Thrombin Recognition Research Articles

Binding-induced activation of CRISPR-Cas12a using PAM-engineered toehold switch and DNA tile architectures for enhanced detection of thrombin

This study presents a novel biosensor that integrates CRISPR-Cas12a technology with DNA tile-based architectures to enhance thrombin detection. Utilizing PAM-engineered toehold switches, the biosensor dynamically activates the CRISPR-Cas12a system upon thrombin recognition, significantly improving both sensitivity and specificity. The label-free electrochemical detection method offers rapid, cost-effective analysis, making it ideal for real-time clinical applications. The biosensor achieved an impressive detection limit of 103 aM, surpassing traditional methods and setting a new standard for thrombin assays. The biosensor's robust performance in complex biological samples, such as diluted human serum, demonstrates its reliability for clinical diagnostics. This is particularly important for the early diagnosis and management of thrombotic and coagulation-related disorders. By combining advanced genetic engineering with traditional electrochemical techniques, the biosensor not only optimizes detection but also expands its potential applications across various biomedical fields. Overall, this biosensor provides a powerful and innovative tool for thrombin detection, offering enhanced diagnostic accuracy, faster patient management, and potential cost savings in healthcare. Its superior performance and adaptability highlight its promise for broader adoption in clinical diagnostics and other biosensing applications.

An electrochemical biosensor for the amplification of thrombin activity by perylene-mediated photoinitiated polymerization

BackgroundThrombin, a coagulation system protease, is a key enzyme involved in the coagulation cascade and has been developed as a marker for coagulation disorders. However, the methods developed in recent years have the disadvantages of complex operation, long reaction time, low specificity and sensitivity. Meanwhile, thrombin is at a lower level in the pre-disease period. Therefore, to accurately diagnose the disease, it is necessary to develop a fast, simple, highly sensitive and specific method using signal amplification technology. ResultsWe designed an electrochemical biosensor based on photocatalytic atom transfer radical polymerization (photo-ATRP) signal amplification for the detection of thrombin. Sulfhydryl substrate peptides (without carboxyl groups) are self-assembled to the gold electrode surface via Au–S bond and serve as thrombin recognition probes. The substrate peptide is cleaved in the presence of thrombin to generate –COOH, which can form a carboxylate-Zr(IV)-carboxylate complex via Zr(IV) and initiator (α-bromophenylacetic acid, BPAA). Subsequently, an electrochemical biosensor was prepared by introducing polymer chains with electrochemical signaling molecules (ferrocene, Fc) onto the electrode surface by photocatalytic (perylene, Py) mediated ATRP using ferrocenylmethyl methacrylate (FMMA) as a monomer. The concentration of thrombin was evaluated by the voltammetric signal generated by square wave voltammetry (SWV), and the result showed that the biosensor was linear between 1.0 ng/mL ∼ 10 fg/mL, with a lower detection limit of 4.0 fg/mL (∼0.1 fM). Moreover, it was shown to be highly selective for thrombin activity in complex serum samples and for thrombin inhibition screening. SignificanceThe biosensor is an environmentally friendly and economically efficient strategy while maintaining the advantages of high sensitivity, anti-interference, good stability and simplicity of operation, which has great potential for application in the analysis of complex samples.

A specific fluorescence resonance energy quenching–based biosensor for measuring thrombin activity in whole blood

BackgroundAt sites of vessel injury, thrombin acts as the central mediator of coagulation by catalyzing fibrin clot formation and platelet activation. Thrombin generation is most frequently measured in plasma samples using small-molecule substrates; however, these have low specificity for thrombin and limited utility in whole blood. Plasma assays are limited because they ignore the hemostatic contributions of blood cells and require anticoagulation and the addition of supraphysiological concentrations of calcium. ObjectivesTo overcome these limitations, we designed and characterized a fluorescence resonance energy quenching–based thrombin sensor (FTS) protein. MethodsThe fluorescence resonance energy quenching pair of mAmetrine and tTomato, separated by a thrombin recognition sequence, was developed. The protein was expressed using Escherichia coli, and purity was assessed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The cleavage of FTS was monitored by fluorescence using excitation at 406 nm and emission at 526 nm and 581 nm. ResultsCompared with small-molecule substrates, the FTS demonstrated high specificity for thrombin; it is not cleaved by thrombin or inhibited by α2-macroglobulin and interacts with thrombin’s anion-binding exosite I. The FTS can effectively measure thrombin generation in plasma and in finger-prick whole blood, which allows it to be developed into a point-of-care test of thrombin generation. The FTS does not inhibit standard thrombin-generation assays. Lastly, FTS-based thrombin generation in nonanticoagulated finger-prick blood was delayed but enhanced compared with that in citrated plasma. ConclusionThe FTS will broaden our understanding of thrombin generation in ways that are not attainable with current methods.

