Research article Received: 12 May 2015,
Revised: 9 October 2015,
Accepted: 9 October 2015
Published online in Wiley Online Library: 5 November 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.3058
A simple and sensitive label-free fluorescence sensing of heparin based on Cdte quantum dots B. Rezaei,* M. Shahshahanipour and Ali A. Ensafi ABSTRACT: 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. Keywords: heparin; CdTe quantum dot; fluorescence spectroscopy; label-free
Introduction
958
Heparin is an important natural biomolecule that is normally extracted and purified from animal tissues, especially from porcine and bovine (1). This drug can be used as an anti-coagulant, antithrombotic, anti-lipemic, anti-atherosclerosis, anti-phlogistic and anti-allergic product (2). The use of heparin avoids the formation of clots in blood vessels before or after surgery or during certain medical procedures. Also, it is used to treat certain blood, heart, and lung disorders and helps in the diagnosis and treatment of certain bleeding disorders. It has been widely used in clinical therapy for more than 60 years and it is still regarded as the first option to avoid thrombosis and cure urgent vein thrombus (3). High doses of heparin can counteract some undesirable effects (such as internal bleedings, puke, loss of consciousness and headache) (4). Beyond the requirement for simple, accurate and real-time determinations of heparin levels in patient serum during surgery and postoperative remedy period, there is also the need for detection methods that can check the heparin levels in infusion solutions to prevent the risk of human errors in dosing. Conventional clinical methods for heparin detection rely on the measurement of activated clotting time or activated partial thromboplastin time (5). Different methods have been developed for heparin measurement, including resonance Rayleigh scattering (6), fluorimetry (3,6-8), electrochemical sensor (2,9-11), capillary electrophoresis (12), high performance liquid chromatography (HPLC) (13) and colorimetric methods (4,14,15). These methods are not sufficiently reliable and exact for clinical settings because of their lack of specificity and possible interference with other factors (16). The main limitations and disadvantages of HPLC are the cost of equipment, the use of environmentally dangerous solvents and the co-elution of compounds. The major drawback of capillary electrophoresis is its complex assay validation. Electrochemical sensors have a number of limitations such as the electroactivity of certain
Luminescence 2016; 31: 958–964
species and as such electrochemically active interference in the sample (17). The main limitation of colorimetric methods is its low sensitivity. In addition, some of these methods are not appropriate for use outside the laboratory or for field monitoring. Fluorescence sensors have many attractive advantages, including high sensitivity, remote control, inexpensiveness, easy recognition, and an especially suitable diagnostic device for analytical concerns. A few techniques have also been reported for determination of heparin applying QDs. Cao and et al. utilized the Ru complex, which quenched CdTe QD fluorescence (18). Heparin addition removed the quencher from the QD surface and led to fluorescence reclamation by the CdTe QDs. Zhang and coworkers also used MPA (3-mercaptopropionic acid) capped Mn-doped ZnS quantum dots and polybrene (hexadimethrine bromide) for the application of a room temperature phosphorescence (RTP) determination of heparin (19). In their study, the RTP intensity of QDs was strongly enhanced after the addition of polybrene. Heparin could remove polybrene from the surface of QDs, thus the RTP intensity of Mn-doped ZnS QDs was decreased with increasing heparin concentration. In another study, Liu and coworkers applied L-cysteine-capped CuInS2 QDs (20). Heparin could aggregate the QDs via electrostatic force and therefore decreased the intensity
* Correspondence to: B. Rezaei, Department of Chemistry, Isfahan University of Technology, Isfahan 84156–83111, Iran. E-mail: [email protected] Department of Chemistry, Isfahan University of Technology, Isfahan8415683111, Iran Abbreviations: DLS, dynamic light scattering; HPLC, high performance liquid chromatography; IUT, Isfahan University of Technology; QY, quantum yields; RTP, room temperature phosphorescence; TEM, transmission electron microscopy; XRD, X-ray diffraction.
Copyright © 2015 John Wiley & Sons, Ltd.
A simple and sensitive label-free heparin sensing of QD fluorescence. The addition of heparinase to this system caused de-aggregation of the L-cysteine capped CuInS2 QDs and increased the fluorescence intensity. Most of these assays require several laborious steps and several intermediate materials, therefore they are moderately expensive and time consuming. Quantum dots are also known as zero-dimensional particles, and semiconductor nanocrystals (21). QDs are brightly luminescent nanoparticles that have found remarkable applications in recent decades in sensing events as luminescent probes, because of excellent photophysical properties such as their wide absorption spectra and narrow photoluminescence spectrum (21,22). Colloidal nanocrystals QDs have about 10–20 times more brilliant fluorescence and their photodurability is 100–200 times better than that of organic dyes. The emission wavelengths of QDs can be set by size, shape and composition, leading to high flexibility in the choice of emission wavelength (23). SHINER photoluminescence is the result of great quantum yields assisted with large molar extinction coefficients. One of the ordinary methods used for dispersing QDs in aqueous solution is to modify their exterior surface with anionic carboxylate groups. At a suitable basic pH, electrostatic repulsion between QDs indicated an immutable colloidal suspension (21). In this study water-soluble glutathione-capped CdTe QDs were used as a fluorescent probe as they display very stable luminescence under physiological conditions depending on their particle size, and are synthesized easily, are biocompatible and are monodispersed. Glutathione-capped CdTe QDs also have higher quantum yields (QYs) than the CdTe QDs capped with other thiol compounds, without any post-preparative treatment. These values were comparable with, or even better than, most QDs prepared because of a better surface access of the CdTe crystalline lattice by glutathione (24-26). The unique advantages of this method are its very fast process, its low cost, and its ease of production
and the high sensitivity switch in methods for heparin sensing in human serum samples.
