Anion-Exchange-Based Amperometric Assay for Heparin Using

Publication Date (Web): February 28, 2013 ... 2013, 85, 6, 3439-3445 ... Monitoring of Heparin Activity in Live Rats Using Metal–Organic Framework ...
1 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/ac

Anion-Exchange-Based Amperometric Assay for Heparin Using Polyimidazolium as Synthetic Receptor Hetong Qi, Li Zhang, Lifen Yang, Ping Yu,* and Lanqun Mao* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, The Chinese Academy of Sciences (CAS), Beijing 100190, China S Supporting Information *

ABSTRACT: This study demonstrates a facile yet effective strategy for amperometric assay of electrochemically inactive heparin based on an anionexchange mechanism with polyimidazolium (Pim) as the synthetic receptor. The rationale for the amperometric heparin assay is essentially based on the different binding affinity of the synthetic Pim receptor toward electrochemically active ferricyanide (Fe(CN)63−) and electrochemically inactive heparin. To accomplish the amperometric assay, Pim is first synthesized and used as the artificial receptor to recognize the anions (i.e., Fe(CN)63− and heparin). The stronger binding affinity of the synthetic Pim receptor toward heparin than toward Fe(CN)63− essentially validates the amperometric heparin assay through an anion-exchange mechanism with the decrease in the redox peak current of Fe(CN)63− adsorbed onto the Pim film as the signal readout. The anion exchange between Fe(CN)63− and heparin on the Pim receptor is verified by cyclic voltammetry and Fourier transform IR and UV−visible spectroscopies. The ratio of the current decrease shows a linear relationship with heparin concentration with a concentration range from 0.5 to 10 μM. With animal experiments by dosing intraperitoneally and collecting the serum sample, the method is demonstrated to be potentially useful for investigating heparin metabolism in the biological system. This study not only provides a simple yet effective route to a heparin assay but also opens a new way to developing amperometric methods for electrochemically inert species by fully utilizing the supramolecular principles.

W

As one kind of electrochemically inactive molecule under normal experimental conditions, heparin plays important roles in regulation of various normal physiological and pathological processes such as blood coagulation and inflammatory response, cell growth, immune defense, lipid transport, and metabolism.5 Simple quantitative monitoring of heparin during surgery and anticoagulant therapy is of crucial significance for understanding the molecular basis of physiological and pathological events.6 So far, some methods have been developed for the heparin assay including the traditional activated coagulation time, the activated partial thromboplastin time, the protamine complexation method, and the recently developed colorimetric and fluorescence methods.7 More strikingly, some elegant potentiometric methods have previously been demonstrated for the heparin assay, since this kind of method exhibits analytical advantages in, for example, method simplicity, high sensitivity, and ease in automation.8 Nevertheless, it still remains a great challenge for the direct amperometric assay of heparin with electrochemical mechanisms9 because of the poor electrochemical property of heparin and no specific natural receptors available for heparin.

ith the rapid development and formulation of supramolecular principles, recognition and sensing of anionic species by using synthetic receptors has recently emerged as one of the key research fields, since the efficient combination of supramolecular principles with analytical mechanisms (i.e., signal readout) brings new opportunities for the development of analytical chemistry.1 For example, by combining synthetic receptors with the fluorescence mechanism, some elegant methods have been developed for sensing and recognizing various anionic species.2 On the other hand, as one of the most important research communities in analytical chemistry, electrochemical methods have been well documented to be particularly attractive for various analytical purposes because of their high sensitivity, a wide linear range, and low-cost instrumentation.3 One of the longstanding challenges in electroanalytical chemistry lies in its limitation in the amperometric assay of electrochemically inactive species. In this regard, introducing supramolecular principles into electroanalytical chemistry would potentially broaden the analytical targets of electrochemical methods into the species that are electrochemically inactive under normal experimental conditions presumably based on the principles of supramolecular recognition.4 In spite of this great potentiality, the uses of supramolecular principles to develop new electroanalytical methods have scarcely been reported so far. © 2013 American Chemical Society

Received: January 20, 2013 Accepted: February 28, 2013 Published: February 28, 2013 3439

dx.doi.org/10.1021/ac400201c | Anal. Chem. 2013, 85, 3439−3445

Analytical Chemistry

Article

the anion exchange between surface-confined Fe(CN)63− and heparin occurring onto the electrode surface. Such a property eventually decreases the electrochemical response of surfaceconfined Fe(CN)63−, which could form a straightforward basis for the amperometric heparin assay. With the animal experiments by dosing intraperitoneally, the method is demonstrated to be potentially useful for investigating the metabolism of heparin in biological systems. As far as we know, this is the first example on direct amperometric sensing of electrochemically inactive heparin with an electrochemical mechanism, which not only offers a new and simple route to the investigation on the heparin metabolism but also opens a new way to developing amperometric methods for the electrochemically inactive molecules based on supramolecular principles.

