Heparin In Serum Research Articles

Dual-mode upconversion sensors for detecting differently charged biotargets based on the oxidase-mimicking activity of Ce4+ and electrostatic control

Heparin is a highly negatively charged sulfated linear polymer glycosaminoglycan that has been widely used as an anticoagulant in medicine. Protamine is a cationic protein rich in arginine that is used to treat the blood-brain barrier during excess heparin surgery. Trypsin is the most important digestive enzyme-encoding generated by the pancreas and can specifically cleave the carboxyl ends of arginine and lysine residues. Heparin, protamine, and trypsin interact and constrain each other, and their fluctuations reflect the body's dysfunction. Therefore, it is necessary to develop a fast, sensitive, and highly selective assay for regularly monitoring the levels of heparin, protamine, and trypsin in serum. Herein, a fluorescent and colorimetric dual-mode upconversion nanoparticle (UCNP) biosensor was used for the determination of heparin, protamine, and trypsin based on the oxidase-mimicking activity of Ce4+ and electrostatic control. The biosensor exhibited sensitive detection of heparin, protamine, and trypsin with low limits of detection (LODs) of 16 ng/mL, 87 ng/mL and 31 ng/mL, respectively. Furthermore, the designed biosensor could eliminate autofluorescence, which not only effectively increased the accuracy of the sensor but also provided a new sensing pathway for the detection of differently charged biotargets.

2‐(N‐Morpholino)ethanesulphonic acid mediated facile and rapid one‐pot synthesis of gold nanoparticles and its application for colorimetric detection of heparin in human serum

AbstractA facile and rapid method for one‐pot synthesis of gold nanoparticles (AuNPs) has been developed. It employed 2‐(N‐morpholino)ethanesulphonic acid (MES) to act as reducing agent, buffering agent, and capping ligand to perform the reduction of the gold precursor (HAuCl4) into AuNPs. The AuNPs were synthesized by a simple mixing of MES buffer (pH 6.0) and HAuCl4 solution under a vortex at room temperature, and the process was completed within 1 min, which not only significantly simplifies the preparation procedure without heating or cooling but also greatly shortens the reaction time. Moreover, a novel colorimetric approach for sensitive detection of heparin was fabricated based on the prepared AuNPs. The detection limit was determined to be 3.1 μg/ml (i.e., 0.465 U/ml) with a linear response range of 3.25–130 μg/ml. Additionally, the sensor was also successfully applied for the detection of heparin in human blood serum samples, demonstrating its potential applicability for disease diagnosis in the future.

Nanozyme catalysis-assisted ratiometric multicolor sensing of heparin based on target-specific electrostatic-induced aggregation

Monitoring the level of heparin in clinical matrices is significant because of its pivotal role in preventing thrombosis. Compared to traditional single-signal sensors, multi-signal ratiometric detection can provide anti-interference results especially in complicated environments. However, fabricating an easy-to-operation, low-cost and robust sensor for the ratiometric detection of heparin still remains challenging. Here we propose a novel nanosensor for the ratiometric multicolor sensing of heparin with high performance. The sensor is based on the specific electrostatic interaction between the target and a positively charged species generated from nanozyme catalysis. FeMoO4 nanorods are explored as an oxidase mimic for the first time, showing a high activity at neutral pH to catalyze the colorless 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation to blue TMBox with an absorbance at 652 nm. Heparin can induce the rapid aggregation of the produced TMBox intermediate with rich positive charges due to their strong electrostatic interaction, leading to the formation of a purple Heparin-TMBox complex providing a signal at 565 nm. With the increase of heparin, the color changes from blue to indigo and further purple, enabling the multicolor sensing of the target. As a result, ultrasensitive determination of heparin was obtained with a very low detection limit. The fabricated nanosensor could differentiate heparin from complex species with no interferences, and it provided reliable analytical results for heparin in both serum and plasma. With robust performance, low cost and facile fabrication, the sensor holds great potential in monitoring heparin for clinical applications.