Experimental Chemicals Glutathione, Na2TeO3, CdCl2 and NaBH4 were purchased from Aldrich (Darmstadt, Germany). Heparin sodium salt was obtained from PanReac AppliChem (Darmstadt, Germany). All chemicals were of analytical reagent grade (with the highest degree of purity available) and deionized water was used throughout all experiments. A 50 μg mL–1 stock solution of heparin was prepared by dissolving an appropriate amount of heparin sodium salt into a 100-mL standard flask. Lower heparin concentrations were obtained by sequential dilution of the stock solution. A 0.040 mol L–1 solution CdCl2 and a Na2TeO3 solution (0.010 mol L–1) for use as precursors were prepared in deionized water.
Apparatus UV–vis light absorption spectra and luminescence spectra were obtained using a Jasco V-570 UV/Vis/NIR spectrophotometer and a Jasco FP-750 spectrofluorometer (Tokyo, Japan), respectively. Transmission electron microscopy (TEM) experiments were carried out with a Philips CM30 300 kV TEM (Eindhoven, The Netherlands). The particle size distribution was determined using a Malvern ZEN3600 dynamic light scattering instrument (Birmingham, UK). X–ray diffraction (XRD) analyses were carried out with a Bruker D8/Advance X-ray diffractometer with Cu-Ka radiation (Washington, USA).
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Figure 1. The absorption and fluorescence spectra of synthesized glutathione-capped CdTe QDs (a). HRTEM images of synthesized glutathione-capped CdTe QDs (b). XRD pattern of synthesized glutathione-capped CdTe QDs (c). DLS analysis of synthesized glutathione-capped CdTe QDs (d).
B. Rezaei et al. Preparation of CdTe QDs The water-soluble glutathione-capped CdTe QDs were prepared according to the published protocol (23). In brief, 2.0 mL of 0.04 mol L–1 CdCl2 were diluted to 50 mL and trisodium citrate dihydrate (0.0500 g), glutathione (0.0250 g), Na2TeO3 (2.0 mL, 0.01 mol L–1) and NaBH4 (0.0250 g) were added under strong stirring. After reaction for 2 h at room temperature, the mixture was refluxed for 12 h at 90 °C. CdTe QDs could be stored in a dark container at 4 °C for several weeks. The absorption and fluorescence spectra are shown in Fig. 1(a). The absorption spectrum in Fig. 1(a) displays the broad absorption area and the peak of fluorescence emission in the same figure was noted at 615 nm. The sizes of the CdTe QDs were verified through TEM as shown in (Fig. 1b), this image demonstrates that the particles were rounded with an average particle size of 7 nm. Figure 1(c) shows XRD patterns of the as-prepared glutathionecapped CdTe QDs. For these nanoparticles, three characteristic peaks occur at 2θ of 111, 220 and 311. Dynamic light scattering (DLS) of the as-prepared glutathione-capped CdTe QDs in aqueous solution confirmed that the QDs have a narrow size distribution (see Fig. 1d). Absorption spectrum was applied for the calculation of CdTe QDs concentration (27). The final concentration of CdTe QDs was calculated to be 0.34 μmol L–1.
Measurement procedure For the determination of heparin, a freshly prepared mixture containing 30 μL of 34.0 nmol L–1 CdTe QD and an appropriate volume of sample or standard heparin solution were added to the vial and the final volume was made up to 3.0 mL with phosphate-buffered saline (pH 8.0). Then, the solution was transferred to a fluorimetric cell and its fluorescence spectra were recorded at the excitation wavelength of 400 nm in the wavelength range of 470–700 nm. The response function (ΔF = F – F0) values of the solution were used as an analytical signal, where F and F0 referred to the fluorescence intensity at 615 nm in the presence and absence of heparin, respectively.
Sample preparation Fresh human plasma samples of healthy volunteers without heparin treatment were taken from the Health Center of Isfahan University of Technology. A standard addition method was used for the determination of heparin in these plasma samples. After the dilution of plasma (10 times) an appropriate amount of standard heparin solution was spiked into plasma samples and then 1.0 mL of each sample was transferred into a vial, followed by the above measurement procedure.