Aptasensors based on silicon nanowire field-effect transistors for electrical detection of thrombin

Arrays of silicon nanowire field-effect transistors (Si NWFETs) were built to detect thrombin (a model biomarker) electrically. The Si NWFETs were created using a conventional top-down CMOS process, allowing them to be co-integrated with CMOS readout circuits in the future. EHTES organosilane was then used to graft aptamer probes onto the HfO2 gate oxide of Si nanowires. We investigated the influence of aptamer grafting and thrombin recognition on the electrical transfer capabilities of Si NWFET aptasensors in details. Our technique was evaluated on a significant number of Si NWFETs, including two distinct chips with 30 aptasensors apiece. According to the findings, aptamer grafting increased the threshold voltage by a positive range of +28.8 mV to +87.7 mV, depending on the aptasensor employed. Thrombin identification, on the other hand, resulted in a negative shift of the threshold voltage between −26.6 and − 23.8 mV. These opposing voltage shifts coincide with the aptamer probes' and thrombin molecules' electric charges, respectively. These findings provide unique demonstration of Si NWFETs manufactured utilizing typical top-down CMOS processing methods, allowing these devices to be used in various biomedical and biosensing applications.

Interface Engineering of "Clickable" Organic Electrochemical Transistors toward Biosensing Devices.

"Clickable" organic electrochemical transistors (OECTs) allow the reliable and straightforward functionalization of electronic devices through the well-known click chemistry toolbox. In this work, we study various aspects of the click chemistry-based interface engineering of "clickable" OECTs. First, different channel architectures are investigated, showing that PEDOT-N3 films can properly work as a channel of the transistors. Furthermore, the Cu(I)-catalyzed click reaction of ethynyl-ferrocene is studied under different reaction conditions, endowing the spatial control of the functionalization. The strain-promoted and catalyst-free cycloaddition of a dibenzocyclooctyne-derivatized poly-l-lysine (PLL-DBCO) is also performed on the OECTs and validated by a fiber optic (FO)-SPR setup. The further immobilization of an azido-modified HD22 aptamer yields OECT-based biosensors that are employed for the recognition of thrombin. Finally, their performance is evaluated against previously reported architectures, showing higher density of the immobilized HD22 aptamer, and originating similar KD values and higher maximum signal change upon analyte recognition.

