Thrombin In Human Serum Research Articles

Electrochemical biosensor using direct electron transfer and an antibody–aptamer hybrid sandwich for target detection in complex biological samples

Direct electron transfer (DET) between an electrode and redox labels is feasible in electrochemical biosensors using small aptamer–aptamer sandwiches; however, its application is limited in biosensors that rely on larger antibody–antibody sandwiches. The development of sandwich-type biosensors utilizing DET is challenged by the scarcity of aptamer–aptamer sandwich pairs with high affinity in complex biological samples. Here, we introduce an electrochemical biosensor using an antibody–aptamer hybrid sandwich for detecting thrombin in human serum. The biosensor enables rapid DET through an antibody–aptamer hybrid configuration comprising (i) an antibody capture probe that provides high and specific affinity to the target in human serum, (ii) the target thrombin, and (iii) an aptamer detection probe that facilitates convenient terminal conjugation with long flexible spacer DNA and polylinker peptide containing multiple amine-reactive phenazine ethosulfate (arPES) redox labels, allowing the conjugated labels to easily approach the electrode. Rapid repeated DET using arPES-catalyzed NADH oxidation strongly enhanced the electrochemical signals. Properly sized spacer and polylinker provided low nonspecific adsorption of the aptamer probe conjugated with multiple arPESs and low interference with the binding of the aptamer probe. Methods for immobilizing thiol-terminated antibodies on Au electrodes were compared and optimized. The developed biosensor using the antibody–aptamer hybrid sandwich exhibited high sensitivity and selectivity in detecting thrombin, surpassing the limitations of an aptamer–aptamer sandwich owing to the low affinity of thrombin aptamers in human serum. The calculated detection limit of the biosensor was ∼1.5 pM in buffer and ∼2.7 nM in human serum.

Aptamer functionalized fluorescent probe for detection of thrombin in human serum via static quenching

Thrombin plays a pivotal role in blood coagulation, wound healing, and tumor metastasis. In this study, we developed a fluorescent probe (Apt15@QDs) by functionalizing quantum dots (QDs) with an aptamer 15 to specifically recognize thrombin. The aptamer Apt15 binds specifically to thrombin by adopting a G-quadruplex conformation, resulting in the formation of a binary complex that quenches the fluorescence of the probe. Analysis using the classical fluorescence quenching equations Stem-Volmer and Lineweaver-Burk revealed that the mechanism of quenching is static in nature. The established method was successfully applied for detecting thrombin in human serum samples. As shown from the results, the established method exhibited high sensitivity, with a limit of detection (LOD) of 8 nM, and possessed a wide linear range within the concentration span of 20 to 200 nM. Additionally, this method circumvented the need for intricate experimental procedures. Furthermore, it offered a short analysis time, obviated the requirement for additional reagents, and proved suitable for high-throughput analysis of biological samples. This study presents a sensitive, straightforward, and rapid approach for detecting thrombin in complex biological matrices.

Label-free electrochemical aptasensor for ultrasensitive thrombin detection using graphene nanoplatelets and carbon nano onion-based nanocomposite

Thrombin (TB) is a protease enzyme functioning in the blood coagulation cascade. It plays an important function in a number of diseases, including thromboembolic disease, Alzheimer's disease, and cancer.Therefore, precise, and sensitive detection of TB in clinical samples is of utmost importance. In this work, we have first synthesized a nanocomposite comprised of carboxyl-functionalized graphene nanoplatelets (GNPs), carbon nano-onions (CNOs), and chitosan (CS), which was used to develop an electrochemical aptasensor for selective detection of TB. This nanocomposite modified GCE exhibited a remarkable electrochemical response and a greater surface area (216 %) than the bare GCE. The morphology of the nanomaterials and nanocomposite were characterized using FESEM, TEM, and Energy-Dispersive X-ray Spectroscopy (EDS). The Layer-by-layer fabrication of the aptasensor was studied using the CV and DPV in Fe[CN]6]3/4− redox probeat pH 7.4. The designed aptasensor displayed sensitive detection of TB from 26.74 pM to 26.74 fM with a LOD of 8.61fM. Finally, this aptasensor showed promising potential in the quantitative determination of TB in human serum. This proposed aptasensor displayed excellent sensitivity, high specificity, and satisfactory reproducibility and can be useful in biomedical applications as well as for clinical purposes.