The key point to develop the anion sensors based on supramolecular principles is the design of the synthetic receptors. Among the various types of anion receptors, imidazolium-based receptors have received a great deal of attention because they can form strong (C−H)+···X− ionic hydrogen bonds with various anions.10 Moreover, the physical and chemical properties of imidazolium-based synthetic receptors could be easily tuned by varying the substituent groups and/or degree of polymerization. More importantly, the cation−π and hydrophobic interactions between imidazolium and carbon nanotubes could make this kind of receptor very attractive for the development of electrochemical methods.11 This is because, on one hand, the nanocomposite formed by carbon nanotubes and imidazolium is very easily to be stably confined onto an electrode surface to form the electrochemical sensors. On the other hand, the existence of carbon nanotubes well facilitates the electron transfer, which is very favorable for the electrochemical analysis. In this study, we demonstrate a facile yet effective amperometric method for the heparin assay with polyimidazolium (Pim) as the synthetic receptor. To accomplish the assay for electrochemically inactive heparin, Pim is first synthesized and used as the artificial receptor to recognize the anions (i.e., Fe(CN)63− and heparin). As shown in Scheme 1, Pim could be

■

EXPERIMENTAL SECTION Reagents and Solutions. Heparin was purchased from Sigma and used as supplied. Potassium ferricyanide (K3Fe(CN)6) was obtained from Beijing Chemical Co. (Beijing, China). Phosphate-buffered solution (PBS, 0.10 M, pH 7.0) was prepared with Na2HPO4 and NaH2PO4. Azo-bis-isobutryonitrile (AIBN), 1-vinylimidazole, and 1-chlorobutane were obtained from Aldrich. Multiwalled carbon nanotubes (MWCNTs) were purchased from Shenzhen Nanotech Port Co., Ltd. (Shenzhen, China) and used as received. Other chemicals were of at least analytical grade reagents and were used as received. All aqueous solutions were prepared with Milli-Q water. All electrochemical experiments were performed at room temperature. Synthesis of Pim. Synthesis of poly(1-vinyl-3-butylimidazolium chloride) (i.e., Pim) was performed according to a previous report.12 Briefly, 1-vinylimidazole (9.6 g, 102 mM) and 1-chlorobutane (29.6 g, 320 mM) were mixed into a threenecked flask, and the mixture was then stirred vigorously for 75 h in an oil bath of 70 °C under nitrogen atmosphere. After cooling to room temperature, the top phase was decanted, and the bottom viscous liquid was first washed by ethyl acetate for three times and then filtered and dried under vacuum at 50 °C overnight to obtain [Vbim][Cl] as a white solid. [Vbim][Cl]: 1 H NMR (400 MHz, D2O) δ = 0.92 (t, 3H), 1.34 (m, 2H), 1.88 (m, 2H), 4.23 (t, 2H), 5.42 (dd, 1H), 5.80 (dd, 1H), 7.14 (dd, 1H), 7.57 (s, 1H), 7.76 (s, 1H) (NMR data, Figure S1, Supporting Information). Pim was synthesized by free radical polymerization, as reported previously.11 Typically, [Vbim][Cl] (3.0 g), AIBN (0.015 g), and chloroform (30 mL) were mixed into a three-necked flask in an oil bath of 60 °C under nitrogen atmosphere, and the mixture was then stirred for 18 h. After that, the mixture was cooled to room temperature and allowed to precipitate with the addition of ethyl ether. The resulting precipitate was redissolved into water, and the solution was dialyzed (cutoff, 10 000) in water for 2 days. The solution left in the dialysis bag was concentrated with a rotary evaporator at 45 °C, and the obtained product was dried under vacuum at 50 °C overnight to give poly(1-vinyl-3-butylimidazolium chloride) (NMR data, Figure S2, Supporting Information). Electrode Preparation and Apparatus. Glassy carbon electrodes (GC, 3 mm in diameter) were first polished on emery paper, then with aqueous slurries of alumina powder (0.3 and 0.05 μm) on a polishing cloth, and finally rinsed with MilliQ water under an ultrasonic bath for 10 min. To prepare the Pim/MWCNT nanocomposite, 1 mg of MWCNTs and 3 mg of synthetic Pim receptor were mixed into 1 mL of water, and

Scheme 1. (A) Schematic Illustration for the Amperometric Heparin Assay Based on the Anion-Exchange Mechanism with Imidazolium-Based Polycation as the Synthetic Receptor; (B) Chemical Structures of Heparin and Pim

stably immobilized onto an electrode surface by forming a nanocomposite with carbon nanotubes, on which the electroactive probe Fe(CN)63− could be stably confined due to the electrostatic and hydrogen-bonding interactions between Fe(CN)63− and Pim, as reported in our previous work.11a We herein find that the synthetic Pim receptor exhibits a stronger affinity toward heparin than toward Fe(CN)63−. This strong binding affinity between Pim and heparin essentially results in 3440

dx.doi.org/10.1021/ac400201c | Anal. Chem. 2013, 85, 3439−3445

Analytical Chemistry

Article

Figure 1. (A) UV−vis spectra of pure 200 μM K3Fe(CN)6 (black curve) and the mixture of 0.2 mg/mL Pim and 200 μM K3Fe(CN)6 in the absence (red curve) and presence (blue curve) of 1 mM heparin. Inset, digital pictures of the mixture containing Pim and K3Fe(CN)6 with the presence of 1 mM heparin (vial 1), the mixture of Pim and heparin (vial 2), and the mixture of Pim and K3Fe(CN)6 (vial 3). These mixtures were centrifuged at 5000 rpm for 5 min. (B) Cyclic voltammograms (CVs) obtained at the bare GC electrode in 1 mM K3Fe(CN)6 (black curve) and in the mixture of 0.5 mg/mL Pim and 1 mM K3Fe(CN)6 in the absence (red curve) and the presence (blue curve) of 1 mM heparin. Red and blue curves were recorded after the addition of Pim and heparin into the solution for 15 min.