A Novel Substrate for Assessing Thrombin Activity in Whole Blood

At sites of vessel injury, thrombin acts as the central mediator of coagulation through catalyzing fibrin clot formation and platelet activation. Currently, thrombin generation is measured using small molecule substrates. These substrates are limited in that they detect both active thrombin and thrombin bound to its inhibitor α2-macroglobulin (α2-M) and have limited utility in whole blood (Ninivaggi et al. Clin Chem 2012). In addition, these substrates contain short thrombin recognition sequences ranging from 3-4 amino acids and are unable to form additional exosite interactions with thrombin, which limit their specificity (Gallwitz et al. Plos One 2012). Furthermore, they are unfeasible for in vivo use because their hydrophobicity allows them to enter cells. However, plasma assays are limited because they ignore the hemostatic contributions of red blood cells, platelets and leukocytes and require anticoagulation and the addition of exogenous coagulation activators (Swystun & Liaw. Blood 2016 & do Amaral et al. Stem Cells Int 2016). Thus, we have developed a fluorescence resonance energy transfer (FRET)-based thrombin sensor to overcome these limitations. We cloned and expressed the construct encoding the FRET-based thrombin substrate in Escherichia coli and purified the substrate using affinity chromatography. We confirmed using Sodium dodecyl sulfate−polyacrylamide gel electrophoresis that the substrate was successfully purified and that thrombin cleavage of the purified substrate changes its FRET efficiency by monitoring its spectrum of emission. To determine whether the FRET-based thrombin substrate can form interactions with thrombin's anion binding exosites and assess its potential for in vivo use, we measured the kinetics parameters of the substrate for thrombin in the presence of exosite inhibitors and thrombin's exosite variants as well as mouse thrombin and plasmin. We then compared the resulting kinetics parameters with commercially available thrombin substrates S2238 and technothrombin. We show that unlike S2238 and technothrombin, our FRET-based thrombin sensor forms strong interactions with thrombin's anion binding exosite I and displays preference for free thrombin compared to thrombomodulin (TM) bound thrombin which implied its preference for procoagulant thrombin rather than anticoagulant TM bound thrombin that activates protein C. Furthermore, our FRET-based thrombin substrate has potential to be used in mouse models because it can be efficiently cleaved by mouse thrombin and is relatively plasmin resistant. To assess the specificity of the FRET-based thrombin substrate for thrombin, we compared the rate of hydrolysis of the substrate by thrombin and several other enzymes in circulation and compared the results to S2238 and technothrombin. Our results indicated that when compared to commercially available thrombin substrates, our FRET-based thrombin substrate is highly specific for thrombin. Since the thrombin inhibitor α2-M is present in circulation, we also compared the rate of hydrolysis of the 3 substrates for α2-M bound thrombin and free thrombin. Unlike S2238 and technothrombin, our FRET-based thrombin substrate is uncleaved by α2-M bound thrombin. The key properties of the FRET-based thrombin substrate in comparison to S22238 and technothrombin are summarized in Table 1. We then measured thrombin generation in plasma using our FRET-based thrombin substrate and showed that it is sensitive to direct oral anticoagulants in ways that are consistent with literature findings where thrombin generation was measured using technothrombin (Sivaraja et al. Plos One 2018 & Douxfils et al. Thromb Res 2012). We also confirmed that the FRET-based thrombin substrate itself is not a coagulation inhibitor since its presence did not alter thrombin generation parameters measured using technothrombin. Lastly, we confirmed that the FRET-based thrombin substrate can measure thrombin generation in whole blood since it successfully produced thrombin generation curves. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Controlled Decomposable Hydrogel Triggered with a Specific Enzyme.

In this study, superabsorbent polyelectrolyte hydrogels were synthesized by cross-linking a nondegradable poly (allylamine hydrochloride) (PAH) and a recombinant protein with a specific enzymatic cleavage site. The recombinant protein was produced by E. coli with the pET-32b(+) plasmid, which is featured with the thioredoxin (Trx) gene containing a thrombin recognition site and a T7/lac hybrid promoter for high expression of recombinant protein. The swelling test shows that the composite hydrogel still maintained a high swelling ratio to 900% when 15% recombinant protein was cross-linked with PAH. The degradation test shows that such a PAH composite hydrogel could be decomposed by the addition of specific enzyme thrombin, which might lead to new biomedical applications of hydrogels needed to be decomposable by specific time not determined by the time period.

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.

Charge-Transfer Interactions Stabilize G-Quadruplex-Forming Thrombin Binding Aptamers and Can Improve Their Anticoagulant Activity.

In the search for optimized thrombin binding aptamers (TBAs), we herein describe the synthesis of a library of TBA analogues obtained by end-functionalization with the electron-rich 1,5-dialkoxy naphthalene (DAN) and the electron-deficient 1,8,4,5-naphthalenetetra-carboxylic diimide (NDI) moieties. Indeed, when these G-rich oligonucleotides were folded into the peculiar TBA G-quadruplex (G4) structure, effective donor–acceptor charge transfer interactions between the DAN and NDI residues attached to the extremities of the sequence were induced, providing pseudo-cyclic structures. Alternatively, insertion of NDI groups at both extremities produced TBA analogues stabilized by π–π stacking interactions. All the doubly-modified TBAs were characterized by different biophysical techniques and compared with the analogues carrying only the DAN or NDI residue and unmodified TBA. These modified TBAs exhibited higher nuclease resistance, and their G4 structures were markedly stabilized, as evidenced by increased Tm values compared to TBA. These favorable properties were also associated with improved anticoagulant activity for one DAN/NDI-modified TBA, and for one NDI/NDI-modified TBA. Our results indicated that TBA pseudo-cyclic structuring by ad hoc designed end-functionalization represents an efficient approach to improve the aptamer features, while pre-organizing and stabilizing the G4 structure but allowing sufficient flexibility to the aptamer folding, which is necessary for optimal thrombin recognition.