Construction and application of thrombin-activated fluorescence-SERS dual-mode optical nanoprobes

Thrombin (TB) plays a key role in the pathological and physiological coagulation of diseases. In this work, a TB-activated fluorescence-surface-enhanced Raman spectroscopy (SERS) dual-mode optical nanoprobe (MRAu) was constructed by linking rhodamine B (RB)-modified magnetic fluorescent nanospheres with AuNPs through TB-specific recognition peptides. In the presence of TB, the polypeptide substrate could specifically be cleaved by TB, resulting in the weakening of SERS hotspot effect and the reduction of Raman signal. Meanwhile, the fluorescence resonance energy transfer (FRET) system was destroyed, and the RB fluorescence signal originally quenched by AuNPs was recovered. Using MRAu, SERS and fluorescence methods were combined to extend the TB detection range to 1–150 pM, and the detection limit was as low as 0.35 pM. In addition, the ability to detect TB in human serum also verified the effectiveness and practicality of the nanoprobe. The probe was also successfully employed to evaluate the inhibitory effect against TB of active components in Panax notoginseng. This study provides a new technical means for the diagnosis and drug development of abnormal TB-related diseases.

Aptamer-functionalized smart photonic hydrogels: application for the detection of thrombin in human serum

Smart photonic hydrogels based on two-dimensional photonic crystals (2DPC) provide a promising sensing platform for constructing novel chemical and biological sensors due to their facile optical signal readout and highly sensitive responsivity toward target analytes. Aptamers, as recognition elements with high selectivity and affinity, are extensively used to construct a variety of sensors. Herein, we developed two partially base complementary aptamer-functionalized 2DPC hydrogels as aptasensors for the detection of thrombin (TB) in human serum. The photonic hydrogel aptasensors swelled upon exposure to TB solution, leading to an increase in the particle spacing of the 2DPCs. The particle spacing changes were acquired by simply measuring the diameters of the Debye ring diffracted by the 2DPCs without the requirement of sophisticated instruments. The aptasensor swelling resulted from the decrease in the hydrogel cross-linking density induced by the specific binding between one of the aptamers and TB and the increase in hydrogel mixing free energy induced by the introduction of TB. The particle spacing increase of the optimized aptasensor was linear over the TB concentration range of 1–500 nM, and the limit of detection was 0.64 nM. The constructed 2DPC hydrogel aptasensor was used to detect TB in human serum and achieved recoveries of 95.74–104.21% and a relative standard deviation of 2.52–6.58%, showing the practicability and accuracy of the sensor. The aptamer-actuated 2DPC hydrogel biosensor provides a new strategy for designing other target molecule-sensitive aptasensors, showing great potential for development into home kits.

An antifouling electrochemical aptasensor based on hyaluronic acid functionalized polydopamine for thrombin detection in human serum

Accurate detection of disease markers in a complex biological media is a major challenge because of serious biofouling and non-specific protein adsorption. Herein, a universal strategy for sensitive and low-fouling detection of thrombin in human serum was developed based on hyaluronic acid functionalized polydopamine. The material hyaluronic acid with hydroxyl groups was grafted to the polydopamine modified electrode surface through the connection of 6-mercapto-1-hexanol to exert antifouling performance, and the hyaluronic acid also provided a good substrate for the immobilization of aptamers specific for thrombin. The constructed aptasensor showed good sensitivity and selectivity toward the detection of thrombin with a detection limit as low as 0.03 pM. Moreover, thanks to the presence of hyaluronic acid within the sensing interface, the aptasensor was able to assay thrombin in diluted human serum with markedly decreased side effect of non-specific adsorption.

Electrochemical aptasensor based on proximity binding-induced DNA networked for enzyme-free and ultrasensitive detection of thrombin

The abnormal change of thrombin concentration is closely related with various diseases. By using proximity binding-induced DNA network assembled by nonlinear hybridization chain reaction (HCR), we here developed an enzyme-free and ultrasensitive electrochemical strategy for analyzing thrombin in human serums. The binding of the two different aptamers to a thrombin enhanced the local concentration of the aptamers and facilitates the formation of an intact strand. Subsequently, the formed strand caused nonlinear HCR, forming DNA network. The DNA network bound to the capture probes (CP)-modified electrode, producing electrochemical signal of MB. Benefiting from the proximity binding-induced DNA network, the proposed biosensor exhibited a wide linear range from 1.0 pM to 1.0 nM with a low detection limit of 0.56 pM and high specificity. More importantly, the developed enzyme-free electrochemical biosensor has been used to detect thrombin in serum samples with good performance. This makes such a strategy possess potential promise in the application of clinical diagnosis.