MWCNT-modified GC electrodes were first immersed in 0.10 M PBS (pH 7.0) containing different concentrations of heparin for 15 min and then taken out of the solution and rinsed with water. The electrodes were subsequently immersed into 0.10 M pure PBS for the electrochemical measurements. UV−visible (UV−vis) spectra were recorded on a TU-1900 spectrometer (Beijing, China), and Fourier transform IR (FT-IR) spectra were obtained on a Tensor-27 FT-IR spectrometer (Bruker) with KBr pellet. Amperometric Assay of Heparin in Rat Serum. Animal experiments were performed as reported previously.13 Briefly, adult male Sprague−Dawley rats (250−300 g) obtained from Health Science Center, Peking University, were housed on a 12:12 h light−dark schedule with food and water ad libitum. Heparin was dosed intraperitoneally with 2 mL of a 100 μM solution in normal saline (0.9%). After the dosing for 40 min, 3 mL of blood was first taken from the abdominal aorta of the rats and transferred to a centrifuge tube and then centrifuged for 15 min at 3000 rpm. The resulting upper liquid−serum was frozen at −20 °C. A 1 mL amount of serum was diluted with 3 mL of 0.10 M PBS before electrochemical determination. Throughout the surgery, the body temperature of the animals was maintained at 37 °C with a heating pad.

the resulting mixture was then sonicated for 1 h to form a homogeneous dispersion. A 5 μL amount of the as-formed dispersion was dip-coated onto GC electrodes. The nanocomposite was found to be stably confined onto the electrode surface. The electrodes (denoted as Pim/MWCNT-modified GC electrodes, hereafter) were then dried at ambient temperature and used for electrochemical measurements. The Pim/MWCNT-modified GC electrodes were subjected to consecutive potential scanning from −0.2 to 0.6 V for 60 cycles at the scan rate of 50 mV s−1 in 0.10 M PBS containing 1 mM K3Fe(CN)6 (Figure S3 B, Supporting Information). The resulting electrodes (i.e., Fe(CN)63−/Pim/MWCNT-modified GC electrodes) were then taken out of the solution and consecutively scanned in 0.10 M pure PBS within a potential window from −0.2 to 0.6 V at the scan rate of 50 mV s−1 until a stable peak current was obtained for the surface-confined Fe(CN)63− (typically 20 cycles, Figure S4 A, Supporting Information). The electrodes were finally dried at room temperature before electrochemical measurements. For amperometric sensing of heparin, the Fe(CN)63−/Pim/MWCNTmodified GC electrodes was first immersed in the heparin solution with different concentrations for a certain time and then taken out of the solution and rinsed with ultrawater to remove the physically adsorbed heparin. The resulting electrode was then immersed into the PBS solution for the electrochemical determination. Cyclic voltammetry with the Fe(CN)63−/Pim/MWCNT-modified GC electrodes in 0.10 M PBS after the electrodes were immersed into 1 μM heparin for different times demonstrates that the anion-exchange between surface-confined Fe(CN)63− and heparin could reach a balance in 15 min (Figure S5, Supporting Information). Therefore, 15 min was used as the exchange time for the heparin assay in this study. Electrochemical measurements were carried out using a CHI 660C electrochemical workstation (Shanghai Chenhua Instrument Co. Ltd., China) with a conventional three-electrode system, in which the modified GC electrodes were used as the working electrode, platinum wire as the auxiliary electrode, and Ag/AgCl (KCl-saturated) as the reference electrode. For the amperometric heparin assay, the prepared Fe(CN)63−/Pim/

■

RESULTS AND DISCUSSION Anion Exchange between Fe(CN)63− and Heparin with Synthetic Pim Receptor. To investigate the anion exchange between Fe(CN)63− and heparin with the synthetic Pim receptor, the influence of heparin on the stability of the Fe(CN)63−/Pim nanocomposite was first studied by UV−vis spectroscopy and cyclic voltammetry. As reported in our earlier work, Fe(CN)63− and Pim could form a stable nanocomposite in water.11a As shown in Figure 1A, the aqueous solution of Fe(CN)63− exhibits several absorption peaks at about 260, 302, and 420 nm (black curve).14 The addition of Pim into the Fe(CN)63− solution results in a redshift of the absorption peaks to 264, 305, and 422 nm (red curve), whereas the addition of heparin into the mixture of Pim and K3Fe(CN)6 leads to the absorption peak of K3Fe(CN)6 shifted back to the position of pure Fe(CN)63− solution (blue curve). These results essentially 3441

dx.doi.org/10.1021/ac400201c | Anal. Chem. 2013, 85, 3439−3445

Analytical Chemistry

Article

Figure 2. (A) FT-IR spectra of pure heparin (red curve), pure Pim (black curve), and the composite of heparin and Pim (blue curve). (B) FT-IR spectra for pure K3Fe(CN)6 (red curve), the nanocomposite of Fe(CN)63−/Pim (black curve), and the nanocomposite of Fe(CN)63−/Pim after exchanging with heparin (blue curve).