The Modified Recombinant Proinsulin: A Simple and Efficient Way to Produce Insulin Glargine in E. coli

Background: Recombinant insulin glargine, a long-acting analog of insulin, is expressed as proinsulin in the host cell and, after purification and refolding steps, is processed to mature insulin by using trypsin and carboxypeptidase B. Because of several internal residues of arginine and lysine in the proinsulin B and C chains, several unwanted products are formed after treatment with these enzymes. To overcome this problem, we introduced three thrombin recognition sites into the proinsulin encoding sequence. Materials and methods: After the design, the modified proinsulin encoding sequence containing the 5′ His-Tag tail and three thrombin recognition sites located between the His-Tag and B chain, B and C chains, and C and A chains, respectively, was synthesized by overlap extension Polymerase Chain Reaction (PCR) using seven specific primers in multiple sequential PCR reactions. The final amplified fragment was cloned in the pGEM-5zf vector by the EcoRV enzyme. After sequencing, the modified proinsulin encoding sequence was subcloned into the pET-26b(+) vector using NdeI and XhoI enzymes. Finally, the modified proinsulin was expressed in E. coli BL-21(DE3) by induction with Isopropyl β-d-1-thiogalactopyranoside (IPTG). Results: The accuracy of the synthesized modified proinsulin sequence was confirmed by DNA sequencing. The modified proinsulin cloning was evaluated by PCR with specific primers and digestion with specific restriction enzymes. In this study, the modified proinsulin protein was expressed up to 40%. The modified proinsulin protein expression was assessed using sodium dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and western blotting. Conclusions: This modified proinsulin can be used to easily and efficiently produce insulin glargine without any impurities after processing with thrombin in one step in a nickel chromatographic column.

Structure of the platelet glycoprotein Ib receptor in complex with a novel antithrombotic agent

AbstractAgkisacucetin, a snake C-type lectin-like protein isolated from the venom of Deinagkistrodon acutus (formerly Agkistrodon acutus), is a novel antithrombotic drug candidate in phase 2 clinical trials. Agkisacucetin specifically recognizes the platelet surface receptor glycoprotein Ib α chain (GPIbα) to block GPIb and von Willebrand factor (VWF). In this study, we solved the crystal structure of the GPIbα N-terminal domain (residues 1-305) in complex with agkisacucetin to understand their molecular recognition mechanism. The crystal structure showed that agkisacucetin primarily contacts GPIbα at the C-terminal part of the conserved leucine-rich repeat (LRR) domain (LRR-6 to LRR-8) and the previously described “β-switch” region through the β chain. In addition, we found that agkisacucetin α chain contacts part of the GPIbα C-terminal peptide after the LRR domain through complementary charge interactions. This C-terminal peptide plays a key role in GPIbα and thrombin recognition. Therefore, our structure revealed that agkisacucetin can sterically block the interaction between the GPIb receptor and VWF and thrombin proteins to inhibit platelet function. Our structural work provides key molecular insights into how an antithrombotic drug candidate recognizes the GPIb receptor to modulate platelet function to inhibit thrombosis.

Monoclonal Antibody HlyIIC‑15 to C-End Domain HlyII B. cereus Interacts with the Trombin Recognition Site.

Among the panel of monoclonal antibodies to the recombinant protein HlyIICTD Bacillus cereus an antibody was found capable of forming an immune complex with a thrombin recognition region, the amino acid sequence of which is located inside the recombinant HlyIICTD. Localization of the epitope was carried out using peptide phage display methods, as well as enzyme immunoassay and immunoblotting for interaction with recombinant proteins, either containing or not containing individual components HlyIICTD. The identified epitope is located in the region of the thrombin site and retains the ability to interact with the antibody after the proteolyotic attack of the protein by thrombin.

