Reversible fluorescence modulation of BSA stabilised copper nanoclusters for the selective detection of protamine and heparin.

Protamine and heparin are the most important polyionic drugs used during surgeries and extracorporeal therapies. In this article, a selective and sensitive fluorescence method for the detection of both protamine and heparin was developed by using bovine serum albumin stabilised copper nanoclusters. Blue emitting fluorescent copper nanoclusters were synthesized in aqueous solution using bovine serum albumin as a capping agent and a reducing agent. A one pot microwave assisted method was adopted to synthesize fluorescent copper nanoclusters showing emission at 410 nm upon excitation at 330 nm. The fluorescence of copper nanoclusters was found to be enhanced after the addition of protamine and the limit of detection obtained is 0.12 ng mL-1. The significant enhancement in fluorescence can be attributed to the electrostatic interactions between the copper nanocluster and protamine. In contrast, the enhanced fluorescence intensity of the copper nanocluster with protamine added was decreased after the addition of heparin, and the copper nanocluster regained its original fluorescence intensity. This can be attributed to the strong interaction of protamine with heparin and the limit of detection was calculated as 0.0406 ng mL-1. The selectivity and sensitivity of the sensor for both protamine and heparin were also determined in the presence of potentially co-existing biomolecules, cations, and anions and satisfactory results were obtained. Additionally the validity of the proposed protamine and heparin sensor was attested in real sample matrices such as human urine samples and human blood serum samples. The results exhibited that the recovery percentage of protamine and heparin reached 98-99% and 92-99% in urine samples and 97-99% in serum samples.

Heparin-protamine balance after neonatal cardiopulmonary bypass surgery.

Essentials Heparin-protamine balance (HPB) modulates bleeding after neonatal cardiopulmonary bypass (CPB). HPB was examined in 44 neonates undergoing CPB. Post-operative bleeding occurred in 36% and heparin rebound in 73%. Thrombin-initiated fibrin clot kinetic assay and partial thromboplastin time best assessed HPB. SUMMARY: Background Neonates undergoing cardiopulmonary bypass (CPB) are at risk of excessive bleeding. Blood is anticoagulated with heparin during CPB. Heparin activity is reversed with protamine at the end of CPB. Paradoxically, protamine also inhibits blood coagulation when it is dosed in excess of heparin. Objectives To evaluate heparin-protamine balance in neonates undergoing CPB by using research and clinical assays, and to determine its association with postoperative bleeding. Patients/Methods Neonates undergoing CPB in the first 30 days of life were studied. Blood samples were obtained during and after surgery. Heparin-protamine balance was assessed with calibrated automated thrombography, thrombin-initiated fibrin clot kinetic assay (TFCK), activated partial thromboplastin time (APTT), anti-FXa activity, and thromboelastometry. Excessive postoperative bleeding was determined by measurement of chest tube output or the development of cardiac tamponade. Results and Conclusions Of 44 neonates enrolled, 16 (36%) had excessive postoperative bleeding. The TFCK value was increased. By heparin in neonatal blood samples, but was only minimally altered by excess protamine. Therefore, it reliably measured heparin in samples containing a wide range of heparin and protamine concentrations. The APTT most closely correlated with TFCK results, whereas anti-FXa and thromboelastometry assays were less correlative. The TFCK and APTT assay also consistently detected postoperative heparin rebound, providing an important continued role for these long-established coagulation tests in the management of postoperative bleeding in neonates requiring cardiac surgical repair. None of the coagulation tests predicted the neonates who experienced postoperative bleeding, reflecting the multifactorial causes of bleeding in this population.

Impact of protamine I on colon cancer proliferation, invasion, migration, diagnosis and prognosis.