results essentially indicate that there is a strong interaction between the imidazole ring in the Pim receptor and the SO3− in heparin, such as the electrostatic interaction and/or hydrophobic interactions between the polymers. As described above, Fe(CN)63− could also form a stable nanocomposite with the Pim receptor, possibly through electrostatic and hydrogenbonding interactions, according to our previous report.11a This strong interaction could also induce the shift of the characteristic peak of Pim and Fe(CN)63−, as displayed in Figure 2B. Typically, the characteristic peak for K3Fe(CN)6 at 2117 cm−1, which was assigned to the stretching vibration of CN of K3Fe(CN)6,18 redshifts to 2108 cm−1 for the nanocomposite of Pim and Fe(CN)63− (red curve and black curve). To verify the different affinities of heparin and Fe(CN)63− with Pim, the anion-exchange experiments were conducted by separately adding the heparin and Fe(CN)63− into the dispersion of Fe(CN)63−/Pim and heparin/Pim, respectively. As demonstrated above, the addition of heparin into the Fe(CN)63−/Pim aggregate turns the initial yellow aggregates into the white aggregates. Figure 2B shows the FT-IR spectra of the obtained white aggregates (blue curve), in which the typical peaks for Pim and heparin at 1650, 1557, 1236, and 1027 cm−1 were observed, essentially indicating that the white aggregates were formed by Pim and heparin. Although the characteristic peak of the aggregates of Pim and Fe(CN)63− was also observed at 2108 cm−1, this could be elucidated by the incomplete exchange between Fe(CN)63− and heparin on the Pim receptor. Differently, while adding the K3Fe(CN)6 into the heparin/Pim aggregate, no solution color change was observed. Meanwhile, the characteristic peak of Fe(CN)63− was not observed in the FT-IR spectra (Figure S6, Supporting Information), essentially demonstrating that the anion exchange between Fe(CN)63− and heparin was irreversible. All these results strongly suggest that, although both Fe(CN)63− and heparin could interact with the synthetic Pim receptor forming the aggregates in the aqueous solution, the higher charge density of heparin and the hydrophobicity of Pim might endow a stronger affinity with heparin toward Pim than that of Fe(CN)63− toward the synthetic Pim receptor. This stronger affinity also results in the irreversible anion exchange between heparin and Fe(CN)63−. These properties are particularly useful for the quantitative amperometric assay of heparin through an anion-exchange mechanism between heparin and Fe(CN)63− with the synthetic Pim receptor.

indicate that the existence of heparin leads to the release of Fe(CN)63− from the Fe(CN)63−/Pim nanocomposite, which could be caused by the stronger affinity of the Pim receptor toward heparin than toward Fe(CN)63−. The different affinities of the Pim receptor toward K3Fe(CN)6 and heparin eventually result in the anion exchange of Fe(CN)63− by heparin on the Pim receptor-based interface. The anion exchange was also verified with solution experiments, as displayed in Figure 1A (inset). The addition of Pim into both aqueous solutions of Fe(CN)63− and heparin led to formation of aggregates (Figure 1A, inset, vials 2 and 3), while the addition of heparin into the aggregates formed by Pim and K3Fe(CN)6 turns the initial yellow aggregates (vial 3) into the white aggregates (vial 1). Meanwhile, the solution color was changed into yellow (Figure 1A, inset, vial 1), further demonstrating that Fe(CN)63− was exchanged by heparin from the Pim receptor. Figure 1B shows the typical CVs obtained at bare GC electrodes in 0.10 M PBS containing 1 mM Fe(CN)63−. A pair of well-defined peaks was observed at +0.26 V (black curve), which was ascribed to the redox process of solution-phased Fe(CN)63−.15 The addition of Pim into the Fe(CN)63− solution results in an obvious decrease in the redox peak currents on the bare electrode (red curve), due to the formation of aggregation in the aqueous solutions, while the subsequent addition of heparin to this solution leads to the increase of peak currents (blue curve). This result again demonstrates the anion exchange between Fe(CN)63− and heparin due to their different affinities with the synthetic Pim receptor. To further confirm these different affinities of the synthetic Pim receptor toward heparin and Fe(CN)63−, FT-IR spectroscopy was conducted. Figure 2A displays FT-IR spectra of pure heparin (red curve), pure Pim (black curve), and the nanocomposite formed by heparin and Pim (blue curve). As shown, heparin exhibits strong absorption at 1234 and 1024 cm−1 (red curve), which were assigned to the SO stretching vibration of NSO3− and the CO stretching vibration, respectively,16 while for the nanocomposite of Pim and heparin, these two peaks were blueshifted to 1236 and 1027 cm−1. Meanwhile, the characteristic peaks of pure Pim at 1637, 1550, and 1161 cm−1 (black curve), which were attributed to the stretching vibration of the C−N bonds in the imidazole ring,17 were also blueshifted to 1650, 1557, and 1164 cm−1 (blue curve), after forming the nanocomposite with heparin. These 3442