2D titanium carbide nanosheets based fluorescent aptasensor for sensitive detection of thrombin

The emerging two-dimensional titanium carbides (MXenes) have a large potential in biomedical sensing owing to their excellent electrical and optical properties. Herein, a fluorescence resonance energy transfer (FRET) aptasensor with high sensitivity and specificity was constructed with single layer Ti3C2 MXene for quantitative detection of thrombin. The dye labelled thrombin-binding aptamer (TBA) was deposited on the surface of Ti3C2, and the fluorescence of which was efficiently quenched owing to the FRET between the dye and Ti3C2. The fact that thrombin forms quadruplex with TBA on Ti3C2 surface is due to the high electronic affinity between thrombin and Ti3C2. This process will cause the subsequent detachment of dye from the surface of Ti3C2, resulting in the recovery of fluorescence. Because of the special structure and high fluorescence quenching efficiency of Ti3C2 MXene, the aptasensor shows a high sensitivity with a low detection limit for thrombin at 5.27 pM. Three different aptamers were compared, and the aptamer HD22 is most sensitive for detection of thrombin originated from its great specificity in the human plasma. Importantly, this Ti3C2 MXene-based FRET aptasensor can detect thrombin in human serum accurately. These results suggest that the Ti3C2 MXene-based FRET aptasensor hold a great prospect in clinical diagnosis in the real-world applications.

Electrode immobilization-free and sensitive electrochemical sensing of thrombin via magnetic nanoparticle-decorated DNA polymers

Abstract The development of sensitive and simple sensing strategies for trace proteins has received increasing attentions in the diagnosis and treatment of diseases. We describe here an electrode immobilization-free and homogeneous electrochemical method for sensitively detecting thrombin in human serums. The strategy relies on target-promoted proximity binding hybridization chain reaction (HCR) formation of magnetic nanoparticle (MNP)-decorated DNA polymers. Simultaneous binding of thrombin by two distinct aptamers increases their local concentrations and enables the proximity binding-induced strand displacement reaction, which leads to subsequent initiation of HCR between the methylene blue (MB)- and biotin-labeled hairpins into long DNA polymers. The streptavidin-modified MNPs further bind the biotin moieties to form the MNPs/DNA polymers. Subsequent accumulation of the MNPs/DNA polymers on the AuNP-deposited electrode can thus yield substantially enhanced current, due to the oxidation of the many MB labels, for highly sensitive detection of thrombin ranging from 5 pM∼50 nM with a detection limit of 1.1 pM in a simple electrode immobilization-free way. Selective interrogation of low levels of thrombin in diluted human serums was also verified, revealing its potential for convenient and ultrasensitive monitoring of a variety of protein biomarkers for disease diagnosis at the early stages.

A homogeneous strategy of target-triggered catalytic hairpin assembly for thrombin signal amplification

Sensitive and rapid detection of thrombin is play an important role in biomedical and biological analysis. In this work, tetraferrocene was synthesized and used for the construction of a homogeneous electrochemical aptasensor for sensitive and rapid detection of thrombin in human serum via coupling host-guest recognition of tetraferrocene and β-CD with catalytic hairpin assembly (CHA) signal amplification. The presence of target thrombin causes unfoldment of a large number of double hairpin probes (H1). Subsequently, two tetraferrocenes modified on H1 enter the β-CD’s cavity modified on a gold electrode (AuE) based on host-guest recognition of tetraferrocene and β-CD, which drastically amplifies the current response for thrombin detection. The prepared aptasensor shows a wide linear relationship between 1 pM and 3.125 nM, and detection limit of 0.28 pM. More importantly, this homogeneous strategy provides essential information for the manufacturing of sensors for sensitive and rapid analysis of other biomarkers.

Introduction of a thrombin sensor based on its interaction with dabigatran as an oral direct thrombin inhibitor

Dabigatran (DAB) is a direct thrombin inhibitor used for preventing blood clots and emboli after orthopedic surgery. The DAB - thrombin interaction was followed by fluorescence and UV–Vis spectroscopic methods. The binding of DAB to thrombin was also modeled by the molecular docking method. The obtained experimental results were consistent with theoretical results. The voltammetric method was also tested for DAB - thrombin interaction. Based on voltammetric findings, carbon paste electrode containing graphite powder, paraffin oil, MWCNTs, and DAB was constructed and used for thrombin monitoring after investigation of the DAB oxidation mechanism for the first time. The decrease in the linear sweep voltammetric (LSV) peak current of DAB in the presence of thrombin was utilized for the thrombin analysis. The calibration plot was linear in the concentration range of 1 to 70 nM (R2 = 0.9992) by LSV technique with a detection limit of 0.3 nM. The applicability of the proposed sensor was evaluated by the determination of thrombin in human serum as a real sample.

