Heparin Sensor Research Articles

Ratiometric fluorescence sensor for protamine and heparin based on N,P‐doped carbon dots and Eosin Y

AbstractA novel ratiometric fluorescent assay was proposed for protamine and heparin based on blue‐emitting N,P‐doped carbon dots (N,P‐CDs) and green‐emitting Eosin Y. The N,P‐CDs were prepared using arginine as carbon and nitrogen sources according to hydrothermal method with favorable luminescence intensity, photostability, and good reproducibility. And the N,P‐CDs were used as internal reference fluorescence molecules. The anionic Eosin Y was used as fluorescence signal molecule. Protamine with positive group could interact with Eosin Y through electrostatic effect, leading to the fluorescence quenching of Eosin Y. Upon the addition of heparin, heparin was combined with protamine with stronger affinity through electrostatic interactions and hydrogen bonds, which preventing the bonding of protamine with Eosin Y and obvious fluorescence emissions of Eosin Y at 538 nm could be observed. Meanwhile, the fluorescence signal of N,P‐CDs at 420 nm remained stable during the whole process. A good linear range between the fluorescence emission intensity ratio of I538/I420 was obtained for protamine from 0.01 to 40 μg/mL with a low detection limit of 5.6 ng/mL, and heparin from 0 to 35 μg/mL with a low detection limit of 2.5 ng/mL. Moreover, the method was further applied to detection of heparin with satisfactory results in fetal bovine serum samples.

Ultra-sensitive fiber optic heparin sensor based on phase-modulated configuration with electrostatic self-assembly

Ultra-sensitive fiber optic heparin sensor based on phase-modulated configuration with electrostatic self-assembly

A highly efficient and selective optical detection method for Heparin that works in 100% human serum

Sensing Heparin in 100% serum matrix is a challenging task and is rarely reported in literature. Herein, we present a tetracationic porphyrin based probe that detects Heparin in 100% serum matrix, both, colorimetrically and fluorometrically, with a clinically relevant LOD. The probe also shows high selectivity towards Heparin when compared with its structurally related analogs, chondroitin sulfate and hyaluronic acid along with other relevant analytes. The porphyrin probe-Heparin complex could also be used for the detection of Protamine, which is the only clinically approved antitoxin for Heparin overdose. The easy commercial availability of the probe coupled with its other desirable attributes is expected to significantly improve its prospect in Heparin sensor applications and Heparin related bio-chemical research.

The heparinase-linked differential time method allows detection of heparin potency in whole blood with high sensitivity and dynamic range

Anticoagulation therapy with heparin is an effective treatment against thrombosis. Heparin tends to cause spontaneous bleeding and requires regular monitoring during therapy. Most high-sensitivity heparin sensors have focused on the concentration detection in clarified buffer solution. However, the pharmacodynamics of heparin vary depending on individual patient or disease, while potency detection with high sensitivity and dynamic range outperforms concentration detection in clinical diagnosis. In this study, a novel heparinase-linked differential time (HLDT) method was established with a two-zone of Graphene modified Carbon (GR-C) sensor, which was utilized to evaluate heparin potency in whole blood. It was based on electrochemical measurement of clotting time shifting associated with presence or absence of heparinase. Heparinase inhibits the anticoagulant ability of heparin by forming a heparin–antithrombin–thrombin complex during coagulation. And the intensity and peak time of electrochemical current were associated with thrombin activity and clotting on the electrode. The results demonstrated that the sensor had high selectivity for heparin potency in 10 μL of whole blood with a detection limit of 0.1 U/mL, and the linear detection range was 0.1–5 U/mL. The coefficient of variation (CV) of the peak time was less than 5%, and linear correlation between the GR-C sensor and the TEG-5000 instrument was 0.987. Thus, the HLDT method has better clinical application due to its good repeatability, high sensitivity and wide range in heparin potency evaluation.