dx.doi.org/10.1021/ac400201c | Anal. Chem. 2013, 85, 3439−3445

Analytical Chemistry

Article

Figure 3. (A) CVs obtained at the Pim/MWCNT-modified GC electrodes (black curve) and the Fe(CN)63−/Pim/MWCNT-modified GC electrodes (red curve) in 0.10 M PBS (pH 7.0). Blue curve represents the CV obtained after immersing the Fe(CN)63−/Pim/MWCNT-modified GC electrodes in 0.10 M PBS (pH 7.0) for 40 min. Scan rate, 50 mV s−1. (B) CVs obtained at the Fe(CN)63−‑/Pim/MWCNT-modified GC electrodes in 0.10 M PBS (pH 7.0) before (blue curve) and after (red curve) immersing the electrodes into 1 mM heparin solution for 15 min. Black curve represents CV obtained at the same electrode in 0.10 M PBS (pH 7.0) after the electrode was immersed first in 1 mM heparin solution for 15 min and then in 1 mM K3Fe(CN)6 for 15 min. Scan rate, 50 mV s−1.

Figure 4. (A) DPVs recorded at the Fe(CN)63− /Pim/MWCNT-modified GC electrodes in 0.10 M PBS after the electrodes were immersed into different concentrations of heparin for 15 min. The heparin concentrations were (from outer to inner) 0, 0.5, 1, 4, 6, 8,10, 30, and 70 μM. (B) Linear plot of the ratio of current decrease versus heparin concentration. Inset, plot of the ratio of current decrease versus heparin concentration from 0 to 70 μM.

Moreover, as we reported previously,11a the electrochemical assay with the Fe(CN)63−/Pim/MWCNT-modified electrodes was virtually interference from the physiologically important species in their physiological concentrations. These excellent properties substantially enabled the amperometric heparin assay developed in this study to be conducted under physiological conditions, as described below. Amperometric Heparin Assay with the Anion-Exchange Mechanism. To quantify heparin based on the anion-exchange mechanism by using synthetic Pim as the artificial receptor, the electrochemical property of the Fe(CN)63−/Pim/MWCNT-modified GC electrode was investigated. Figure 3A, black curve, depicts typical CVs obtained at the Pim/MWCNT-modified GC electrode in 0.10 M PBS. Prior to the confinement of Fe(CN)63− onto the electrode, the electrode shows no redox waves within the potential window employed here. Upon the surface confinement of Fe(CN)63−, one pair of well-defined redox peaks was observed at +0.19 V (red curve), which was attributed to the electron transfer

process of Fe(CN)63− adsorbed onto the electrode surface. In addition, the current response of this redox wave was relatively stable; no obvious change in the current response was observed after the Fe(CN)63−/Pim/MWCNT-modified GC electrode was immersed in 0.10 M PBS for 40 min, as typically depicted in Figure 3A, blue curve, suggesting the stable adsorption of Fe(CN)63− onto the electrode surface. However, when the Fe(CN)63−/Pim/MWCNT-modified GC electrodes were immersed into 1 mM heparin solution for 15 min, taken out of solution, and rinsed with water, the current response of the electrode was largely decreased in 0.10 M pure PBS (Figure 3B, red curve), demonstrating that the existence of heparin clearly decreases the current response of Fe(CN)63− through the anion-exchange mechanism demonstrated above. While being immersed into 0.10 M PBS containing 1 mM Fe(CN)63−, this electrode shows no obvious current increase of the redox waves of Fe(CN)63− (Figure 3B, black curve), again demonstrating that the anion exchange between heparin and Fe(CN)63− with the synthetic Pim receptor was irreversible. 3443

dx.doi.org/10.1021/ac400201c | Anal. Chem. 2013, 85, 3439−3445

Analytical Chemistry

Article

For the amperometric heparin assay, differential pulse voltammetry were conducted because of its better resolution and higher signal-to-noise ratio, as compared with cyclic voltammetry. Figure 4A displays typical differential pulse voltammograms (DPVs) recorded at the Fe(CN)63−/Pim/ MWCNT-modified GC electrode in 0.10 M pure PBS (pH 7.0) after the electrodes were immersed into different concentrations of heparin for 15 min. There was an obvious cathodic peak observed at the Fe(CN)63−/Pim/MWCNT-modified GC electrode, which was corresponding to the reduction of Fe(CN)63− on the electrode surface. The cathodic peak current decreases with increasing the concentration of heparin in the immersion solution. To eliminate the electrode-to-electrode variation in the background signal, we used the ratio of current decrease (R) for heparin quantification by the following equation: R = (I0 − I )/I0 × 100%