This paper investigates protamine I (PRM1) expression and its effects on proliferation, invasion and migration of colon cancer cells as well as its function in clinical diagnosis and prognosis. Gene chips were used to screen differentially expressed genes. PRM1 expression was detected by Western blotting and quantitative real time-polymerase chain reaction (qRT-PCR). Hematoxylin and eosin (HE) staining and immunohistochemistry were utilized to compare the expression of PRM1 from multiple differentiation levels of colon cancer tissues. Cell viability, cell apoptosis and cell cycle were tested using the MTT assay and flow cytometry. Cell invasion and migration capability were tested using the Transwell assay and wound healing. In vivo effects of PRM1 on colon cancer were explored using a xenograft model. PRM1 expression in serum was detected by enzyme-linked immunosorbent assay (ELISA). The expression level of PRM1 was significantly higher in colon cancer tissues and the staining degree of PRM1 in poorly-differentiated was stronger. pcDNA3.1-PRM1 decreased cell apoptosis while it increased the proliferation, cell invasion and migration. The si-PRM1 group displayed an opposite tendency. The serum PRM1 level was significantly higher and could serve as a diagnostic biomarker for colon cancer.

Implementing a Statistical Model for Protamine Titration: Effects on Coagulation in Cardiac Surgical Patients.

OBJECTIVES: To implement a statistical model for protamine titration. DESIGN: Prospective randomized trial. SETTING: University hospital. PARTICIPANTS: Sixty (n = 30+30) patients scheduled for elective coronary artery bypass surgery were randomly assigned to 2 groups. INTERVENTIONS: Protamine dose calculated according to an algorithm established from a statistical model or to a fixed protamine-heparin dose ratio (1:1). MEASUREMENTS AND MAIN RESULTS: Both groups demonstrated comparable patient demographics and intraoperative data. Coagulation effects were evaluated using rotational thromboelastometry. Using the statistical model reduced (p<0.01) the protamine dose from 426±43 mg to 251±66 mg, followed by significantly (p<0.01) shorter intrinsic clotting time (208±29 seconds versus 244±52 seconds) and stronger clot firmness (p = 0.01), and effects on indices of extrinsic or fibrinogen coagulation pathways were insignificant. Test of residual heparin was negative in all patients after protamine administration, aligned with insignificant (p = 0.27) intergroup heparinase-verified clotting time differences. CONCLUSIONS: The statistical model for protamine titration is clinically feasible and protects the patient from exposure to excessive doses of protamine, with advantageous effects on coagulation as measured using rotational thromboelastometry. Significance regarding clinical outcome is yet to be defined.

combination effect of hypertonic disease with chronic pancreatitis on the processes maintain homeostasis.

OBJECTIVE: Introduction: Abnormalities comorbidity - a frequent phenomenon in medical practice. This determines the relevance of research processes maintaining homeostasis with a combination of various diseases. The aim of this study was to examine and compare the character of vegetative, antioxidant, kallikrein-kinin system and parameters of endogenous intoxication disorders in the patients with isolated essential hypertension and with combination of hypertonic disease and chronic pancreatitis. PATIENTS AND METHODS: Materials and Methods: Cardiointervalography was used in the research with definition of standard statistical and spectral heart rate variability. Determination of superoxide dismutase, glutathione, catalase, middle molecular peptides, total proteolytic activity of plasma by the hydrolysis of protamine sulfate, prekallikrein, kallikrein, α1 -proteinase inhibitor, α2 -macroglobulin and kininase II was conducted by laboratory methods. RESULTS: Results: Sympathicotonia with the moderate tension of adaptation processes, violation of antioxidant protection, kallikrein-kinin system and displays of endogenous intoxication were found in the patients with isolated hypertension. Reduction of sympathicotonia, reducing total power spectrum, increasing the share of humoral-metabolic effects on heart rate, tendency to asympathicotonia autonomic reactivity, lower levels of superoxide dismutase, glutathione, prekallikrein, α2 -macroglobulin, kininase II, higher levels of catalase, middle molecular peptides, total proteolytic activity of plasma kallikrein were observed upon accession the concomitant chronic pancreatitis. CONCLUSION: Conclusions: The signs of compensatory mechanisms disruption and increased autonomic nervous system imbalance with a decrease in ductility autonomous processes in the load were determined upon accession the concomitant chronic pancreatitis. The combination of pathologies also accompanied by more severe manifestations of endogenous intoxication, significant violations of antioxidant and kallikrein-kinin systems.