Effect of sample solution pH
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One of the most commonly used methods for dispersing QDs in aqueous solutions is to modify their outer surface with anionic carboxylate groups. In the present study, glutathione was capped on the outer surface of CdTe QDs. The fluorescence of CdTe solution was dependent on its pH value. To find the optimum pH value, the response of the sensor was measured at different pH (in the range 5.0–9.0) using solutions containing 25 μL of 34 nmol L–1 CdTe QDs and 0.1 μg mL–1 heparin Fig. 2(a). As can
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–1
Figure 2. Effect of pH on the sensor response to heparin ions. Conditions 3.4 × 10 –1 –1 nmol L CdTe QD, 100.0 ng mL of heparin at different pH values (a). Influence of the concentration of CdTe QD on the sensor response to heparin ions. Conditions: –1 100.0 ng mL of heparin, pH 8 and different amounts of CdTe-QD (b). Incubation time –1 of the sensor at the optimum conditions for heparin at 100.0 ng mL (c).
be seen, pH 8.0 was the best value for the determination of heparin by the sensing method. The zeta potential of the glutathione-capped CdTe QDs was negative at pH 8.0 because the pKa of the –COOH group was 3.6 in glutathione. At acidic pH, heparin was protonated and interacted with the –COO group in glutathione, resulting in fluorescence quenching of QDs. It should be noted that at an adequately basic pH, electrostatic repulsion between QDs affords a stable colloidal suspension and the acidic pH yields insoluble aggregates of QDs (21). Heparin and glutathione molecules have
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2016; 31: 958–964
A simple and sensitive label-free heparin sensing some functional groups that can interact together by hydrogen bond formation on the QD surface. So, in a strong basic medium (pH > 8) OH– increases and can hinder the interaction between heparin and CdTe QDs by binding with the functional groups of heparin and glutathione molecules on the QD surface and form hydrogen bonds with them. Therefore this interaction decreased and the fluorescence response was also decreased. Also, at high pH an excess amount of OH led to the formation of Cd(OH)2 coated onto the surface of the CdTe QDs (28). Therefore, the optimum pH 8.0 was selected for further experiments.
Effect of CdTe QD concentration In order to optimize the CdTe QD concentration, several solutions with different volumes of 34 nmol L–1 CdTe QD (5 to 40 μL) were added to a vial; then the volume made up to 3.0 mL with PBS (pH 8.0) such that the final concentration of heparin was 100 ng mL–1. The results are shown in Fig. 2(b). It was found that the solution containing 3.4 × 10–1 nmol L–1 CdTe QD had the best
Figure 3. The structure of heparin.
response toward heparin. It was clear that decreasing the CdTe QD concentration reduced the sensitivity of the sensor. A higher concentration of CdTe QD could result in self-quenching of the QDs fluorescence and a decrease in sensor sensitivity. Incubation time Incubation time is an important factor for any optical sensor. The duration time between heparin addition into the sensing mixture and the detection of the fluorescence signal at optimum conditions was recorded as the incubation time. The results are shown in Fig. 2(c). The practical incubation time of the sensor for concentrations of 100 ng mL–1 was 5 sec.
Results and discussion Principle of the operation Heparin is not a single well known molecule, but it is rather typically considered as a whole family of polysaccharides with an average molecular weight of 12 000 to 15 000 and an average charge of 75. Under physiological conditions, heparin is a highly negatively charged glycosaminoglycan due to totally ionized sulfate (–OSO3, –NHSO3) and carboxylate (–COO) groups (29,30). The structure of heparin is shown in Fig. 3. As can be seen, heparin is an anionic polymer. It is a member of the glycosaminoglycan family of carbohydrates. Scheme 1 illustrates that the fluorescence of glutathionecapped CdTe QDs was raised in the presence of heparin. As is clear, both heparin and glutathione molecules have many sites that are able to form hydrogen bonds, and heparin is more negatively
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Scheme 1. Schematic illustration for the increasing quantum dots fluorescence in the presence of heparin.
B. Rezaei et al. charged than glutathione and is a heavy molecule. So when heparin is added to a solution containing glutathione-capped QDs, it can bond to glutathione on the QD surface and thereby become connected to it. The negative charges on the QD surface are increased and the repulsion forces between the QDs increase, so QD dispersion increases and aggregation decreases. Conversely, the QDs can be more rigid, consequently the fluorescence intensity of QDs was increased. Fluorescence emission spectra of mixtures containing different heparin concentrations are shown in Fig. 4(a). The response function (F – F0) values of the biosensor were obtained at different heparin concentrations. Figure 4(b) shows the calibration curve of heparin at the optimum conditions.