Corrigendum to “Blood heparin sensor made from a paste electrode of graphite particles grafted with molecularly imprinted polymer” [Sens. Actuators B: Chem. 259 (2018) 455–462

Corrigendum to “Blood heparin sensor made from a paste electrode of graphite particles grafted with molecularly imprinted polymer” [Sens. Actuators B: Chem. 259 (2018) 455–462

Au nanoparticles as label-free competitive reporters for sensitivity enhanced fiber-optic SPR heparin sensor

A label-free Au NPs-enhanced surface plasmon resonance (SPR) sensor was developed for the ultrasensitive detection of heparin based on competitive adsorption behavior of heparin and Au NPs on the poly (dimethyl-diallylammonium chloride) (PDDA)-modified optical fiber surface and the corresponding change in the resonance wavelength of SPR. Due to the high affinity between heparin and PDDA, the present senor shows good analytical performance with respect to heparin detection. Two obvious advantages of the proposed heparin sensor over other reported methods are: its much wider linear concentration range (10−6-10−10 g/mL) and lower limit of detection (0.0257 ng/mL). The analysis of heparin in serum demonstrated that the present sensor exhibited high sensitivity and selectivity. It should be noted that the sensing strategy takes advantage of a portable fiber-optic SPR sensing system and avoids the need for complex processes for labeled-Au NPs, and thus the present sensor promises to be a practical tool for the point-of-care monitoring of heparin.

Fabrication of Orange-Emitting Organic Nanoparticle-Protamine Conjugate: Fluorimetric Sensor of Heparin.

Among the diverse sensing techniques, fluorimetric detection dominates over the other methods because of its rapid signaling, high selectivity and sensitivity, and operational simplicity. This present article delineates fabrication of a fluorescent organic nanoparticle-protamine (FONP-Pro) conjugate for selective and sensitive detection of heparin simply by exploitation of the aggregation-induced emission (AIE) property of the FONPs. Naphthalene diimide-based bola-type amphiphilic molecules (NDI-1) comprise a naphthyl residue and a 3-aminopyridyl unit at both terminals, forming organic nanoparticles in a dimethyl sulfoxide-water binary solvent mixture, and exhibited AIE through excimer formation. The presence of naphthyl residue in the molecular backbone facilitates the intramolecular charge transfer to generate orange-emitting (λem = 594 nm) AIE-luminogen (AIE-gen). The aminopyridine residues within NDI-1 induced negative surface charge on NDI-1 FONPs, which facilitated interaction with positively charged protamine (Pro) to construct FONP-Pro conjugates. Formation of this NDI-1 FONP-Pro conjugate through the interaction between Pro and FONP drastically reduced the orange emission intensity (fluorescence off) of the AIE-gens. Interestingly, addition of heparin to this FONP-Pro conjugate turned on the fluorescence signal of FONPs through unwinding of the Pro from the FONP surface because of a strong binding affinity between heparin and Pro. Formation of the FONP-Pro conjugate and fluorimetric sensing of heparin was investigated by monitoring the change in emission behavior of NDI-1 FONPs. Also, the heparin-sensing was found to be highly selective against many other biomolecules including proteins, enzymes, and DNA. Hence, a selective and efficient heparin sensor (FONP-Pro) was developed having a limit of detection of 12 nM simply by utilizing the fluorescence "turn-off" and "turn-on" mechanism of NDI-1 FONP.

An efficient J-aggregate based fluorescence turn-on and ratiometric sensor for heparin

Heparin is the second most widely used natural drug and an extensively employed blood anticoagulant worldwide. Construction of fluorescent sensors for Heparin has remained heavily dependent on the complex and time-consuming laborious synthetic efforts. However, reports on the direct usage of in-expensive commercially available probe molecule, for sensitive and selective fluorimetric sensing of Heparin, which can greatly simplify the synthetic efforts, has been limited and remains highly desirable. Herein, we report a very sensitive and selective fluorescence turn-on sensor for Heparin, which utilizes a commercially available organic dye molecule, Pseudoisocyanine. The supramolecular interaction between the Pseudoisocyanine and Heparin leads to the J-aggregate formation of Pseudoisocyanine on the surface of Heparin, with an exceptional fluorescence enhancement of ∼400 fold. The J-aggregates of Pseudoisocyanine also display remarkable changes in the absorption features, in the presence of Heparin, which provides a unique advantageous feature of dual sensing of Heparin, by colorimetry and fluorimetry. Pseudoisocyanine also displays high selectivity towards Heparin over its structurally related analogs Chondroitin sulphate and Hyaluronic acid, and other analytes, with a detection limit of 2 nM. More importantly, our sensor system shows good performance in the serum samples, a complex biological media. We further demonstrate that, the interaction of Heparin with its only medically approved antidote, Protamine can also be monitored using Pseudoisocyanine J-aggregates. Thus, our assay is sensitive and selective, rapid, simple, and inexpensive, which can promote the Heparin related biochemical and biomedical research.