Flow Chronopotentiometry with Ion-Selective Membranes for Cation, Anion, and Polyion Detection.

We report here on the development of a chronopotentiometric readout for ion-selective electrodes that allows one to record transition times in continuous flow conditions without the necessity to stop the flow. A sample plug of 150 µL is injected into the carrier solution (0.5 mM NaCl) and subsequently transported to the detection cell (∼20 µL) at moderate flow rates (∼0.5 mL min(-1)), where a short current pulse (5s) is applied between the ionophore-based working electrode and a biocompatible and nonpolarizable Donnan exclusion anion-exchanger membrane reference/counter electrode. Flow conditions bear an influence on the thickness of the aqueous diffusion layer and result in a shift of the chronopotentiometric transition time with respect to stopped flow. Two models based on rotating disk electrodes and flow chronopotentiometry at metal-based electrodes were used to corroborate the data. The method was successfully applied to the determination of calcium, chloride, alkalinity, acidity, and protamine with a range of ion-selective membranes. Because of the limiting exposure time of ca. 20 s of the membranes with the sample, this approach is demonstrated to be useful for the detection of protamine in the therapeutic range of undiluted human blood.

A simple and rapid label-free fluorimetric biosensor for protamine detection based on glutathione-capped CdTe quantum dots aggregation.

A novel fluorescent biosensor is developed, based on glutathione-capped CdTe quantum dots aggregation, for the determination of trace amount of an important drug, protamine. In this method with increasing the protamine concentration, the fluorescence of the quantum dots was quenched due to their aggregation. Different parameters affect the sensitivity, such as pH and the amount of the quantum dots, were optimized. Using the new optical biosensor, under the optimized conditions, protamine could be measured in the range of 2.0-200 ng mL(-1) with a detection limit of 1.0 ng mL(-)(1). The relative standard deviation for five replicates determination of 30.0 ng mL(-)(1) protamine was 1.26%. The influence of common interfering species on the protamine detection was studied. The results showed that the biosensor is highly selective and sensitive for the detection of protamine. The optical biosensor was successfully used for the determination of protamine in real samples.

Reversal of heparin after cardiac surgery: protamine titration using a statistical model.

OBJECTIVE: To establish a statistical model for determination of protamine dose in conjunction with cardiopulmonary bypass. DESIGN: Prospective. SETTING: University hospital. PARTICIPANTS: Ninety consecutive cardiac surgical patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A series of clinically oriented variables were introduced into a statistical model for projection of the protamine dose after cardiopulmonary bypass. The following significant predictors were identified using multivariable regression analysis: The patient's body surface area, the administered dose of heparin, heparin clearance, and the preoperative platelet count. The statistical model projected the protamine dose within 3±23 mg of the point-of-care test used as reference. CONCLUSION: Protamine dosing based on statistical modeling represents an alternative to point-of-care tests.

Facile and sensitive detection of protamine by enhanced room-temperature phosphorescence of Mn-doped ZnS quantum dots.

Quantum dot (QD) nanohybrids provide an effective route to explore the new properties of materials and are increasingly used as highly valuable sensitive (bio) chemical probes. Interestingly, the room-temperature phosphorescence (RTP) of 3-mercaptopropionic acid (MPA)-capped Mn-doped ZnS QDs could be remarkably enhanced by the addition of protamine. Based on the above finding, a simple, sensitive, and selective method for rapid detection of protamine was successfully designed. With this method, protamine as a cationic peptide interacts electrostatically with MPA-capped Mn-doped ZnS QDs to form MPA-capped Mn-doped ZnS QD/protamine complexes, which leads to the aggregation of QDs and enhances the RTP intensity. Under the optimized conditions, the RTP intensity change was linearly proportional to the concentration of protamine in the range 0.2-3.0 µg ml(-1), and the limit of detection was 0.14 µg ml(-1). The proposed method was successfully applied to detect protamine in protamine sulfate injection and human serum samples with satisfactory results, and the recovery ranged from 96.5 to 105.6%.