Table 1. Comparison of analytical data of various methods for the determination of heparin Method Voltammetric Fluorimetric HPLC Colorimetric Colorimetric Colorimetric Fluorimetric
Linear dynamic range (μg mL–1) 0.3–10.0 0.1–1.5 0.5–10.0 0.3–7.0 Up to 10 0.02–0.28 0.01–0.2
Detection limit (DL) (μg mL–1) 0.28 0.02462 0.2 0.1 0.6 0.005 0.002
Ref. 2 7 13 4 14 15 This work
Analytical figures of merit Under optimized conditions, ΔF values were linearly dependent on heparin concentration in the range 10–200 ng mL 1 with a regression equation of ΔF = 0.098C + 2.678 (where C is the concentration of heparin in ng/mL) and R2 = 0.994. The detection limit (DL) (3Sb/m, where S is the blank standard deviation (n = 10), and m is the slope of the calibration curve) was calculated to be 2 ng mL–1. In order to determine the accuracy of these measurements, 100 ng mL–1 of heparin solution was measured 10 times and the relative standard deviation (RSD)% was found to be 0.8. The merits in the present method were compared with other reported methods of heparin determination. The results are given in Table 1. As can be seen in Table 1, the detection limit of the proposed method was superior to that of other methods, because
–1
Selectivity Heparin is a large molecule (1-3) with some functional groups. When it is added to a solution containing glutathione-capped QDs, despite its negative charge, can connect to glutathione on the QD surface via formation of a hydrogen bond. The whole QD surface is covered, therefore the interaction of heparin with QDs is more than that of potentially interfering species. In this study, the pH of the solution was kept stable by the addition of phosphate-buffered saline (pH 8.0), thus the addition of different species such as dopamine could not affect the solution pH. The potential interference of a number of common species was investigated. For this purpose, the fluorescence response in the presence of 3.0 μg mL–1 of different species and 100 ng mL–1 heparin was determined. As can be seen from Fig. 5, the proposed sensor demonstrated good selectivity for heparin over other species. It should be mentioned that some heavy metal ions such as Cu2+ quench the fluorescence of thiol-capped CdTe QDs. The small cations, pass through the shell layer and interact with the core, resulting in chemical displacement of
–1
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Figure 4. Fluorescence spectra of the optical sensor. Conditions: 3.4 × 10 nmol L CdTe QDs contain different heparin concentrations: (1) 0; (2) 0. 01; (3) 0.02; (4) 0.05; (5) –1 0.10; (6) 0.15; or (7) 0.20 μg mL (a). Calibration graph for heparin at the optimum conditions (b).
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fluorescence methods have intrinsic sensitivity; in addition, a low amount of heparin could greatly affect the fluorescence intensity because it is a large molecule and it can cover the surfaces of the QDs and increase repulsion between them; therefore the detection limit was lower than that for other reported methods.
Figure 5. The fluorescence response of the mixture solution containing 30 μL CdTe –1 –1 –1 QD (3.4 × 10 nmol L ) pH 8 in the presence of 0.1 μg mL various species.
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2016; 31: 958–964
A simple and sensitive label-free heparin sensing Table 2. Recovery for the determination of heparin in plasma samples Sample
Heparin concentration(ng mL–1) Added
1
2
3
50.00 100.00 150.00 50.00 100.00 150.00 50.00 100.00 150.00
* Average values deviations.
Recovery (%)
Found* 52.10 ± 0.42 102.46 ± 0.13 147.82 ± 0.35 48.17 ± 2.00 101.38 ± 0.48 151.33 ± 0.33 51.56 ± 0.47 99.19 ± 0.22 149.67 ± 0.49 of
three
104.20 102.46 98.54 96.34 101.14 100.88 103.12 99.19 99.78
Heparin found*, HPLC (ng mL–1) – 93.78 ± 6.5 – – – – – – –
determinations ± standard
the Cd2+ ions at the surface QD core by Cu2+. However, this ion is usually present at very low concentrations (1.0 × 10–7 g/mL) in human plasma. Moreover, the proposed method is highly sensitive for heparin determination (31,32) and thus, this interference can be diminished by diluting the samples. Accordingly, the influence of Cu2+ (5.0 × 10–7 g/mL) on the fluorescence intensity of CdTe QDs was checked and the change in fluorescence intensity was found to be about 4.0%.
Application To further test the possibility of applying the method to a real sample matrix, recovery experiments were carried out by analysing spiked human serum samples. As can be seen in Table 2, the recovery and precision of the proposed method were acceptable. Also, to check the accuracy of the biosensor, HPLC was used to measure heparin in human serum samples. The mean heparin concentrations (ng mL–1) in the human serums sample were found to be 102.46 ± 0.13 (n = 3) by the HPLC method and 93.0 ± 7.8 (n = 3) by the proposed sensor. The t value was equal to 2.30 and table (95%,4) was equal to 2.78. Thus, the accuracy of the sensor was acceptable.
Conclusion
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The authors wish to thank the Isfahan University of Technology (IUT) Research Council and the Center of Excellence in Sensor and Green Chemistry for their support.
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In this study, a new optical sensing method was introduced for the detection of heparin based on CdTe QDs. The fluorescence intensity of this system was found to be enhanced in the presence of heparin. Under the optimized analytical conditions, this method could determine heparin in the range 10–200 ng mL–1, with a low detection limit of 2 ng mL–1. This system exhibited excellent selectivity, remarkable repeatability and a fast response. In contrast with most previous studies, this assay did not need various intermediate materials and did not require several laborious steps. In addition, the procedure was found to be very simple, rapid, and inexpensive. The proposed method was applied successfully to determine heparin in human plasma with suitable recovery in the matrix; therefore, it could be used as a routine method for the analysis of heparin in physiological fluids.