A cellulose-based photoacoustic sensor to measure heparin concentration and activity in human blood samples

Heparin is an indispensable drug in anticoagulation therapy but with a narrow therapeutic window, which dictates regular testing and dose adjustment. However, current monitoring tools have a long turnaround time or are operator intensive. In this work, we describe a cellulose-based photoacoustic sensor for heparin. The sensors have a turnaround time of 6 min for whole blood samples and 3 min for plasma samples regardless of heparin concentration. These sensors have a limit of detection of 0.28 U/ml heparin in human plasma and 0.29 U/ml in whole blood with a linear response (Pearson's r = 0.99) from 0 to 2 U/ml heparin in plasma and blood samples. The relative standard deviation was < 12.5% in plasma and < 17.5% in whole blood. This approach was validated with heparin-spiked whole human blood and had a linear correlation with the activated partial thromboplastin time (aPTT) (r = 0.99). We then studied 16 sets of clinical samples—these had a linear correlation with the activated clotting time (ACT) (Pearson's r = 0.86, P < 0.0001). The photoacoustic signal was also validated against the cumulative heparin dose (Pearson's r = 0.71, P < 0.0001). This approach could have applications in bed-side heparin assays for continuous heparin monitoring.

The cationic dye basic orange 21 (BO21) as a potential fluorescent sensor.

The present study investigates the fluorescence properties of BO21 and their dependence on various intracellular conditions. The results obtained with cell-free solutions indicate that the influences of pH and temperature on the fluorescence spectra are negligible, while viscosity, various proteins and heparin have significant influence. In the presence of heparin, a red shift of the emission spectrum (from 515 to 550 nm) is observed, suggesting that this shift cannot simply be attributed to electrostatic interaction between BO21 and the polyanionic heparin, but rather to aggregation of BO21 on the polyanion. In water, the quantum yield of BO21 was found to be 1000 times lower than that of fluorescein, yet surprisingly its fluorescence polarization (FP) was found to be about 40 times higher (FP = 0.470), even though both have similar structures and molecular weights. A thorough analytical and experimental investigation of these phenomena indicates that the very high FP of BO21 in water is a consequence of its very short lifetime. However, upon the addition of heparin to aqueous BO21, the fluorescence lifetime (FLT) of BO21 increases from τ = 10.35 to 56.5 ps, with a consequent dramatic drop in its fluorescence polarization from 0.470 to 0.230. From its behavior in aqueous glycerol solution, it is hypothesized, with support from theoretical calculations, that BO21 is a molecular rotor. Using these properties, BO21 may be a good candidate as a sensor, for example, of heparin levels in blood or of intracellular viscosity.

A facile four-armed AIE fluorescent sensor for heparin and protamine

In this research a four-armed amino-tailored tetraphenylethene TPE-NH2 was synthesized, and it could detect heparin (Hep) quantitatively, primarily promoted by the electrostatic interactions between its positively charged amino groups and the sulfates, carboxylates of heparin. On account of the aggregation-induced emission (AIE) nature of tetraphenylethene backbone, the aggregation restricted the rotation of CC single bond through rigidifying the conformation of TPE-NH2, thus making it display intense fluorescence with a broad working range (≤2.0 μg/mL) and a low detection limit (down to 35.89 ng/mL) for heparin. Meanwhile, the TPE-NH2/Hep complex can be further disaggregated by protamine due to the stronger affinity between heparin and protamine, consequently leading to the highly sensitive detection of protamine. What’s more, the low toxicity of TPE-NH2 makes it a potential functional sensor for detecting heparin in the matrix of human serum.