Sulfotyrosine-Mediated Recognition of Human Thrombin by a Tsetse Fly Anticoagulant Mimics Physiological Substrates

Despite possessing only 32 residues, the tsetse thrombin inhibitor (TTI) is among the most potent anticoagulants described, with sub-picomolar inhibitory activity against thrombin. Unexpectedly, TTI isolated from the fly is 2000-fold more active and 180Da heavier than synthetic and recombinant variants. We predicted the presence of a tyrosine O-sulfate post-translational modification of TTI, prompting us to investigate the effect of the modification on anticoagulant activity. A combination of chemical synthesis and functional assays was used to reveal that sulfation significantly improved the inhibitory activity of TTI against thrombin. Using X-ray crystallography, we show that the N-terminal sulfated segment of TTI binds the basic exosite II of thrombin, establishing interactions similar to those of physiologic substrates, while the C-terminal segment abolishes the catalytic activity of thrombin. This non-canonical mode of inhibition, coupled with its potency and small size, makes TTI an attractive scaffold for the design of novel antithrombotics.

A Rapid and Ultrasensitive Thrombin Biosensor Based on a Rationally Designed Trifunctional Protein.

Rapid and sensitive detection of thrombin is imperative for the early diagnosis, prevention, and treatment of thrombin-related diseases. Here, an ultrasensitive and rapid thrombin biosensor is developed based on rationally designed trifunctional protein HTs, comprising three functional units, including a far-red fluorescent protein smURFP, hydrophobin HGFI, and a thrombin cleavage site (TCS). smURFP is used as a detection signal to eliminate any interference from the autofluorescence of sample matrix to increase detection sensitivity. HGFI serve as an adhesive unit to allow rapid immobilization of HTs on a multiwall plate. The TCS linking HGFI and smURFP function as a sensing element to recognize and detect thrombin. HTs immobilization is symmetrically optimized and characterized. Thrombin assay reveals the specific recognition of active thrombin in samples and the hydrolysis of the immobilized HTs, resulting in a decrease in the fluorescence intensity of the sample in a thrombin concentration-dependent manner. The limit of detection (LOD) is as low as 0.2 am in the serum. To the authors' knowledge, this is the lowest LOD ever reported for any thrombin biosensor. This study sheds light on the engineering of multifunctional proteins for biosensing.

D-shaped plastic optical fibre aptasensor for fast thrombin detection in nanomolar range

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.

P3. Abstract Title: A Point-of-Care Assay for Measuring Thrombin Activity in Finger-Prick Whole Blood

At sites of vessel injury, thrombin acts as the central mediator of coagulation through catalyzing fibrin clot formation and platelet activation. Thus, numerous commercially available small molecule chromogenic and fluorogenic substrates have been developed to evaluate thrombin activity. These substrates are limited in that they detect both active thrombin and thrombin bound to its inhibitor α2-macroglobulin, have limited specificity for thrombin and can only be used in plasma samples because of signal quenching. But plasma is not reflective of physiological settings because of the lack of red blood cells which can promote coagulation. Furthermore, assays of plasma require anticoagulation using chelating agents followed by recalcification using supraphysiological concentrations of calcium to initiate coagulation. In addition to calcium ions, chelating agents used as anticoagulants also sequester magnesium and zinc ions which alter coagulation. Thus, divalent cations present in physiological settings are removed in plasma assays. Thus, we designed a novel thrombin substrate that allows for thrombin activity assessment in whole blood. The thrombin substrate consists of a mAmetrine and tTomato fluorescence resonance energy transfer (FRET) pair linked by a thrombin recognition sequence (LVPRGVNL). In addition, the thrombin recognition sequence is flanked by short, antiparallel beta sheets that are linked to the FRET pair through a zipper made of arginine and glutamic acid residues. This design allows it to accommodate long thrombin recognition sequences that enhance specificity and ignore inactive thrombin bound to α2-macroglobulin. Thrombin activity is assessed by evaluating for the loss of FRET. Expressed in E.coli, the thrombin substrate contains two hexahistidine tags on its N-terminus and a calmodulin binding protein tag on its C-terminus. Purification based on these tags ensures that only intact substrate is purified; the tags are then removed by a specific bacterial protease. The purity and integrity of the substrate was confirmed using SDS-PAGE and FRET efficiency assessment. The purified substrate demonstrated specificity for thrombin over factor Xa and activated protein C in buffer containing calcium. To determine whether this thrombin substrate can be used as a point-of-care test of thrombin generation, blood (20 µL) obtained from separate finger pricks from the same healthy donor was incubated with the thrombin substrate and reagents (vehicle, APTT reagent, dabigatran) and evaluated in a portable fluorometer. The fluorometer (StellarCase-Fluoro analyzer) uses a 405 nm LED as excitation, a diffraction grating, a PC tablet and is powered by a lithium battery and enclosed in a rugged case. The change in fluorescence emission was analyzed with Excel to obtain a thrombin generation curve. Compared to blood alone, APTT reagent significantly shortened the time to peak thrombin generation. When dabigatran was added to the blood simultaneously with APTT reagent, the time to peak thrombin generation was increased. These results are consistent with published literature using plasma assays that require larger amounts of blood and centrifugation, suggesting that our substrate can be used as a point-of-care thrombin generation test using a single drop of blood. In conclusion, we have developed a thrombin sensor that has specificity for thrombin compared to related proteases and can measure thrombin activity in whole blood. This sensor is currently being developed into a point-of-care test of anticoagulants and has potential to help our understanding of hemostasis through evaluating thrombin generation in whole blood in varied scenarios.