Acknowledgements
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28. Zhang YH, Zhang HS, Ma M, Guo XF, Wang H. The influence of ligands on the preparation and optical properties of water-soluble CdTe quantum dots. Appl Surf Sci 2009;255:4747–53. 29. Langmaier J, Samcov E, Samec Z. Potentiometric. sensor for heparin polyion: transient behavior and response mechanism. Anal Chem 2007;79:2892–900. 30. Bunkoed O, Kanatharana P. Mercaptopropionic acid-capped CdTe quantum dots as fluorescence probe for the determination of salicylic acid in pharmaceutical products. Luminescence 2015. DOI:10.1002/ bio.2862 [Epub ahead of print]. 31. McMillin GA, Travis JJ, Hunt JW. Direct measurement of free copper in serum or plasma ultrafiltrate. Am J Clin Pathol 2009;131:160–5. 32. Kubala-Kukuś A, Banaś D, Braziewicz J, Majewska U, Pajek M, Wudarczyk-Moćko J, Antczak G, Borkowska B, Góźdź S, Smok-Kalwat J. Analysis of copper concentration in human serum by application of total reflection X-ray fluorescence method. Biol Trace Elem Res 2014;158:22–8.
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Luminescence 2016; 31: 958–964
Revised: 9 October 2015,
Accepted: 9 October 2015
Published online in Wiley Online Library: 5 November 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.3058
A simple and sensitive label-free fluorescence sensing of heparin based on Cdte quantum dots B. Rezaei,* M. Shahshahanipour and Ali A. Ensafi ABSTRACT: 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. Keywords: heparin; CdTe quantum dot; fluorescence spectroscopy; label-free
Introduction
958
Heparin is an important natural biomolecule that is normally extracted and purified from animal tissues, especially from porcine and bovine (1). This drug can be used as an anti-coagulant, antithrombotic, anti-lipemic, anti-atherosclerosis, anti-phlogistic and anti-allergic product (2). The use of heparin avoids the formation of clots in blood vessels before or after surgery or during certain medical procedures. Also, it is used to treat certain blood, heart, and lung disorders and helps in the diagnosis and treatment of certain bleeding disorders. It has been widely used in clinical therapy for more than 60 years and it is still regarded as the first option to avoid thrombosis and cure urgent vein thrombus (3). High doses of heparin can counteract some undesirable effects (such as internal bleedings, puke, loss of consciousness and headache) (4). Beyond the requirement for simple, accurate and real-time determinations of heparin levels in patient serum during surgery and postoperative remedy period, there is also the need for detection methods that can check the heparin levels in infusion solutions to prevent the risk of human errors in dosing. Conventional clinical methods for heparin detection rely on the measurement of activated clotting time or activated partial thromboplastin time (5). Different methods have been developed for heparin measurement, including resonance Rayleigh scattering (6), fluorimetry (3,6-8), electrochemical sensor (2,9-11), capillary electrophoresis (12), high performance liquid chromatography (HPLC) (13) and colorimetric methods (4,14,15). These methods are not sufficiently reliable and exact for clinical settings because of their lack of specificity and possible interference with other factors (16). The main limitations and disadvantages of HPLC are the cost of equipment, the use of environmentally dangerous solvents and the co-elution of compounds. The major drawback of capillary electrophoresis is its complex assay validation. Electrochemical sensors have a number of limitations such as the electroactivity of certain
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species and as such electrochemically active interference in the sample (17). The main limitation of colorimetric methods is its low sensitivity. In addition, some of these methods are not appropriate for use outside the laboratory or for field monitoring. Fluorescence sensors have many attractive advantages, including high sensitivity, remote control, inexpensiveness, easy recognition, and an especially suitable diagnostic device for analytical concerns. A few techniques have also been reported for determination of heparin applying QDs. Cao and et al. utilized the Ru complex, which quenched CdTe QD fluorescence (18). Heparin addition removed the quencher from the QD surface and led to fluorescence reclamation by the CdTe QDs. Zhang and coworkers also used MPA (3-mercaptopropionic acid) capped Mn-doped ZnS quantum dots and polybrene (hexadimethrine bromide) for the application of a room temperature phosphorescence (RTP) determination of heparin (19). In their study, the RTP intensity of QDs was strongly enhanced after the addition of polybrene. Heparin could remove polybrene from the surface of QDs, thus the RTP intensity of Mn-doped ZnS QDs was decreased with increasing heparin concentration. In another study, Liu and coworkers applied L-cysteine-capped CuInS2 QDs (20). Heparin could aggregate the QDs via electrostatic force and therefore decreased the intensity
* Correspondence to: B. Rezaei, Department of Chemistry, Isfahan University of Technology, Isfahan 84156–83111, Iran. E-mail: [email protected] Department of Chemistry, Isfahan University of Technology, Isfahan8415683111, Iran Abbreviations: DLS, dynamic light scattering; HPLC, high performance liquid chromatography; IUT, Isfahan University of Technology; QY, quantum yields; RTP, room temperature phosphorescence; TEM, transmission electron microscopy; XRD, X-ray diffraction.
