Article pubs.acs.org/ac
Multidimensional Optical Sensing Platform for Detection of Heparin and Reversible Molecular Logic Gate Operation Based on the Phloxine B/Polyethyleneimine System Yu Ling, Zhong Feng Gao, Qian Zhou, Nian Bing Li,* and Hong Qun Luo* Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China S Supporting Information *
ABSTRACT: A multidimensional optical sensing platform which combines the advantages of resonance Rayleigh scattering (RRS), fluorescence, and colorimetry has been designed for detection of heparin. Phloxine B, a fluorescein derivative showing the special RRS spectrum in the long wavelength region, was selected to develop an easy-to-get system which can achieve switch-on sensing to obtain high sensitivity. The noise level of RRS in the long wavelength region is much weaker, and the reproducibility is much better; in this way, the sensitivity and selectivity can be improved. In the absence of heparin, the phloxine B and polyethyleneimine (PEI) form a complex through electrostatic interaction. Thus, the RRS signal at 554 nm is low; the phloxine B fluorescence is quenched, and the absorption signal is low. In the presence of heparin, competitive binding occurred between phloxine B and heparin toward PEI; then, phloxine B is gradually released from the phloxine B/PEI complex, causing obvious enhancement of the RRS, fluorescence, and absorption signals. Besides, the desorption of phloxine B is less effective for the heparin analogues, such as hyaluronic acid and chondroitin sulfate. In addition, the system presents a low detection limit of heparin to 5.0 × 10−4 U mL−1 and can also be applied to the detection of heparin in heparin sodium injection and 50% human serum samples with satisfactory results. Finally, the potential application of this method in reversible on−off molecular logic gate fabrication was discussed using the triplechannel optical signals as outputs.
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interaction such as intermolecular electrostatic attraction, hydrogen bonding, hydrophobic interaction, and so on.9 This multidimensional optical sensing platform for the detection of heparin combines the advantages of the RRS assay, the high sensitivity of fluorescence assay, and the convenience and low cost of the visual assay. More importantly, the simultaneous optical signal changes can help to enhance the accuracy for detection.2 Additionally, monitoring the RRS and fluorescence signals can be easily achieved on a fluorescence spectrophotometer in different detection modes, thereby greatly simplifying experimental procedures.1 Heparin, which primarily (>70%) consists of a trisulfated disaccharide repeating unit, is a highly sulfated linear acidic polysaccharide.10 Having the highest negative charge density of a naturally occurring biomolecule, heparin has been widely used as an anticoagulant drug and can effectively prevent the formation of clots within the blood.11 The monitoring of heparin levels is essential during cardiovascular surgery or to avoid thrombosis because overdose of heparin can induce adverse effects such as hemorrhaging and thrombocytopenia.12 Traditional methods for detecting heparin, including measuring
ultidimensional sensing devices have drawn intensive attention due to the fact that they can offer more than one transduction channel and thus increase the accuracy and/or diversity.1,2 Phloxine B dye is a color additive for foods, drugs, and cosmetics.3 By taking advantage of the color change, a colorimetric sensor can be developed. In addition, the dye is also widely used as a stain in fluorescence microscopy.4 Thus, it can be used to construct a fluorescent probe and has potential applications in biochemical analysis. Moreover, the phloxine B has not only a resonance Rayleigh scattering (RRS) peak in the short wavelength range but also a special RRS peak in the long wavelength range. Furthermore, phloxine B and other structurally related fluorescein derivatives are anionic and, thus, easily attachable to cationic groups. Hyperbranched polyethyleneimine (PEI) is a cationic water-soluble polymer which contains primary, secondary, and tertiary amino groups and exhibits outstanding adsorption capacity for different substances.5,6 Simultaneously, PEI has been widely used for biological applications.7,8 Herein, we report a multidimensional optical sensing platform based on the simultaneous utilization of the three optical signals (RRS, fluorometric, and colorimetric signals) of the phloxine B/PEI complex for heparin detection. Resonance Rayleigh scattering, an analytical technique developed in recent years, has been known for its sensitivity and simplicity. Moreover, RRS is very sensitive to the molecular © 2015 American Chemical Society
Received: July 10, 2014 Accepted: January 9, 2015 Published: January 9, 2015 1575
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were measured with a pH meter (PHS-3C, Shanghai Leici Instrument Company, Ltd., China). General Procedure for Detection of Heparin. Phloxine B solution (0.1 mM, 20 μL) was dissolved in ultrapure water (840 μL). Subsequently, PEI solution (0.02 mM, 40 μL) was added, and the solution was mixed. Then, heparin (100 μL) with different concentrations was added and mixed well. After 4 min, the RRS, fluorescence emission, and absorption spectra of the mixture (1.0 mL) were recorded, respectively. The RRS spectrum was recorded in the range of 220−650 nm; the fluorescence emission spectrum of the system was collected in the range of 540−650 nm when excited at the maximum excitation wavelength (530 nm), and fluorescence intensity at the maximum emission wavelength (560 nm) was recorded. The absorption spectrum was measured in the range of 475− 580 nm. The absorption at 538 nm was recorded. Appropriate amounts of heparin were added to the heparin sodium injection and 50% human serum. Then, the same procedure was prepared as that of the standard solution detection mentioned above.