Blood heparin sensor made from a paste electrode of graphite particles grafted with molecularly imprinted polymer

A real-time heparin monitor could be used to optimize the dosage of heparin during extracorporeal circulation procedures. This report describes the development of a graphite-paste (GP) electrode with molecularly imprinted polymer (MIP) grafted onto it. Heparin-imprinted poly (methacryloxyethyltriammonium chloride -co- acrylamide -co- methylenebisacrylamide) was grafted directly onto graphite particles. The grafted particles were thoroughly mixed with oil to fabricate the MIP-GP electrode. Traditional cyclic voltammetry was performed with the electrode in physiological saline or bovine whole blood containing 5 mM ferrocyanide and 0–8 units/mL heparin. The current intensity increased with heparin concentration, due to expansion of the effective surface area resulting from heparin-promoted mobility of the oil in the MIP-GP electrode. No significant difference was found in the sensitivity of the current to unfractionated heparin among the electrodes fabricated because of the electrode homogenization resulting from thorough mixing of the MIP-grafted particles and oil. (A previous MIP-grafted indium tin oxide electrode exhibited lower sensitivity in blood than in saline.) Only 60 s were needed to stabilize the current. The current at the MIP-GP electrode was also sensitive to low-molecular-weight heparin in blood, but insensitive to chondroitin sulfate C (CSC), which is a heparin analog. The non-imprinted polymer (NIP)-grafted electrode was insensitive to heparin. Thus, the MIP-GP electrode, which operated through a new heparin-sensing mechanism, is an excellent candidate for application as a disposable sensor to monitor heparin levels in blood.

A Nanoscale Tool for Photoacoustic-Based Measurements of Clotting Time and Therapeutic Drug Monitoring of Heparin.

Heparin anticoagulation therapy is an indispensable feature of clinical care yet has a narrow therapeutic window and is the second most common intensive care unit (ICU) medication error. The active partial thromboplastin time (aPTT) monitors heparin but suffers from long turnaround times, a variable reference range, limited utility with low molecular weight heparin, and poor correlation to dose. Here, we describe a photoacoustic imaging technique to monitor heparin concentration using methylene blue as a simple and Federal Drug Administration-approved contrast agent. We found a strong correlation between heparin concentration and photoacoustic signal measured in phosphate buffered saline (PBS) and blood. Clinically relevant heparin concentrations were detected in blood in 32 s with a detection limit of 0.28 U/mL. We validated this imaging approach by correlation to the aPTT (Pearson's r = 0.86; p < 0.05) as well as with protamine sulfate treatment. This technique also has good utility with low molecular weight heparin (enoxaparin) including a blood detection limit of 72 μg/mL. We then used these findings to create a nanoparticle-based hybrid material that can immobilize methylene blue for potential applications as a wearable/implantable heparin sensor to maintain drug levels in the therapeutic window. To the best of our knowledge, this is the first use of photoacoustics to image anticoagulation therapy with significant potential implications to the cardiovascular and surgical community.

Pyrene-based heparin sensors in competitive aqueous media - the role of self-assembled multivalency (SAMul).

Amine-functionalised pyrene derivatives are reported and their ability to detect heparin via a fluorescent response determined - different responses are observed dependent on whether self-assembled multivalent binding between sensor and analyte takes place, and ratiometric heparin sensors which can detect this surgically-relevant polyanion in competitive media are reported.

“Turn on-off” fluorescent sensor for protamine and heparin based on label-free silicon quantum dots coupled with gold nanoparticles

An ultrasensitive “turn on-off” fluorescent sensor was presented for determination of protamine and heparin based on the high quenching ability of gold nanoparticles (AuNPs) to the fluorescence of silicon quantum dots (SiQDs) as well as high binding affinity of protamine with heparin. The fluorescence of SiQDs was quenched significantly by adding AuNPs into SiQDs solution. Upon addition of protamine, the AuNPs aggregated and the fluorescence of SiQDs was recovered due to the competitive adsorption of protamine and SiQDs on AuNPs. Addition of heparin disturbed the interaction between protamine and AuNPs due to its strong affinity to protamine, resulting in an adsorption of SiQDs on the surface of AuNPs. Thus the fluorescence of SiQDs was quenched. The strategy was simply achieved by measuring the changes in the fluorescence of SiQDs. The research results suggested that the developed method has several advantages such as label-free, high sensitivity, low cost, ease of operation. The linear response range was obtained over the range 0–1.2μg/mL and 0.002–1.4μg/mL with the low detection limit of 6.7ng/mL and 0.67ng/mL for protamine and heparin, respectively. The sensing platform was successfully applied to determination of heparin and protamine in serum samples.