Figure 5. DPVs obtained at the Fe(CN)63−/Pim/MWCNT-modified GC electrode in 0.10 M PBS (black curve) with the presence of normal rat serum (blue curve) or rat serum after intraperitoneal injection of 2 mL of 100 μM heparin for 40 min (red curve).

where I0 was the initial current response recorded at the Fe(CN)63−/Pim/MWCNT-modified GC electrode and I was the current response at the Fe(CN)63−/Pim/MWCNTmodified GC electrodes after the electrodes were treated in the different concentrations of heparin. As shown in Figure 4B, with increasing the heparin concentration, the ratio of current decrease was linearly increased with a dynamic linear concentration range from 0.5 to 10 μM (R = 2.13 + 7.52Cheparin (μM), γ = 0.9982), which could satisfy the requirements of clinical correlative heparin monitoring during postoperative and long-term care (1.7−10 μM).5d,19 Moreover, the validity of the method for the heparin assay in real samples was further evaluated by spiking the standard heparin samples at different levels (i.e., 1, 4, 8, and 10 μM) into the serum samples and then analyzing the samples with the method developed in this study. As summarized in Table 1, good recoveries were achieved for all concentrations of

μA) after the addition of the serum sample from the Sprague− Dawley rats with the intraperitoneal heparin injection (red curve). The ratio of current decrease was 36.7 ± 2.52% (n = 3), corresponding to 4.6 ± 0.05 μM (n = 3) heparin, according to the calibration curve (Figure 4B). The final concentration of heparin in rat serum was calculated to be 18.4 ± 0.20 μM (n = 3) by considering the dilution in our experiments. This result essentially demonstrates that our strategy for the amperometric heparin assay based on the anion-exchange mechanism successfully enables a highly selective and sensitive sensing of heparin in the rat serum.

■

CONCLUSIONS By taking advantages of supramolecular principles, we have successfully demonstrated a simple yet effective strategy for the amperometric assay of heparin by using synthetic Pim as the artificial receptor. The stronger affinity of the Pim receptor toward heparin than toward Fe(CN)63− essentially makes our assay highly selective toward heparin sensing in biological systems. With the animal experiments by dosing intraperitoneally, the method has been demonstrated to be potentially useful for investigating the metabolism of heparin in biological systems. This study not only provides a simple yet effective method for the amperometric assay of electrochemically inactive heparin in biological systems but also opens a new avenue to developing amperometric methods for the electrochemically inert species based on supramolecular principles.

Table 1. Recovery Results in Normal Rat Serum sample no.

spiked conc (μM)

1 2 3 4

1 4 8 10

found (n = 3, μM) 1.10 4.15 7.84 9.70

± ± ± ±

0.06 0.20 0.20 0.40

recovery (%) 110.0 103.8 98.0 97.0

± ± ± ±

6.0 5.0 2.5 4.0

heparin spiked into serum, further validating the amperometric assay developed in this study for the measurements of heparin metabolism in biological systems, as demonstrated below. To further investigate the potential utility of the amperometric method for heparin assay, the metabolism of heparin in rat was conducted. For such a purpose, heparin was dosed intraperitoneally with a solution of 100 μM in 0.9% normal saline, and serum samples were collected after dosing for 40 min and were then diluted 4-fold with 0.10 M PBS for the electrochemical detection. Figure 5 depicts DPV responses obtained at the Fe(CN)63−/Pim/MWCNT-modified GC electrodes in 0.10 M PBS (black curve) with the presence of normal serum from the Sprague−Dawley rats (blue curve) or the serum of Sprague−Dawley rats after intraperitoneal injection (red curve). As shown, the peak current recorded for the Fe(CN)63−/Pim/MWCNT-modified GC electrode was 2.8 μA in 0.10 M PBS and 2.7 μA in the normal rat serum, essentially indicating that there was no interference in the serum. However, the current was remarkably decreased (1.7

■

ASSOCIATED CONTENT

* Supporting Information S

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Fax: +86-10-62559373. E-mail: [email protected] (P.Y.); [email protected] (L.M.). Notes