Aggregation-induced fluorescence of the luminol-terbium(III) complex in polymer nanoparticles for sensitive determination of thrombin.

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 430nm. Hence, thrombin can be fluorometrically determined. The detection limit of thrombin is as low as3.5 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 thestandard 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.

A “signal-on” electrochemical biosensor based on DNAzyme-driven bipedal DNA walkers and TdT-mediated cascade signal amplification strategy

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.

Target-induced structure switching of aptamers facilitates strand displacement for DNAzyme recycling amplification detection of thrombin in human serum.

To monitor the thrombin concentration under the condition of abnormal blood coagulation is of clinical significance for the diagnosis of various diseases. Here, on the basis of the aptamer structure switching induced by the target molecules and the signal amplification strategy via recycling of metal-ion dependent DNAzymes, we have established a sensitive and simple fluorescent aptasensor for detecting thrombin in human serum. The thrombin target specifically binds to the aptamer sequence and causes a corresponding conformational structure switching, which leads to the formation of a toehold sequence to facilitate the strand migration displacement reaction for the generation of functional metal-ion dependent DNAzymes. These DNAzymes further cleave the fluorescently quenched hairpin substrates cyclically to yield substantially amplified fluorescence recovery for sensitively detecting thrombin in the dynamic range from 0.01 nM to 50 nM. Such an aptasensor shows a detection limit of 6.9 pM and can achieve the monitoring of thrombin in diluted human serum with high selectivity, offering a universal sensing strategy for the construction of various sensitive and simple aptasensors to detect different biomarker molecules.

Aptamer proximity recognition-dependent strand translocation for enzyme-free and amplified fluorescent detection of thrombin via catalytic hairpin assembly

By coupling a new aptamer proximity recognition-dependent strand translocation strategy with catalytic hairpin assembly (CHA) signal amplification, we have developed a simple and sensitive method for detecting thrombin in human serums. Simultaneous binding of two engineered aptamer probes to the thrombin target significantly increases the local concentrations of the two probes and facilitates the translocation of a ssDNA strand from one of the probes to the other through toehold mediated strand displacement. Such a strand translocation leads to the generation of a ssDNA tail in the aptamer sequence for subsequent initiation of the assembly of two fluorescently quenched hairpins into many DNA duplexes via CHA. The formation of the DNA duplexes thus results in significant fluorescence recovery for amplified detection of thrombin down to 8.3 pM. The developed method is highly selective to the thrombin target against other interference proteins due to the dual recognition mode, and can be employed to monitor thrombin in human serum samples. With the advantage of simplicity, sensitivity and selectivity, this method can be a universal non-enzymatic and nanomaterial-free amplified sensing platform for detecting different protein molecules.

Ultrasensitive detection of thrombin based on MoS2-aptamer biosensors by resonance light scattering technique

Abstract Rapid and sensitive detection of thrombin has very important significance in biomedical and bioanalytical applications. In this study, a label-free and simple resonance light scattering (RLS) sensor based on MoS2 nanomaterial is developed for ultrasensitive and highly selective detection of thrombin. In the presence of thrombin, the thrombin aptamer can transform into a G-quartet structure and combine with thrombin, resulting in dissociation of the random DNA (rDNA) in the solution. Once MoS2 is introduced, the free rDNA can be adsorbed on the surface of MoS2 to form a stable DNA1–thrombin–MoS2 complex, which increases RLS signals. The sensor shows two linear ranges of 10 pM to 500 pM and 1 nM to 60 nM, and a detection limit of 0:71 pMfor detection of thrombin. Our sensor is a promising tool for clinical diagnosis of thrombin in human serum. Moreover, this work can offer new opportunities for the development of convenient sensor for detecting other proteins.

Molybdenum disulfide sphere-based electrochemical aptasensors for protein detection.