Upconversion-based dual-mode optical nanosensor for highly sensitive and colorimetric evaluation of heparin in serum

Heparin is an important anticoagulant in clinics, which requires regular detection and dose adjustment. Fluorimetry is a powerful tool considering the advantages of fast response and high sensitivity while most fluorescent sensors suffer from spontaneous fluorescence for excitation under short-wavelength light, the occurrence of false positives, complex and time-consuming preparation, as well as expensive detection instruments. Herein, we developed upconversion nanoparticles (UCNPs) based nanosensor for fluorescent and colorimetric detection of heparin. The nanosensor exhibited sensitive detection for heparin with the low limit of detection (LOD) of 0.1 nM in fluorescent mode and 0.3 nM in colorimetric mode. In addition, we used a smartphone-sensing platform for quantitative detection of heparin with the LOD of 2 nM. Depending on long-wavelength excitation and fluorescent-colorimetric dual-response detection signal, the designed nanosensor could eliminate the auto-fluorescence, which effectively increased the accuracy of the sensor, thus extending the applications for bed-side heparin assay and related medical safety.

An electrostatically regulated organic self-assembly for rapid and sensitive detection of heparin in serum.

Heparin (Hep) is a highly negatively charged linear glycosaminoglycan involved in various physiological processes, especially blood coagulation. Hep is also a first-line drug for anticoagulation and prevention of thromboembolism, but its overdose will cause serious side effects. Herein, we designed a long-wavelength double-charged cationic fluorescent probe PYPN, and studied its aggregation state and detection performance for Hep. PYPN was readily synthesized through a one-step reaction without complicated purification. In aqueous medium, PYPN molecules with an amphiphilic structure spontaneously form nano-assemblies, which can be immediately decomposed by Hep due to the formation of a PYPN-Hep complex based on electrostatic attraction. The assembly shows a fast, sensitive and ratiometric fluorescence response to Hep, without being obviously interfered by other compounds. In various serum matrices, the fluorescence intensity ratio F610/F470 has a good linearity with Hep concentration (0-12 μg mL-1), and the detection limit (0.11-0.12 U mL-1) is lower than the minimum concentration (0.2 U mL-1) used in clinical treatment. Our study provides an easy-to-prepare and feasible tool for the selective and sensitive quantification of Hep in serum.

Micellization-induced amplified fluorescence response for highly sensitive detection of heparin in serum

Fluorescence-based assays should be feasible in aqueous media for effectively detecting the biological factors. However, numerous sensors have limited signal transductions and low fluorescence quantum yields due to the ingerently reduced excited state energy of fluorophores in aqueous solution, which reduces their sensitivity. This necessitates a smart sensing approach with an amplified fluorescence response for analytes in aqueous solution. Herein, a new building block which self-assembles in aqueous media, giving a micellar sturcuture with the hydrophobic π-extended conjugated system at the core and hydrophilic groups at the periphery, was devised for the first time. We demonstrated that the aggregated fluorophores in a micelle induce amplified fluorescence quenching, in which the excited electron efficiently migrates through π-extended conjugated system in a micelle, as in a polymeric system. Such feature differentiates this sensing approach from the numerous fluorescence-based tools previously developed for sensitive detection. This new system exhibited highly sensitive signal transduction for specific analytes even under actual bioanalytical conditions.

Glycosaminoglycan‐Induced Emissive J‐Aggregate Formation in a meso‐Ester BODIPY Dye

AbstractThe analyte‐directed formation of emissive J‐aggregates of a meso‐ester BODIPY dye is explored in the design of fluorescence “light‐up” probes. The heparin‐specific pyridinium‐functionalized meso‐ester BODIPY dye C10‐Py+ self‐assembles in aqueous buffer into spherical aggregated nanostructures with spectroscopic features indistinguishable from the monomeric dye (signal off). In the presence of heparin, the aggregated assemblies rearrange and associate with heparin through charge‐pairing interactions, triggering the spontaneous formation of emissive J‐type aggregates (signal on). The heparin‐induced J‐aggregate formation of C10‐Py+ provides an excellent “turn‐on” signal with large spectral shifts. The response is highly selective for heparin over other closely related glycosaminoglycan (GAG) analogues, sensitive, fast, and operationally simple. Assays for heparin in human serum, and for the adulterant OSCS in heparin are demonstrated, confirming the reliability and robustness of the J‐aggregation approach using meso‐ester BODIPY dyes in the design of aggregation‐induced emission (AIE)‐based fluorogenic probes.