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A simple and sensitive label-free heparin sensing of QD fluorescence. The addition of heparinase to this system caused de-aggregation of the L-cysteine capped CuInS2 QDs and increased the fluorescence intensity. Most of these assays require several laborious steps and several intermediate materials, therefore they are moderately expensive and time consuming. Quantum dots are also known as zero-dimensional particles, and semiconductor nanocrystals (21). QDs are brightly luminescent nanoparticles that have found remarkable applications in recent decades in sensing events as luminescent probes, because of excellent photophysical properties such as their wide absorption spectra and narrow photoluminescence spectrum (21,22). Colloidal nanocrystals QDs have about 10–20 times more brilliant fluorescence and their photodurability is 100–200 times better than that of organic dyes. The emission wavelengths of QDs can be set by size, shape and composition, leading to high flexibility in the choice of emission wavelength (23). SHINER photoluminescence is the result of great quantum yields assisted with large molar extinction coefficients. One of the ordinary methods used for dispersing QDs in aqueous solution is to modify their exterior surface with anionic carboxylate groups. At a suitable basic pH, electrostatic repulsion between QDs indicated an immutable colloidal suspension (21). In this study water-soluble glutathione-capped CdTe QDs were used as a fluorescent probe as they display very stable luminescence under physiological conditions depending on their particle size, and are synthesized easily, are biocompatible and are monodispersed. Glutathione-capped CdTe QDs also have higher quantum yields (QYs) than the CdTe QDs capped with other thiol compounds, without any post-preparative treatment. These values were comparable with, or even better than, most QDs prepared because of a better surface access of the CdTe crystalline lattice by glutathione (24-26). The unique advantages of this method are its very fast process, its low cost, and its ease of production
and the high sensitivity switch in methods for heparin sensing in human serum samples.
Experimental Chemicals Glutathione, Na2TeO3, CdCl2 and NaBH4 were purchased from Aldrich (Darmstadt, Germany). Heparin sodium salt was obtained from PanReac AppliChem (Darmstadt, Germany). All chemicals were of analytical reagent grade (with the highest degree of purity available) and deionized water was used throughout all experiments. A 50 μg mL–1 stock solution of heparin was prepared by dissolving an appropriate amount of heparin sodium salt into a 100-mL standard flask. Lower heparin concentrations were obtained by sequential dilution of the stock solution. A 0.040 mol L–1 solution CdCl2 and a Na2TeO3 solution (0.010 mol L–1) for use as precursors were prepared in deionized water.
Apparatus UV–vis light absorption spectra and luminescence spectra were obtained using a Jasco V-570 UV/Vis/NIR spectrophotometer and a Jasco FP-750 spectrofluorometer (Tokyo, Japan), respectively. Transmission electron microscopy (TEM) experiments were carried out with a Philips CM30 300 kV TEM (Eindhoven, The Netherlands). The particle size distribution was determined using a Malvern ZEN3600 dynamic light scattering instrument (Birmingham, UK). X–ray diffraction (XRD) analyses were carried out with a Bruker D8/Advance X-ray diffractometer with Cu-Ka radiation (Washington, USA).
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Figure 1. The absorption and fluorescence spectra of synthesized glutathione-capped CdTe QDs (a). HRTEM images of synthesized glutathione-capped CdTe QDs (b). XRD pattern of synthesized glutathione-capped CdTe QDs (c). DLS analysis of synthesized glutathione-capped CdTe QDs (d).
B. Rezaei et al. Preparation of CdTe QDs The water-soluble glutathione-capped CdTe QDs were prepared according to the published protocol (23). In brief, 2.0 mL of 0.04 mol L–1 CdCl2 were diluted to 50 mL and trisodium citrate dihydrate (0.0500 g), glutathione (0.0250 g), Na2TeO3 (2.0 mL, 0.01 mol L–1) and NaBH4 (0.0250 g) were added under strong stirring. After reaction for 2 h at room temperature, the mixture was refluxed for 12 h at 90 °C. CdTe QDs could be stored in a dark container at 4 °C for several weeks. The absorption and fluorescence spectra are shown in Fig. 1(a). The absorption spectrum in Fig. 1(a) displays the broad absorption area and the peak of fluorescence emission in the same figure was noted at 615 nm. The sizes of the CdTe QDs were verified through TEM as shown in (Fig. 1b), this image demonstrates that the particles were rounded with an average particle size of 7 nm. Figure 1(c) shows XRD patterns of the as-prepared glutathionecapped CdTe QDs. For these nanoparticles, three characteristic peaks occur at 2θ of 111, 220 and 311. Dynamic light scattering (DLS) of the as-prepared glutathione-capped CdTe QDs in aqueous solution confirmed that the QDs have a narrow size distribution (see Fig. 1d). Absorption spectrum was applied for the calculation of CdTe QDs concentration (27). The final concentration of CdTe QDs was calculated to be 0.34 μmol L–1.
Measurement procedure For the determination of heparin, a freshly prepared mixture containing 30 μL of 34.0 nmol L–1 CdTe QD and an appropriate volume of sample or standard heparin solution were added to the vial and the final volume was made up to 3.0 mL with phosphate-buffered saline (pH 8.0). Then, the solution was transferred to a fluorimetric cell and its fluorescence spectra were recorded at the excitation wavelength of 400 nm in the wavelength range of 470–700 nm. The response function (ΔF = F – F0) values of the solution were used as an analytical signal, where F and F0 referred to the fluorescence intensity at 615 nm in the presence and absence of heparin, respectively.
Sample preparation Fresh human plasma samples of healthy volunteers without heparin treatment were taken from the Health Center of Isfahan University of Technology. A standard addition method was used for the determination of heparin in these plasma samples. After the dilution of plasma (10 times) an appropriate amount of standard heparin solution was spiked into plasma samples and then 1.0 mL of each sample was transferred into a vial, followed by the above measurement procedure.