the activated clotting time and activated partial thromboplastin time,13−15 other methods including nuclear magnetic resonance, capillary electrophoresis, etc., are also reported.16,17 A recently developed electrochemical method is attractive for heparin detection because of its sensitivity, a wide linear range, and low cost,18 but the testing of the interference of the coexisting analogues has been neglected. In addition, a large amount of interest has focused on colorimetric and fluorescent approaches to heparin monitoring based on supramolecule,11,19,20 cationic polymer,21,22 molecular beacon,10 gold nanoparticle,23 benzimidazolium dye library,24 quantum dots,25 and the polyadenosine−coralyne complex.26 It is obvious that these methods have made great progress in heparin sensing. However, highly sensitive, fast, time-saving, and economical methods are more beneficial and practical. Hence, much attention still focuses on the ongoing need for simple and commercially available systems which can achieve switch-on sensing to obtain high sensitivity and operate in human serum/ plasma or, even better, in whole blood.16 In this study, the complex of commercially available phloxine B and PEI is utilized as a simple RRS, fluorescent, and colorimetric platform to assay heparin with a good selectivity, and the detection limit is as low as 5.0 × 10−4 U mL−1. Compared to other reports, the special RRS peak in the long wavelength range was utilized for determination, because it suffered little interference and showed better reproducibility; in this way, the sensitivity and selectivity can be improved. In addition, this sensor has been applied to the detection of heparin in heparin sodium injection and 50% human serum with satisfactory results. Therefore, we established a method that is capable of multidimensional, highly sensitive, selective, and rapid detection of heparin. In addition, molecular logic gates have attracted significant attention in the field of molecular-scale computers, electronics, and “autonomously regulated” chemical systems.27−29 On the basis of Boolean logic, the IMPLICATION logic26 gate with PEI and heparin as inputs is developed.
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RESULTS AND DISCUSSION Optimization of the Conditions. We scanned more than 30 kinds of dyes to optimize the suitable reporter (Table S1 in the Supporting Information). Given the electrostatic interaction between the reporter dye and the cationic polymer PEI, anionic dyes were preferred. Moreover, it is implied that only fluorescein derivatives or analogues have the xanthene ring (conjugate structure) and show the special RRS spectrum in the long wavelength region. Although the RRS peak in the short wavelength region is stronger than that in the long wavelength region, the peak in the long wavelength is located near the absorption band, which is a key factor for producing the RRS spectrum.30 Besides, the noise level in the long wavelength region is much weaker and the reproducibility is much better. Compared to the previous report based on RRS,30 in which the combination of heparin with some basic cationic dyes can result in a significant enhancement of RRS, our method utilizes the anionic dye/PEI complex and shows better sensitivity. The structures of fluorescein derivatives which comprise eosin Y, erythrosine, rose bengal, and phloxine B are shown in Figure S1 in the Supporting Information. PEI could not decrease the RRS signal of sodium fluorescein, because the unsubstituted xanthene ring has low negative charge density. Interestingly, phloxine B and eosin Y whose four hydrogen atoms on the xanthene ring are substituted for bromide atoms display higher sensitivity to heparin than erythrosine and rose bengal whose hydrogen atoms on the xanthene ring are substituted for iodine atoms. On the one hand, iodine atoms show remarkable steric effects. On the other hand, bromide atoms have stronger electron withdrawing effects than iodine atoms. However, the phenyl ring can rotate freely around the single bond, so steric effects of chlorine atoms can be neglected. Moreover, the electron-withdrawing effects of chlorine atoms on the phenyl ring play an important role in increasing the charge density and further improving the electrostatic binding ability. Therefore, phloxine B shows a better selectivity for heparin over other glycosaminoglycans and was selected as the optical probe in subsequent experiments. To establish the sensitive detection of heparin, a series of tests was performed to optimize the sensing parameters, such as phloxine B concentration, the molar ratio of PEI/phloxine B, pH, electrolyte concentration, temperature, and reaction time.