Highly Sensitive and Selective Detection of Heparin in Serum Based on a Long-Wavelength Tetraphenylethylene-Cyanopyridine Aggregation-Induced Emission Luminogen.

A positively charged aggregation-induced emission luminogen (AIEgen), TPE-P+, was constructed by linking a pyridyl cation to tetraphenylethylene (TPE) via a cyanoethylene bond. TPE-P+ can realize the identification of heparin (Hep) by aggregating with negatively charged Hep via electrostatic interactions. Upon addition of Hep, TPE-P+ exhibited 36-fold fluorescence enhancement in less than 5 s, exhibiting quick and sensitive response to Hep with a low detection limit down to 4 nM. Among various biological substances, even Hep analogs like chondroitin 4-sulfate and hyaluronic acid, TPE-P+ showed the most significant fluorescence enhancement to Hep only, demonstrating its excellent selectivity for Hep. In particular, with long-wavelength emission near 600 nm and large stocks shift (∼160 nm), TPE-P+ enabled minimization of autofluorescence interference from a complex biological matrix and provided more accurate results. Finally, TPE-P+ was successfully applied for sensitive and selective detection of Hep in serum. Notably, there existed a good linear relationship in a serum assay when the Hep concentration ranging from 0 to 4 μM (R2 = 0.9934) covered the clinical dosage level during both cardiovascular surgery and long-term care, suggesting the potential clinical practice for quantifying Hep in serum. Moreover, TPE-P+-Hep complex can be further disaggregated by protamine (PRTM) due to the stronger affinity between Hep and PRTM, thereby leading to further detection of PRTM effectively. Last, but not least, the "off-on-off" system designed for both Hep and PRTM detection proved to be reversible.

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.

A visualized colorimetric detection strategy for heparin in serum using a metal-free polymer nanozyme

A metal-free polymer nanozyme has been developed for the first time simply via the one-pot covalent reaction of hyperbranched polyethyleneimine (PEI) and redox dihydroxybenzaldehyde (DHB). The yielded PEI-DHB nanoparticles could display the peroxidase-like catalysis activity, so as to act as the metal-free nanozyme in catalyzing the chromogenic reactions of 3,3′,5,5′-tetramethylbenzidine and H2O2. More interestingly, the catalysis activity of the polymer nanozymes could be specifically inhibited by heparin oppositely charged. A facile visualized colorimetric method was thereby developed for sensing heparin through combining the specific catalysis and electrostatic attraction of PEI-DHB nanozyme. It was found that the developed colorimetric method can enable the detection of heparin in serum over the linear concentration range of 0.050–1.0 μM, promising the wide applications for the detection of heparin in the clinical laboratory for the disease diagnostics and pharmacological monitoring. More importantly, such a green synthesis method may be tailored for fabricating various metal-free polymer nanozymes for the wide catalysis applications.

Mallard Blue binding to heparin, its SDS micelle-driven de-complexation, and interaction with human serum albumin: A combined experimental/modeling investigation

Heparin is a sulfated glycan widely used as anticoagulant in medicine. Mallard Blue (MalB), a small cationic dye developed in our laboratories, is able to detect heparin in serum and plasma in a dose-response manner, with performance superior to its direct competitors. However, many aspects of MalB/heparin binding still remain to be explored which, once solved, may foster the clinical use of MalB. Among these, the characterization of the energetics that drives the MalB/heparin binding process, the competition for MalB binding by other polyanions (e.g., negatively-charged surfactant micelles), and the interaction of MalB with serum proteins are of particular interest. This work fills this gap by means of a combination of experimental investigations (UV-visible spectroscopy and isothermal titration calorimetry), and computational approaches based on molecular dynamics (MD) simulation techniques. In combination, the results obtained show that MalB efficiently binds to both heparin and SDS, with the binding being enthalpic in nature; yet, SDS is able to extract MalB from its complex with heparin when the surfactant is in its self-assembled form, the driving force underlying SDS-induced MalB/heparin de-complexation being entropic in nature as the two enthalpies of binding effectively cancel each other out. Once bound to SDS, the dye remains electrostatically bound to the micellar surface and does not penetrate the micelle palisade layer, as verified by steered molecular dynamics/umbrella sampling simulations. Finally, the affinity of MalB for human serum albumin (HSA), the most abundant plasma protein, is found to be lower than that for heparin, confirming the ability of the dye to work in complex physiological environments.