Serological features of antibodies to protamine inducing thrombocytopenia and thrombosis.

BACKGROUND: A significant proportion of patients undergoing cardiopulmonary bypass develop anti-protamine antibodies, with or without the association of thromboembolic events. METHODS: We extensively investigated the serological features of protamine antibodies, which developed in six patients who were clinically suspected to have heparin-induced thrombocytopenia (HIT). Three patients had thrombotic events. Sera were tested by four different commercially available immunoassays, a heparin-platelet aggregation test, and for their binding properties to heparin, platelet factor 4 (PF4), complex heparin-PF4, protamine, and protamine complex with heparin. Sera from four patients were also tested for the capability to induce platelet activation and the formation of platelet-monocyte heterotypic aggregates. RESULTS: The ELISA assay Zymutest HIA was strongly positive in all cases, the HPIA Asserachrome was borderline, and the gel centrifugation test PaDGIA was positive in two tested patients. Platelet aggregation tests were negative. Using a variation of the Zymutest HIA we demonstrate that IgG antibodies bound only to protamine or protamine complex with heparin, but not to heparin or PF4 only. Sera-induced platelet P-selectin expression and the formation of platelet-monocyte aggregates. Blood samples from one patient proofed positive concomitantly with the thromboembolic event. However, serological characteristics did not differ between antibodies associated with thromboembolic events from those without. CONCLUSIONS: These data show that protamine-induced antibodies are specific and may induce platelet activation, which explains their association with thromboembolic events.

Protamine requirements in cardiac surgery: effect of changes in the heparin reference standard.

OBJECTIVE: UFH (unfractionated heparin) and protamine are integral to cardiac surgery, and inappropriate dosing can predispose to coagulopathy and hemorrhage. The FDA (Food and Drug Administration) recently has instituted changes to UFH formulation and it is not known if this has influenced its susceptibility to neutralization by protamine. Hence, the authors sought to compare 2 commercial preparations of UFH (old and new) with regard to their neutralization by protamine in patients undergoing cardiopulmonary bypass (CPB). DESIGN: Prospective, observational, cohort study. SETTING: Tertiary care university hospital and associated research laboratory PARTICIPANTS: Twenty adult patients undergoing elective cardiac surgery with CPB. INTERVENTIONS: Blood samples were drawn preinduction, prior to, and 5 and 30 minutes following protamine, and 0 and 2 hours after ICU admission. Protamine titration assays were conducted in vitro on samples drawn prior to and following protamine administration. Anti-IIa and anti-Xa activity were assayed in all samples. RESULTS: Anti-IIa and anti-Xa activity were detected ubiquitously at all time points following CPB, and there were no differences in susceptibility to protamine neutralization between the 2 groups. In vitro protamine titration studies revealed that anti-IIa was more resistant to protamine neutralization compared to anti-Xa activity. CONCLUSIONS: The 'old' and 'new' formulations of UFH evaluated in this study were similar in their susceptibility to protamine neutralization. Circulating UFH is detected as early as 5 minutes after protamine administration and anti-IIa is more resistant to protamine neutralization as compared to anti-Xa activity. Further studies are required to quantify the precise dose of protamine following CPB.

Ionophore-based ion-selective optical nanosensors operating in exhaustive sensing mode.

Ion selective optical sensors are typically interrogated under conditions where the sample concentration is not altered during measurement. We describe here an alternative exhaustive detection mode for ion selective optical sensors. This exhaustive sensor concept is demonstrated with ionophore-based nanooptodes either selective for calcium or the polycationic heparin antidote protamine. In agreement with a theoretical treatment presented here, linear calibration curves were obtained in the exhaustive detection mode instead of the sigmoidal curves for equilibrium-based sensors. The response range can be tuned by adjusting the nanosensor loading. The nanosensors showed average diameters of below 100 nm and the sensor response was found to be dramatically faster than that for film-based optodes. Due to the strong binding affinity of the exhaustive nanosensors, total calcium concentration in human blood plasma was successfully determined. Optical determination of protamine in human blood plasma using the exhaustive nanosensors was attempted, but was found to be less successful.