The authors declare no competing financial interest. 3444

dx.doi.org/10.1021/ac400201c | Anal. Chem. 2013, 85, 3439−3445

Analytical Chemistry

■

Article

N.; Wang, H.; Gao, X.; Yang, R.; Chan, W. Anal. Chem. 2010, 82, 4628−4636. (f) Kim, S. K.; Lee, D. H.; Hong, J. I.; Yoon, J. Acc. Chem. Res. 2008, 42, 23−31. (11) (a) Zhuang, X.; Wang, D.; Lin, Y.; Yang, L.; Yu, P.; Jiang, W.; Mao, L. Anal. Chem. 2012, 84, 1900−1906. (b) Park, M. J.; Lee, J. K.; Lee, B. S.; Lee, Y. W.; Choi, I. S.; Lee, S. g. Chem. Mater. 2006, 18, 1546−1551. (c) Wang, J.; Chu, H.; Li, Y. ACS Nano 2008, 2, 2540− 2546. (d) Bellayer, S.; Gilman, J. W.; Eidelman, N.; Bourbigot, S.; Flambard, X.; Fox, D. M.; De Long, H. C.; Trulove, P. C. Adv. Funct. Mater. 2005, 15, 910−916. (12) (a) Mu, X. D.; Meng, J. Q.; Li, Z. C.; Kou, Y. J. Am. Chem. Soc. 2005, 127, 9694−9695. (b) Xie, Y.; Zhang, Z.; Jiang, T.; He, J.; Han, B.; Wu, T.; Ding, K. Angew. Chem., Int. Ed. 2007, 46, 7255−7258. (c) Zhou, X.; Wu, T.; Hu, B.; Yang, G.; Han, B. Chem. Commun. 2010, 46, 3663−3365. (13) (a) Jee, J.-P.; Nam, S.-H.; Park, Y.; Lee, H.-J.; Park, Y.; Maeng, H.-J.; Kim, C.-K. Arch. Pharmacal Res. 2012, 35, 1107−1114. (b) Berna, M.; Murphy, A. T.; Wilken, B.; Ackermann, B. Anal. Chem. 2002, 74, 1197−1201. (c) Zhang, Z.; Zhao, L.; Lin, Y.; Yu, P.; Mao, L. Anal. Chem. 2010, 82, 9885−9891. (d) Kim, J.-Y.; Shim, G.; Choi, H.-W.; Park, J.; Chung, S. W.; Kim, S.; Kim, K.; Kwon, I. C.; Kim, C.-W.; Kim, S. Y.; Yang, V. C.; Oh, Y.-K.; Byun, Y. Biomaterials 2012, 33, 4424−4430. (14) (a) Harish, S.; Joseph, J.; Phani, K. Electrochim. Acta 2011, 56, 5717−5721. (b) Duan, C.; Cui, H.; Zhang, Z.; Liu, B.; Guo, J.; Wang, W. J. Phys. Chem. C 2007, 111, 4561−4566. (15) (a) Yao, H.; Hu, N. J. Phys. Chem. B 2010, 114, 9926−9933. (b) Peng, Y.; Zhang, D.; Li, Y.; Qi, H.; Gao, Q.; Zhang, C. Biosens. Bioelectron. 2009, 25, 94−99. (c) Liu, J.; Jin, Y.; Wu, A.; Li, Z.; Dong, S. Electroanalysis 2004, 16, 1931−1937. (16) (a) Yang, J.; Cai, J.; Wu, K.; Li, D.; Hu, Y.; Li, G.; Du, Y. Int. J. Biol. Macromol. 2012, 50, 1158−1164. (b) Park, I.-K.; Kim, Y. J.; Tran, T. H.; Huh, K. M.; Lee, Y. K. Polymer 2010, 51, 3387−3393. (17) (a) Shi, J.; Wu, P.; Yan, F. Langmuir 2010, 26, 11427−11434. (b) Zhou, Y.; Schattka, J. H.; Antonietti, M. Nano Lett. 2004, 4, 477− 481. (c) Lian, J.; Duan, X.; Ma, J.; Peng, P.; Kim, T.; Zheng, W. ACS Nano 2009, 3, 3749−3761. (d) Zhu, J.; Shen, Y.; Xie, A.; Qiu, L.; Zhang, Q.; Zhang, S. J. Phys. Chem. C 2007, 111, 7629−7673. (18) Combellas, C.; Kanoufi, F.; Sanjuan, S.; Slim, C.; Tran, Y. Langmuir 2009, 25, 5360−5370. (19) Hirsh, J.; Raschke, R. Chest 2004, 126, 188S−203S.

ACKNOWLEDGMENTS This research was financially supported by the NSF of China (grant nos. 20975104, 20935005, 21127901, 21210007, and 91213305 for L.M. and 91132708 for P.Y.), the National Basic Research Program of China (973 programs, 2010CB33502, 2013CB933704), and The Chinese Academy of Sciences (KJCX2-YW-W25 and Y2010015).