Effect of sample solution pH
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One of the most commonly used methods for dispersing QDs in aqueous solutions is to modify their outer surface with anionic carboxylate groups. In the present study, glutathione was capped on the outer surface of CdTe QDs. The fluorescence of CdTe solution was dependent on its pH value. To find the optimum pH value, the response of the sensor was measured at different pH (in the range 5.0–9.0) using solutions containing 25 μL of 34 nmol L–1 CdTe QDs and 0.1 μg mL–1 heparin Fig. 2(a). As can
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Figure 2. Effect of pH on the sensor response to heparin ions. Conditions 3.4 × 10 –1 –1 nmol L CdTe QD, 100.0 ng mL of heparin at different pH values (a). Influence of the concentration of CdTe QD on the sensor response to heparin ions. Conditions: –1 100.0 ng mL of heparin, pH 8 and different amounts of CdTe-QD (b). Incubation time –1 of the sensor at the optimum conditions for heparin at 100.0 ng mL (c).
be seen, pH 8.0 was the best value for the determination of heparin by the sensing method. The zeta potential of the glutathione-capped CdTe QDs was negative at pH 8.0 because the pKa of the –COOH group was 3.6 in glutathione. At acidic pH, heparin was protonated and interacted with the –COO group in glutathione, resulting in fluorescence quenching of QDs. It should be noted that at an adequately basic pH, electrostatic repulsion between QDs affords a stable colloidal suspension and the acidic pH yields insoluble aggregates of QDs (21). Heparin and glutathione molecules have
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A simple and sensitive label-free heparin sensing some functional groups that can interact together by hydrogen bond formation on the QD surface. So, in a strong basic medium (pH > 8) OH– increases and can hinder the interaction between heparin and CdTe QDs by binding with the functional groups of heparin and glutathione molecules on the QD surface and form hydrogen bonds with them. Therefore this interaction decreased and the fluorescence response was also decreased. Also, at high pH an excess amount of OH led to the formation of Cd(OH)2 coated onto the surface of the CdTe QDs (28). Therefore, the optimum pH 8.0 was selected for further experiments.
Effect of CdTe QD concentration In order to optimize the CdTe QD concentration, several solutions with different volumes of 34 nmol L–1 CdTe QD (5 to 40 μL) were added to a vial; then the volume made up to 3.0 mL with PBS (pH 8.0) such that the final concentration of heparin was 100 ng mL–1. The results are shown in Fig. 2(b). It was found that the solution containing 3.4 × 10–1 nmol L–1 CdTe QD had the best
Figure 3. The structure of heparin.
response toward heparin. It was clear that decreasing the CdTe QD concentration reduced the sensitivity of the sensor. A higher concentration of CdTe QD could result in self-quenching of the QDs fluorescence and a decrease in sensor sensitivity. Incubation time Incubation time is an important factor for any optical sensor. The duration time between heparin addition into the sensing mixture and the detection of the fluorescence signal at optimum conditions was recorded as the incubation time. The results are shown in Fig. 2(c). The practical incubation time of the sensor for concentrations of 100 ng mL–1 was 5 sec.
Results and discussion Principle of the operation Heparin is not a single well known molecule, but it is rather typically considered as a whole family of polysaccharides with an average molecular weight of 12 000 to 15 000 and an average charge of 75. Under physiological conditions, heparin is a highly negatively charged glycosaminoglycan due to totally ionized sulfate (–OSO3, –NHSO3) and carboxylate (–COO) groups (29,30). The structure of heparin is shown in Fig. 3. As can be seen, heparin is an anionic polymer. It is a member of the glycosaminoglycan family of carbohydrates. Scheme 1 illustrates that the fluorescence of glutathionecapped CdTe QDs was raised in the presence of heparin. As is clear, both heparin and glutathione molecules have many sites that are able to form hydrogen bonds, and heparin is more negatively
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Scheme 1. Schematic illustration for the increasing quantum dots fluorescence in the presence of heparin.
B. Rezaei et al. charged than glutathione and is a heavy molecule. So when heparin is added to a solution containing glutathione-capped QDs, it can bond to glutathione on the QD surface and thereby become connected to it. The negative charges on the QD surface are increased and the repulsion forces between the QDs increase, so QD dispersion increases and aggregation decreases. Conversely, the QDs can be more rigid, consequently the fluorescence intensity of QDs was increased. Fluorescence emission spectra of mixtures containing different heparin concentrations are shown in Fig. 4(a). The response function (F – F0) values of the biosensor were obtained at different heparin concentrations. Figure 4(b) shows the calibration curve of heparin at the optimum conditions.