Counter electrode based on an ion-exchanger Donnan exclusion membrane for bioelectroanalysis.

Ion-exchanger based Donnan exclusion membranes (IEDEM) are studied here as separators for counter and pseudo-reference electrodes in bioelectroanalysis. Since the potential across the membrane remains indifferent for a wide range of current densities in contact with electrolyte solutions, IEDEM behave as ideally non-polarizable membranes. Consequently, such membranes may be suitable with counter or reference electrode, depending on the adopted cell configuration (three- or two-electrode system). Four configurations were characterized in order to establish the limitations of commercial anion-exchanging membranes, using chronopotentiometry as readout protocol. Three- and two-electrode configurations with and without membrane exhibited similar characteristics in terms of drift and reproducibility (observed drift and RSD were 0.0007 s(1/2) per scan number and 1.71%, respectively). Several currents amplitudes were applied to evaluate the upper current limits for the membranes, which was found at about 10 mA [42.8 mA cm(-2)]. This value is significantly above those typically used in chronopotentiometric experiments, which involve hundreds of µA. Three different analytes were measured in human whole blood using an IEDEM as a counter electrode. A divalent cation (calcium), a polyion (protamine), and an anion (chloride) were successfully determined in blood and compared to reference methods. Finally, the obtained results suggest that such membranes may be used in bioelectrochemical sensing approaches to replace expensive but less appropriate electrode materials for the measurement in matrices that contain lipids and proteins.

A polyadenosine-coralyne complex as a novel fluorescent probe for the sensitive and selective detection of heparin in plasma.

This study presents the development of a simple, label-free, sensitive, and selective detection system for heparin based on the use of a complex of 20-repeat adenosine (A20) and coralyne. Coralyne emits relatively weak fluorescence in an aqueous solution. In the presence of A20, coralyne molecules complexed with A20 through A2-coralyne-A2 coordination. An increase in the fluorescence of coralyne was observed because coralyne remained separate from water in the hydrophobic environment of the folded A20. The presence of heparin and the formation of the coralyne-heparin complex caused coralyne to be removed from the A20-corlayne complex. Because heparin promoted coralyne dimerization, the fluorescence of coralyne decreased as a function of the concentration of added heparin. This detection method is effective because the electrostatic attraction between heparin and coralyne is substantially stronger than the coordination between A20 and coralyne in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at pH 7.0. Under optimal conditions (5 µM coralyne, 1 µM poly A20, and 10mM HEPES), this probe exhibited high selectivity (>90-fold) toward heparin over hyaluronic acid and chondroitin sulfate. The probe׳s detection limit for heparin was determined to be 4 nM (75 ng/mL) at a signal-to-noise ratio of 3. This study validates the practicality of using the A20-corlayne complex to determine the concentration of heparin in plasma.

Gold nanoparticle coupled with fluorophore for ultrasensitive detection of protamine and heparin.

Here we report a novel label-free fluorescent sensor for ultrasensitive detection of protamine and heparin based on the high quenching ability of gold nanoparticles to the fluorescence of fluorescein. The fluorescence was significantly quenched when fluorescein molecules were attached to the surface of gold nanoparticles by electrostatic interaction. Upon addition of protamine, the fluorescein molecules were detached from the surface of the gold nanoparticles due to the stronger adsorption of protamine on the surface of AuNPs, and resulting in the recovery of the fluorescein molecules fluorescence. Heparin is able to bind with Protamine specifically. In the presence of heparin, the interaction of heparin with protamine makes the AuNPs de-aggregate and the fluorescein molecules re-attach to the AuNPs, which lead to marked fluorescence quench again. By measuring the changes in the fluorescence of the fluorescein molecules, the concentration of protamine and heparin were sequentially determined. The linear response range was obtained over the concentration range from 0 to 0.8 µg/mL and 4 to 1.6 µg/mL with the low detection limit 0.0067 µg/mL and 0.0013 µg/mL for protamine and heparin, respectively.