ChemInform Abstract: Heparin Sensing and Binding — Taking Supramolecular Chemistry Towards Clinical Applications

AbstractReview: 101 refs.

Mallard Blue: A High-Affinity Selective Heparin Sensor That Operates in Highly Competitive Media

We report the simple synthesis and full investigation of a novel heparin binding dye, mallard blue, an arginine-functionalized thionine. This dye binds heparin in highly competitive media, including water with high levels of competitive electrolyte, buffered aqueous solution and human serum. The dye reports on heparin levels by a significant change in its UV-vis spectroscopic profile. Molecular dynamics modeling provides detailed insight into the binding mode. Heparin binding is shown to be selective over other glycosaminoglycans, such as hyaluronic acid and chondroitin sulfate. Importantly, we demonstrate that, in the most competitive conditions, mallard blue outperforms standard dyes used for heparin sensing such as azure A.

Heparin sensing and binding – taking supramolecular chemistry towards clinical applications

Heparin is a vital biomolecule in widespread clinical use as an anti-coagulant. Heparin sensors have potential applications in the bedside detection of heparin levels in human blood during surgery, while high-affinity heparin binders may enable the development of effective heparin reversal agents for use in patients once surgery is complete. However, human blood is a challenging medium in which to achieve selective high-affinity molecular recognition, and as such, this system provides a fascinating challenge to supramolecular chemists. This has encouraged research using a variety of different systems and is stimulating new approaches to the application of molecular recognition. This review article provides an overview of research from both clinical and supramolecular communities towards heparin binding and sensing and considers how this area may develop in the future.

Application of the ‘gate effect’ of a molecularly imprinted polymer grafted on an electrode for the real-time sensing of heparin in blood

Heparin is the most important anticoagulant drug used during surgeries and extracorporeal therapies. Although the blood levels of heparin should be monitored continuously during the procedure to ensure the safety of the patient, there is currently no technique for measuring heparin in real time. This study describes the use of a molecularly imprinted polymer (MIP) as a recognition element in the development of a heparin sensor for real-time monitoring. An indium tin oxide (ITO) electrode grafted with a heparin-specific MIP was used as a working electrode to perform cyclic voltammetry of ferrocyanide. The anodic current was found to be dependent on heparin concentration, probably due to the "gate effect", which is a change in the accessibility of the MIP-modified electrode to ferrocyanide, triggered by specific interaction between MIP and heparin. The kinetics of heparin interaction with the MIP-grafted electrode was evaluated using potentiostatic chronoamperometry of ferrocyanide in an electrochemical flow cell. The response time to stepwise changes in heparin concentration between 0 and 0.04 units per mL was estimated at 20 s, which is remarkably shorter than that achieved using conventional methods for monitoring heparin. The MIP-grafted electrode demonstrated exceptional sensitivity and could detect heparin in whole blood samples (0-6 units per mL) diluted 100-fold with physiological saline containing ferrocyanide. Therefore, the MIP-grafted electrode is suitable for real-time monitoring of heparin in blood. Another advantage is that a very small volume of blood is needed, which is very important, especially when regular measurements are required.

Designing of molecularly imprinted polymer-based potentiometric sensor for the determination of heparin

A novel potentiometric sensor with high selectivity and sensitivity has been developed for the determination of heparin, based on the modification of heparin-imprinted polymer film onto a glassy carbon. The performance of the developed heparin sensor was evaluated, and the results indicated that a sensitive potentiometric sensor could be fabricated. The obtained heparin sensor shows high-selectivity monitoring of heparin, shorter response time (<4min), wider linear range (0.003–0.7μM), lower detection limit (0.001μM), and satisfactory long-term stability (>2months). The potentiometric sensor was successfully applied to the determination of heparin in heparin sodium injection with recoveries between 97.1% and 110.0%.