A simple and sensitive label-free fluorescence sensing of heparin based on Cdte quantum dots.

A rapid, simple and sensitive label-free fluorescence method was developed for the determination of trace amounts of an important drug, heparin. This new method was based on water-soluble glutathione-capped CdTe quantum dots (CdTe QDs) as the luminescent probe. CdTe QDs were prepared according to the published protocol and the sizes of these nanoparticles were verified through transmission electron microscopy (TEM), X-ray diffraction (XRD) and dynamic light scattering (DLS) with an average particle size of about 7 nm. The fluorescence intensity of glutathione-capped CdTe QDs increased with increasing heparin concentration. These changes were followed as the analytical signal. Effective variables such as pH, QD concentration and incubation time were optimized. At the optimum conditions, with this optical method, heparin could be measured within the range 10.0-200.0 ng mL(-1) with a low limit of detection, 2.0 ng mL(-1) . The constructed fluorescence sensor was also applied successfully for the determination of heparin in human serum. Copyright © 2015 John Wiley & Sons, Ltd.

A novel label-free upconversion fluorescence resonance energy transfer-nanosensor for ultrasensitive detection of protamine and heparin

A novel label-free fluorescence nanosensor was developed for ultrasensitive detection of protamine and heparin based on fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The FRET system was formed by the electrostatic adsorption of AuNPs on UCNPs, and the fluorescence of UCNPs was significantly quenched. When protamine was added to the mixture of UCNPs–AuNPs, the AuNPs interacted with protamine and then desorbed from the surface of UCNPs and aggregated, resulting in the recovery of the fluorescence of UCNPs. On the addition of both protamine and heparin, the FRET system formed owing to the stronger interaction between heparin and protamine than that with AuNPs, leading to a marked fluorescence quenching of UCNPs. The concentrations of protamine and heparin were proportional to the changes of the fluorescence of UCNPs. The linear response range was obtained over the concentration ranges of 0.02 to 1.2μg/ml and 0.002 to 2.0μg/ml with low detection limits of 6.7 and 0.7ng/ml for protamine and heparin, respectively. Simultaneous measurement of protamine and heparin in human serum can be achieved, suggesting that the nanosensor can be used in a complex biological sample matrix.

Selective room temperature phosphorescence detection of heparin based on manganese-doped zinc sulfide quantum dots/polybrene self-assembled nanosensor

A selective system was developed to detect heparin in aqueous solutions by using MPA(3-Mercaptopropionic Acid)-capped Mn-doped ZnS quantum dots (QDs)/polybrene (hexadimethrine bromide) hybrids as a sensitive room temperature phosphorescence (RTP) nanosensor. In this system, the RTP intensity of QDs was remarkably enhanced via electrostatic self-assembly after the addition of polybrene. The addition of heparin into the system was competitively bound to polybrene and enable to deprive it from the surface of QDs, as a result, the RTP intensity of Mn-doped ZnS QDs/polybrene hybrids was reduced with the increased of heparin concentration. Based on this effect, a selective system was proposed to detect heparin. Under the optimal conditions, the change of RTP intensity was proportional to the heparin concentration from 0.05 to 1.4UmL−1 (about 0.38–10.76μgmL−1) and the limit of detection (LOD) was 0.021UmL−1 (about 0.16μgmL−1). This proposed nanosensor is simple and relatively free of interference from coexisting substances, which can be applied to detect heparin in heparin injection and human serum. In addition, a new pathway was also provided based on the assembly of QDs with other cationic homopolymers for further design of biosensors and detection of biomolecules.