In this work, we report the development of an ultrasensitive sandwich-type electrochemical aptasensor for protein detection. The aptasensor is fabricated by using nitrogen-doped graphene oxide (N-GO) and Au nanoparticles (AuNPs) as sensing substrates, molybdenum disulfide (MoS2) spheres as the hybridization chain reaction (HCR) platform, and thrombin as the model protein. When the hybridization reaction is initiated through two biotinylated hairpin probes, vast horseradish peroxidases are immobilized on the long duplex by the biotin-avidin reaction. An electrochemical-chemical-chemical redox cycling reaction then takes place in the detection system, which contains p-dihydroxybenzene, ferrocene carboxylate and tris(2-carboxyethyl)phosphine. Benefiting from the good conductivity and high specific surface area of N-GO/AuNPs and MoS2 spheres, signal amplification of the HCR and detection system, and excellent selectivity of the aptamer and sandwich-type strategy, the proposed assay shows a wide linear range of 10 fM-0.1 nM towards thrombin with a detection limit of 27 aM (S/N = 3) along with clear distinction from different proteins. The proposed assay is successfully used to detect thrombin in human serum, which would have promising prospects for disease diagnosis and therapy.

Target-induced catalytic hairpin assembly formation of functional Y-junction DNA structures for label-free and sensitive electrochemical detection of human serum proteins

The construction of simple and highly sensitive aptasensors will significantly advance the monitoring of proteins. In this work, we have developed a convenient and label-free protein-induced catalytic hairpin assembly signal amplification approach for aptamer-based sensitive electrochemical detection of thrombin in human serums. The target thrombin binds the aptamer-containing hairpin probes immobilized on the sensor electrodes and triggers catalytic assembly of other two hairpins to form many G-quadruplex Y-junction DNA structures in situ. These G-quadruplex structures further associate with hemin to form G-quadruplex/hemin complexes, which are then subject to electrochemical measurement and generate substantially amplified current output for label-free and sensitive detection of thrombin. The developed aptasensor shows a dynamic range of 0.01–1.0nM and a detection limit of 6.0p.m for thrombin detection. The sensor can also discriminate the target thrombin against other non-target proteins and exhibits promising results for detecting thrombin in human serums. With the successful construction of the thrombin aptasensor, our signal amplified method can be potentially applied to detect other target molecules by choosing appropriate aptamer/ligand pairs.

Target-triggered catalytic hairpin assembly and TdT-catalyzed DNA polymerization for amplified electronic detection of thrombin in human serums

Specific and sensitive detection of protein biomarkers is of great importance in biomedical and bioanalytical applications. In this work, a dual amplified signal enhancement approach based on the integration of catalytic hairpin assembly (CHA) and terminal deoxynucleotidyl transferase (TdT)-mediated in situ DNA polymerization has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. The presence of the target thrombin leads to the unfolding and capture of a significant number of hairpin signal probes with free 3′-OH termini on the sensor electrode. Subsequently, TdT can catalyze the elongation of the signal probes and formation of many G-quadruplex sequence replicates with the presence of dGTP and 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 completely label-free fashion. The sensor shows a linear range of 0.5pM–10.0nM and a detection limit of 0.12pM for thrombin. Moreover, the developed sensor can selectively discriminate the target thrombin against other non-target proteins and can be employed to monitor thrombin in human serum samples.

Proximity Binding and Metal Ion-Dependent DNAzyme Cyclic Amplification-Integrated Aptasensor for Label-Free and Sensitive Electrochemical Detection of Thrombin.

Thrombin plays important roles for the diagnosis of neurodegenerative and cardiovascular diseases. By integrating proximity binding-induced strand displacement and metal ion-dependent DNAzyme recycling amplification, we demonstrate here the development of a simple and sensitive strategy for the detection of thrombin in human serums. The binding of the two distinct aptamers to the thrombin targets increases the local concentration of the aptamers and facilitates the release of the enzymatic sequences through proximity binding-induced strand displacement. The liberated enzymatic sequences further hybridize with the G-quadruplex containing and hairpin-structured substrate sequences on the sensor electrode to form the metal-ion dependent DNAzymes. Subsequently, the metal ions catalyze the cleavage of the substrate sequences to unlock the G-quadruplex forming sequences and to release the enzymatic sequences to trigger another cleavage cycle. Such metal ion-dependent DNAzyme recycling amplification leads to the formation of many active G-quadruplex forming sequences, which associate with hemin to form G-quadruplex/hemin complexes on the electrode surface. Direct electron transfer of hemin to the electrode during the potential scan can thus generate significantly amplified current for sensitive detection of thrombin at the low picomolar level. The work demonstrated here can thus offer new opportunities for the development of convenient signal amplification strategies for detecting various protein targets.