■

REFERENCES

(1) (a) Beer, P. D. Acc. Chem. Res. 1998, 31, 71−80. (b) Rekharsky, M. V.; Inoue, Y. J. Am. Chem. Soc. 2002, 124, 813−826. (c) Biros, S. M.; Bergman, R. G.; Raymond, K. N. J. Am. Chem. Soc. 2007, 129, 12094−12095. (d) Bazzicalupi, C.; Bencini, A.; Giorgi, C.; Valtancoli, B.; Lippolis, V.; Perra, A. Inorg. Chem. 2011, 50, 7202−7216. (e) Lemonnier, J. F.; Floquet, S.; Marrot, J.; Cadot, E. Eur. J. Inorg. Chem. 2009, 2009, 5233−5239. (2) (a) Chung, Y. M.; Raman, B.; Kim, D. S.; Ahn, K. H. Chem. Commun. 2006, 186−188. (b) Kubo, Y.; Ishihara, S.; Tsukahara, M.; Tokita, S. J. Chem. Soc., Perkin Trans. 2002, 1455−1460. (c) Jain, R. K.; Hamilton, A. D. Org. Lett. 2000, 2, 1721−1723. (d) Zhang, T.; Anslyn, E. V. Org. Lett. 2006, 8, 1649−1652. (e) Dwight, S. J.; Gaylord, B. S.; Hong, J. W.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 16850−16859. (3) (a) Zhang, M.; Yu, P.; Mao, L. Acc. Chem. Res. 2012, 45, 533− 543. (b) Qi, H.; Chang, J.; Abdelwahed, S. H.; Thakur, K.; Rathore, R.; Bard, A. J. J. Am. Chem. Soc. 2012, 134, 16265−16274. (c) Chi, Y. S.; Hwang, S.; Lee, B. S.; Kwak, J.; Choi, I. S.; Lee, S. G. Langmuir 2005, 21, 4268−4271. (4) (a) Golub, E.; Pelossof, G.; Freeman, R.; Zhang, H.; Willner, I. Anal. Chem. 2009, 81, 9291−9298. (b) Casas-Solvas, J. M.; OrtizSalmerón, E.; Fernández, I.; García-Fuentes, L.; Santoyo-González, F.; Vargas-Berenguel, A. Chem.Eur. J. 2009, 15, 8146−8162. (5) (a) Capila, I.; Linhardt, R. J. Angew. Chem., Int. Ed. 2002, 41, 390−412. (b) Whitelock, J. M.; Iozzo, R. V. Chem. Rev. 2005, 105, 2745−2764. (c) Mackman, N. Nature 2008, 451, 914−918. (d) Zhan, R.; Fang, Z.; Liu, B. Anal. Chem . 2010, 82, 1326−1333. (6) (a) Dai, Q.; Liu, W.; Zhuang, X.; Wu, J.; Zhang, H.; Wang, P. Anal. Chem. 2011, 83, 6559−6564. (b) Yan, H.; Wang, H. F. Anal. Chem. 2011, 83, 8589−8595. (c) Wright, A. T.; Zhong, Z. L.; Anslyn, E. V. Angew. Chem., Int. Ed. 2005, 44, 5679−5682. (d) Hudalla, G. A.; Koepsel, J. T.; Murphy, W. L. Adv. Mater. 2011, 23, 5415−5418. (e) Gemene, K. L.; Meyerhoff, M. E. Anal. Chem. 2010, 82, 1612− 1615. (f) Doneanu, C. E.; Chen, W.; Gebler, J. C. Anal. Chem. 2009, 81, 3485−3499. (7) (a) Maurer, J.; Haselbach, S.; Klein, O.; Baykut, D.; Vogel, V.; Mäntele, W. J. Am. Chem. Soc. 2010, 133, 1134−1140. (b) Pu, K. Y.; Liu, B. Macromolecules 2008, 41, 6636−6640. (c) Cheng, T. J.; Lin, T. M.; Wu, T. H.; Chang, H. C. Anal. Chim. Acta 2001, 432, 101−111. (d) Hu, P.; Fang, L.; Chess, E. K. Anal. Chem. 2009, 81, 2332−2343. (e) Sommers, C. D.; Mans, D. J.; Mecker, L. C.; Keire, D. A. Anal. Chem. 2011, 83, 3422−3430. (f) Beccati, D.; Roy, S.; Lech, M.; Ozug, J.; Schaeck, J.; Gunay, N. S.; Zouaoui, R.; Capila, I.; Kaundinya, G. V. Anal. Chem. 2012, 84, 5091−5096. (8) (a) Crespo, G. A.; Afshar, M. G.; Bakker, E. Angew. Chem., Int. Ed. 2012, 51, 12575−12578. (b) Shvarev, A.; Bakker, E. Anal. Chem. 2005, 77, 5221−5228. (c) Shvarev, A.; Bakker, E. J. Am. Chem. Soc. 2003, 125, 11192−11193. (d) Gemene, K. L.; Meyerhoff, M. E. Anal. Chem. 2010, 82, 1612−1615. (e) Ma, S. C.; Yang, V. C.; Fu, B.; Meyerhoff, M. E. Anal. Chem. 1993, 65, 2078−2084. (f) Yuan, Y.; Amemiya, S. Anal. Chem. 2004, 76, 6877−6886. (g) Langmaier, J.; Samcová, E.; Samec, Z. Anal. Chem. 2007, 79, 2892−2900. (9) Gadzekpo, V. P. Y.; Xiao, K. P.; Aoki, H.; Bühlmann, P.; Umezawa, Y. Anal. Chem. 1999, 71, 5109−5115. (10) (a) Xu, Z.; Kim, S. K.; Yoon, J. Chem. Soc. Rev. 2010, 39, 1457− 1466. (b) Zeng, L.; Wang, P.; Zhang, H.; Zhuang, X.; Dai, Q.; Liu, W. Org. Lett. 2009, 11, 4294−4297. (c) Yoon, J.; Kim, S. K.; Singh, N. J.; Kim, K. S. Chem. Soc. Rev. 2006, 35, 355−360. (d) Zapata, F.; Caballero, A.; White, N. G.; Claridge, T. D. W.; Costa, P. J.; Félix, V. T.; Beer, P. D. J. Am. Chem. Soc. 2012, 134, 11533−11541. (e) Shao, 3445

dx.doi.org/10.1021/ac400201c | Anal. Chem. 2013, 85, 3439−3445