Table 1. Comparison of analytical data of various methods for the determination of heparin Method Voltammetric Fluorimetric HPLC Colorimetric Colorimetric Colorimetric Fluorimetric
Linear dynamic range (μg mL–1) 0.3–10.0 0.1–1.5 0.5–10.0 0.3–7.0 Up to 10 0.02–0.28 0.01–0.2
Detection limit (DL) (μg mL–1) 0.28 0.02462 0.2 0.1 0.6 0.005 0.002
Ref. 2 7 13 4 14 15 This work
Analytical figures of merit Under optimized conditions, ΔF values were linearly dependent on heparin concentration in the range 10–200 ng mL 1 with a regression equation of ΔF = 0.098C + 2.678 (where C is the concentration of heparin in ng/mL) and R2 = 0.994. The detection limit (DL) (3Sb/m, where S is the blank standard deviation (n = 10), and m is the slope of the calibration curve) was calculated to be 2 ng mL–1. In order to determine the accuracy of these measurements, 100 ng mL–1 of heparin solution was measured 10 times and the relative standard deviation (RSD)% was found to be 0.8. The merits in the present method were compared with other reported methods of heparin determination. The results are given in Table 1. As can be seen in Table 1, the detection limit of the proposed method was superior to that of other methods, because
–1
Selectivity Heparin is a large molecule (1-3) with some functional groups. When it is added to a solution containing glutathione-capped QDs, despite its negative charge, can connect to glutathione on the QD surface via formation of a hydrogen bond. The whole QD surface is covered, therefore the interaction of heparin with QDs is more than that of potentially interfering species. In this study, the pH of the solution was kept stable by the addition of phosphate-buffered saline (pH 8.0), thus the addition of different species such as dopamine could not affect the solution pH. The potential interference of a number of common species was investigated. For this purpose, the fluorescence response in the presence of 3.0 μg mL–1 of different species and 100 ng mL–1 heparin was determined. As can be seen from Fig. 5, the proposed sensor demonstrated good selectivity for heparin over other species. It should be mentioned that some heavy metal ions such as Cu2+ quench the fluorescence of thiol-capped CdTe QDs. The small cations, pass through the shell layer and interact with the core, resulting in chemical displacement of
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Figure 4. Fluorescence spectra of the optical sensor. Conditions: 3.4 × 10 nmol L CdTe QDs contain different heparin concentrations: (1) 0; (2) 0. 01; (3) 0.02; (4) 0.05; (5) –1 0.10; (6) 0.15; or (7) 0.20 μg mL (a). Calibration graph for heparin at the optimum conditions (b).
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fluorescence methods have intrinsic sensitivity; in addition, a low amount of heparin could greatly affect the fluorescence intensity because it is a large molecule and it can cover the surfaces of the QDs and increase repulsion between them; therefore the detection limit was lower than that for other reported methods.
Figure 5. The fluorescence response of the mixture solution containing 30 μL CdTe –1 –1 –1 QD (3.4 × 10 nmol L ) pH 8 in the presence of 0.1 μg mL various species.
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A simple and sensitive label-free heparin sensing Table 2. Recovery for the determination of heparin in plasma samples Sample
Heparin concentration(ng mL–1) Added
1
2
3
50.00 100.00 150.00 50.00 100.00 150.00 50.00 100.00 150.00
* Average values deviations.
Recovery (%)
Found* 52.10 ± 0.42 102.46 ± 0.13 147.82 ± 0.35 48.17 ± 2.00 101.38 ± 0.48 151.33 ± 0.33 51.56 ± 0.47 99.19 ± 0.22 149.67 ± 0.49 of
three
104.20 102.46 98.54 96.34 101.14 100.88 103.12 99.19 99.78
Heparin found*, HPLC (ng mL–1) – 93.78 ± 6.5 – – – – – – –
determinations ± standard
the Cd2+ ions at the surface QD core by Cu2+. However, this ion is usually present at very low concentrations (1.0 × 10–7 g/mL) in human plasma. Moreover, the proposed method is highly sensitive for heparin determination (31,32) and thus, this interference can be diminished by diluting the samples. Accordingly, the influence of Cu2+ (5.0 × 10–7 g/mL) on the fluorescence intensity of CdTe QDs was checked and the change in fluorescence intensity was found to be about 4.0%.
Application To further test the possibility of applying the method to a real sample matrix, recovery experiments were carried out by analysing spiked human serum samples. As can be seen in Table 2, the recovery and precision of the proposed method were acceptable. Also, to check the accuracy of the biosensor, HPLC was used to measure heparin in human serum samples. The mean heparin concentrations (ng mL–1) in the human serums sample were found to be 102.46 ± 0.13 (n = 3) by the HPLC method and 93.0 ± 7.8 (n = 3) by the proposed sensor. The t value was equal to 2.30 and table (95%,4) was equal to 2.78. Thus, the accuracy of the sensor was acceptable.
Conclusion
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The authors wish to thank the Isfahan University of Technology (IUT) Research Council and the Center of Excellence in Sensor and Green Chemistry for their support.
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In this study, a new optical sensing method was introduced for the detection of heparin based on CdTe QDs. The fluorescence intensity of this system was found to be enhanced in the presence of heparin. Under the optimized analytical conditions, this method could determine heparin in the range 10–200 ng mL–1, with a low detection limit of 2 ng mL–1. This system exhibited excellent selectivity, remarkable repeatability and a fast response. In contrast with most previous studies, this assay did not need various intermediate materials and did not require several laborious steps. In addition, the procedure was found to be very simple, rapid, and inexpensive. The proposed method was applied successfully to determine heparin in human plasma with suitable recovery in the matrix; therefore, it could be used as a routine method for the analysis of heparin in physiological fluids.
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