Hollow core photonic crystal fiber as a reusable Raman biosensor

We report that a single hollow core photonic crystal fiber (HC-PCF) can be used for repetitive characterization of multiple samples by Raman spectroscopy. This was achieved by integrating the HC-PCF to a differential pressure system that allowed effective filling, draining and re-filling of samples into a HC-PCF under identical optical conditions. Consequently, high-quality and reliable spectral data could be obtained which were suitable for multivariate analysis (partial least squares). With the present scheme, we were able to accurately predict different concentrations of heparin and adenosine in serum. Thus the detection scheme as presented here paves a path for the inclusion of HC-PCFs in point-of-care technologies and environmental monitoring where rapid sample characterization is of utmost importance.

Raman spectroscopy for clinical-level detection of heparin in serum by partial least-squares analysis

Heparin is the most widely used anti-coagulant for the prevention of blood clots in patients undergoing certain types of surgeries including open heart surgeries and dialysis. The precise monitoring of heparin amount in patients' blood is crucial for reducing the morbidity and mortality in surgical environments. Based upon these considerations, we have used Raman spectroscopy in conjunction with partial least squares (PLS) analysis to measure heparin concentration at clinical level which is less than 10 United States Pharmacopeia (USP) in serum. The PLS calibration model was constructed from the Raman spectra of different concentrations of heparin in serum. It showed a high coefficient of determination (R2>0.91) between the spectral data and heparin level in serum along with a low root mean square error of prediction ~4 USP/ml. It enabled the detection of extremely low concentrations of heparin in serum (~8 USP/ml) as desirable in clinical environment. The proposed optical method has the potential of being implemented as the point-of-care testing procedure during surgeries, where the interest is to rapidly monitor low concentrations of heparin in patient's blood.

Polyelectrolyte‐Functionalized Gold Nanoparticle Scaffold for the Sensing of Heparin and Protamine in Serum

We describe a shape-controlled synthesis of polyelectrolyte-functionalized flowerlike and polyhedral Au nanoparticles and the development of a nanoarchitectured platform for the selective and highly sensitive detection of protamine and heparin by voltammetric, impedimetric, and microgravimetric techniques. The functionalized Au nanoparticles were chemically synthesized in aqueous solution at room temperature in the presence of the polyelectrolyte (either protamine or heparin). The charge on the polyelectrolyte controlled the shape and surface morphology of the nanoparticles. The negatively charged heparin-functionalized Au nanoparticles have multiple branched flowerlike shapes with an average size of 50 nm, whereas the cationic protamine-functionalized nanoparticles are of polyhedral shape with an average size of 25 nm. Both flowerlike and polyhedral nanoparticles have (111), (200), (220), and (311) planes of a face-centered cubic lattice of Au. Voltammetric, impedimetric, and microgravimetric sensing platforms based on functionalized Au nanoparticles have been developed for the sensing of heparin and protamine. The sensing platforms are developed by self-assembling the functionalized nanoparticles on a thiol-functionalized three-dimensional silicate network. The microgravimetric sensing platform shows very high sensitivity and it can detect heparin and protamine at concentrations as low as 0.05 μg mL(-1). The selectivity of the sensing platform towards heparin was examined with potential interferents such as hyaluronic acid (HA) and chondroitin-4-sulfate (CS). Both HA and CS did not interfere with the measurement of heparin. The practical application of the sensing platform was demonstrated by measuring the concentration of heparin and protamine in human serum samples. The sensing platform could successfully quantify the concentration of heparin and protamine in the real serum samples with excellent recovery. The sensing platform was robust and could be used for repeated measurement without compromising the sensitivity.

