, 36 h and 15 days after bite from 28 dogs envenomated by Vbb, and from 28 healthy controls at a single timepoint. Thrombin generation (initiated with and without exogenous phospholipids and tissue factor), thrombin-antithrombin (TAT)-complexes and the procoagulant activity of phosphatidylserine (PS)-expressing extracellular vesicles (EVs), expressed as PS-equivalents, were measured. RESULTS: At presentation the envenomated dogs were hypercoagulable compared to controls, measured as increased thrombin generation, TAT-complexes and PS-equivalents. The hypercoagulability decreased gradually but compared to controls thrombin generation and PS-equivalents were still increased at day 15. The discrepancy in peak thrombin between envenomated dogs and controls was greater when the measurement was phospholipid-dependent, indicating that PS-positive EVs contribute to hypercoagulability. Lag time was shorter in non-antivenom treated dogs, compared to antivenom treated dogs <24 h after envenomation. CONCLUSIONS: Hypercoagulability was measured in dogs up to 15 days after Vbb envenomation. Dogs treated with antivenom may be less hypercoagulable than their non-antivenom treated counterparts. Thrombin generation is a promising diagnostic and monitoring tool for Vbb envenomation.

Development of an Innovative Quantification Assay Based on Aptamer Sandwich and Isothermal Dumbbell Exponential Amplification.

Detecting blood biomarkers such as proteins with high sensitivity and specificity is of the utmost importance for early and reliable disease diagnosis. As molecular probes, aptamers are raising increasing interest for biosensor applications as an alternative to antibodies, which are used in classical enzyme-linked immuno-sorbent assays (ELISA). We have developed a sensitive and antibody-free molecular quantification assay that combines the specificity of aptamers and the sensitivity of the loop-mediated isothermal amplification (LAMP). For the proof-of-concept, we consider two types of biomarkers: (i) a model of oligonucleotide mimicking nucleic acid targets and (ii) the thrombin involved in the complex coagulation cascade as a model protein for which two relevant aptamers form a stable sandwich. The assay protocol is based on a few successive steps, similar to sandwich ELISA. First, aptamer-coated magnetic beads are added to the sample to specifically capture the targets. Then, the sandwich complex is formed by adding the second aptamer. This secondary aptamer is integrated in a larger oligonucleotide dumbbell sequence designed for LAMP detection using only two primers. After a proper rinsing step, the isothermal dumbbell exponential amplification is performed to detect and quantify a low amount of targets (limit of detection ∼ 1 pM for the oligonucleotide and ∼100 pM for thrombin). This study demonstrates that our innovative aptamero-LAMP assay could be relevant for the detection of different types of biomarkers and their quantification at physiological levels. This may also pave the way for antibody-free molecular assays.