Electrochemical aptasensor based on conductive supramolecular polymer hydrogels for thrombin detection with high selectivity

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

Electrochemical determination of thrombin with molecularly imprinted polymers and multiwalled carbon nanotubes

The preparation and application of reagentless electrochemical thrombin molecularly imprinted sensors were studied using multiwalled carbon nanotubes as sensitivity-enhanced materials. The molecularly imprinted polymer film was prepared by the electropolymerization of o-phenylenediamine with thrombin as the template molecule onto the surface of multiwalled carbon nanotubes modified glassy carbon electrode. After removing thrombin, the poly-o-phenylenediamine molecularly imprinted film was obtained with specific recognition for thrombin. Using the poly-o-phenylenediamine molecularly imprinted polymers as the electron probe, the electrochemical molecularly imprinted sensor was fabricated for the detection of the protein thrombin. Under optimized experimental conditions, the sensor exhibited a good linear response from 10.0 fg/mL to 1.0 μg/mL for thrombin, with correlation coefficient 0.999 and a low detection limit of 1.7 fg/mL. The fabricated molecularly imprinted sensor can be applied to the detection of thrombin in actual sample bovine serum with satisfactory results.

Click chemistry-based aptasensor for highly sensitive electrochemical detection of thrombin

In this study, a click chemistry-based electrochemical aptasensor is reported for the simple and fast detection of thrombin. Briefly, the thiol-terminated aptamer1 (Apt1) was first self-assembled on a gold electrode for the specific recognition of thrombin in the following step. After this, the azido-labeled aptamer2 (Apt2) was attached to the electrode via the formation of a sandwich structure with Apt1 and thrombin. The azido terminal was designed for the subsequent labeling of Apt2 with ethynylferrocene via electrochemically mediated Cu(I)-catalyzed azide–alkyne cycloaddition (eCuAAC), in which the active Cu(I) was electrochemically generated in situ. Finally, the quantitatively labeled ethynylferrocene probes could be detected on the basis of differential pulse voltammetry (DPV) for the electrochemical detection of thrombin. Under optimal conditions, this aptasensor could detect thrombin down to 84 pM with a good linear response over the range from 0.1 nM to 1000 nM. In addition, it exhibited excellent specificity in the quantitative detection of thrombin and also showed good detection reliability in serum samples.

Engineering Permissive Insertion Sites in the Bacteriophage Phi29 DNA-Linked Terminal Protein.

Many different DNA delivery vehicles have been developed and tested, all with their advantages and disadvantages. The bacteriophage phi29 terminal protein (TP) is covalently linked to the 5’ ends of the phage genome during the DNA replication process. Our approach is to utilize this TP as a platform to incorporate different protein or peptide modules that can target the DNA to the interior of the cell, to the nucleus, or even to subcellular compartments. In order to be able to insert different peptide modules on the TP sequence to endow it with desired functions and/or eliminate unwanted regions of the protein, we have carried out a transposition screening to detect insertion-permissive points on the sequence of the TP. We report the functional characterization of 12 insertion mutants of the TP, and the identification of one site at position 38 that allows the insertion of peptides up to 17 amino acids in length while maintaining the ability of the TP to support DNA amplification in vitro. A protein with one insertion at that position containing a cysteine residue, a linker, and a thrombin recognition site was purified and its amplification activity was optimized.