Ratiometric Fluorescence Sensor Based on a Pyrene Derivative and Quantification Detection of Heparin in Aqueous Solution and Serum

A ratiometric fluorescence sensor based on pyrene was designed for selective detection of heparin in HEPES (N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid) buffer and serum sample. Pyrene and long-chain alkanes were linked through bisquaternary functionality in the sensor which could interact with heparin via supramolecular assembly. A ratiometric fluorescent signal change of the sensor can be observed because of the specific monomer-excimer conversion of pyrene which is modulated by the supramolecular self-assembly of sensor and heparin. Upon addition of heparin, the excimer emission of the sensor at 489 nm is observed and the monomer emission intensity at 395 nm decreases concomitantly. Addition of heparin derivatives with very similar structure such as chondroitin 4-sulfate or hyaluronic acid to the same sensor solution only leads to very smaller changes in intensity ratios probably because of lower charge density and more distant spatial distribution of anions (or disadvantageous spatial orientation of anions) as compared to those of heparin. The novel sensor can effectively differentiate heparin from its derivatives with relatively low background interference and wide linear response in HEPES and serum. A linear calibration curve is obtained from 0 to 3.4 μM for heparin quantification in serum.

New Fluorescent Assay for the Direct Quantification of Heparin and Low-Molecular-Weight Heparins In Clinical Samples

AbstractAbstract 3325Heparin, a linear polydisperse polysaccharide, is used to prevent blood clotting in various diseases. It consists predominantly of a trisulfated disaccharide repeating unit and has a high negative charge density. Currently accepted methods to control blood levels of heparin or of its more commonly used low-molecular-weight (LMW) fractions include chromogenic substrate assays that analyze the biological inhibition of blood coagulation factor Xa (FXa). There are no established methods to directly determine the concentration of heparins in clinical samples.A modified positively charged perylene diimide (Heparin Red) was developed that binds specifically to heparin and LMW-heparin (Szelke H et al., Chem. Commun. 2010;46:1667). A red fluorescence signal is dose dependently quenched in the presence of heparin and LMW-heparins. LMW-Heparin was added to human serum, plasma or urine. The chromogenic S2222 substrate assay was used to determine the anti-FXa activity of the anticoagulants. Fluorescence was recorded with a portable fluorimeter, equipped with a 550 nm excitation filter and a 610 nm emission filter.For plasma samples spiked with 0.6–1.0 IU/mL LMW-heparin, the intraday (n=5) and interday (n=5) coefficient of variations (CVs) of the fluorimetric Heparin Red assay were in the range of 2.9–6.1% and 4.3–13.8%, respectively. The CVs of the chromogenic S2222 assay for same plasma samples were the range of 3.3–18.8% (intraday) and 6.2–13.7 (interday).In serum and plasma samples of patients treated with LMW-heparin, correlation between the Heparin Red assay and the chromogenic anti-FXa test (S-2222) was r = 0.66. The correlation between the Heparin Red assay and the anti-FXa test in urine samples was r = 0.83. This is probably due to its lower protein concentration in urine compared to plasma and serum samples.The fluorescent perylene diimide Heparin Red allows sensitive quantification of heparin and LMWHs in plasma, serum and urine samples. Ease of application combined with matrix-independent response make this heparin probe attractive for both routine laboratory and fast point-of-care testing as well as high-throughput pharmacokinetic studies, ideally without the need for patient-specific calibration. Disclosures:No relevant conflicts of interest to declare.

Naked-Eye Detection and Quantification of Heparin in Serum with a Cationic Polythiophene

A strategy for naked-eye detection and quantification of heparin in biological media, such as fetal bovine serum (FBS) is demonstrated by monitoring the absorbance change of a water-soluble cationic polythiophene. The negatively charged heparin interacts with positively charged polythiophene through electrostatic interaction, which leads to polymer conformation and color change from yellow to orange in solution. Under optimized conditions, addition of heparin derivatives, such as hyaluronic acid or chondroitin 4-sulfate to the same polymer solution leads to less change in polymer conformation and solution color due to their lower charge density as compared to that of heparin. Increasing the detection temperature or simply adding some organic solvent to the aqueous media reduces the polymer-polymer interchain pi stacking, and the polymer color change can be used to clearly differentiate heparin from its analogues in homogeneous solutions. Quantification of heparin is also demonstrated by correlating the changes in polymer absorbance to the heparin concentration. A linear calibration curve is observed in the 0-6.7 U/mL and 0-2.2 U/mL ranges for heparin quantification in pure water and in FBS, respectively.