Protamine titration.

Protamine titration is the gold standard method for the measurement of unfractionated heparin (UFH) concentration in plasma. Protamine titration produces reliable and reproducible results; however it is -generally not considered a convenient assay for current clinical management of UFH as it is not readily automated (Olson et al. Arch Pathol Lab Med 122(9):782-798, 1998). Early clinical trials of UFH therapy determined that a heparin concentration of 0.2-0.4 U/ml by protamine titration correlated to an APTT of 1.5-2.5 times higher compared to baseline values produced desirable UFH safety and efficacy outcomes (Hull et al. N Engl J Med 315(18):1109-1114, 1986; Hull et al. N Engl J Med 322:1260-1264, 1990; Turpie et al. N Engl J Med 320:352-357, 1989; Brill-Edwards et al. Ann Intern Med 119(2):104-109, 1993; Hull Int Angiol 14(1):32-34, 1995). Such studies paved the way to the current view that it is no longer ideal to manage UFH based solely upon a 1.5-2.5 times prolongation of the "normal" APTT. Most advisory bodies recommend therapeutic APTTs be determined by correlating APTT results with therapeutic UFH levels as measured by anti-Xa assay (0.35-0.7 U/ml) or protamine titration (0.2-0.4 U/ml) (Hirsh and Raschke. Chest 126(3):188S-203S, 2004) (see Note 1). The concentration of UFH in a sample is measured by determining the amount of protamine required to return the thrombin clotting time (TCT) test (prolonged by UFH) to a pre-UFH level (Laffan and Manning. Dacie and Lewis: practical haematology. Churchill Livingstone: London, 2001).

Reversible sensing of the anticoagulant heparin with protamine permselective membranes.

A permselective membrane electrode allows the rapid and operationally reversible detection of the polycationic polypeptide protamine in physiological samples. Anticoagulant levels of heparin can be measured in undiluted whole blood by adding a known excess of its antidote protamine to discrete blood samples.

Anticoagulation monitoring during extracorporeal circulation with the Hepcon/HMS device.

OBJECTIVE: The objective of our study was to compare the standard protocol of anticoagulation to the Hepcon/HMS. METHOD: This study included forty-four patients who underwent coronary bypass grafting surgery (CABG), or biological aortic valve replacement (AVR). Unfractionated heparin (UH) was used for patients who underwent operations in the control group (n = 22) (300U/Kg of UH with a goal of an ACT of 400s). The heparin was antagonized dose/dose by protamine. For the patients who underwent operations in the HMS group (n = 22), the heparin and protamine doses were assessed by the Hepcon/HMS device. RESULTS: The sex ratio amounted to 1.93 (29 men and 15 women) and the mean age was 70 ± 11 years. The patients in the HMS group had a chest closure time that was significantly shorter than patients in the control group. The times were, respectively, 42 ± 15 minutes and 68 ± 27 minutes (p = 0.001). The protamine/heparin ratio was significantly lower in the HMS group (0.62 ± 0.13 vs. 1 ± 0.11) (p = 0.0001). The postoperative bleeding amounted to 804 ± 729 ml in the HMS group versus 1416 ± 1103 in the control group (p = 0.016). In multivariate linear regression analysis, only two independent factors were significantly associated with bleeding: the Hepcon/HMS (OR = 0.1-p = 0.03) and the preoperative hemoglobin rate (OR = 1.4 - p = 0.05). Postoperatively, within 72 hours, the red blood cell transfusion was 1.04 ± 1.5 units for the HMS group and 2.1 ± 1.87 units for the control group (p = 0.05). CONCLUSION: During cardiac surgery under CPB, heparin and protamine titration with the Hepcon/HMS device could predict a lower protamine dose and lower postoperative bleeding without higher thromboembolic events, and lower perioperative red blood cell transfusion with a shorter chest closure time.

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.