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EXPERIMENTAL SECTION Reagents and Solutions. Phloxine B (Guaranteed Reagent, Dye content ≥97%), PEI (Mw = 10 000, 99%), hyaluronic acid (HA), chondroitin 4-sulfate (ChS), and heparin (Hep, 185 U mg−1) were supplied by Aladdin Reagent Co., Ltd. (Shanghai, China). The molecular weight of Hep was determined by disaccharide (644.2 g mol−1), and 1 μM Hep corresponded to 0.12 U mL−1. Human serum sample was obtained from healthy volunteers in the local hospital. All other chemicals not mentioned here were of analytical grade reagents, were used as received, and were supplied by Chengdu Kelong Chemical Reagents Factory (Sichuan, China). All aqueous solutions were prepared with ultrapure water (18.2 MΩ cm). The PEI stock solution (0.2 mM) was prepared by dissolving 0.0940 g of PEI in 1.0 mL of ultrapure water and stored in the dark at −4 °C. The working solution was prepared by diluting the stock solution with ultrapure water. Apparatus. The RRS and fluorescence measurements were recorded using an F-2700 fluorescence spectrophotometer (Hitachi, Japan). The slit width was 10 and 10 nm for excitation and emission, respectively, and the photomultiplier tube (PMT) voltage was set at 400 V. The ultraviolet−visible (UV−vis) absorption spectra were recorded with a UV-2450 spectrophotometer (Shimadzu, Japan). The Fourier transform infrared (FT-IR) spectra were obtained on a Bruker IFS (Germany) 113v spectrometer. The pH values of solutions 1576
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Analytical Chemistry When the phloxine B concentration was fixed at 1.0 μM, we studied the influence of molar ratio of PEI to phloxine B (Figure S2 in the Supporting Information). The change of RRS intensities (ΔIRRS) of the phloxine B/PEI complex at 554 nm upon addition of 0.1 U mL−1 heparin was used to optimize the sensing system (ΔIRRS = I0RRS − IRRS, where I0RRS and IRRS represent the RRS intensity of the phloxine B/PEI complex in the absence and presence of heparin, respectively). The value of ΔIRRS increased with increasing molar ratio of PEI to phloxine B from 0.02:1 to 0.2:1, and the maximum occurs in the molar ratio range of 0.2:1 to 5:1. However, when the molar ratio is further increased, the change of RRS intensities decreased. Sufficient PEI is essential to decrease the background signal and improve the sensitivity, but excess PEI would lead to a difficult release of phloxine B from PEI. Therefore, the optimum molar ratio of PEI to phloxine B is 0.4:1. The pH of the solutions was adjusted with HNO3 or NaOH solution in order to avoid the electrolyte-induced perturbations by buffer solutions. The RRS and fluorescence signals of phloxine B were greatly decreased by PEI over the pH range from 2 to 8, while they slightly decreased at alkaline pH values. Furthermore, upon addition of heparin, the RRS and fluorescence intensities had greater recovery in the pH range of 5−8 and reached the maximum at pH 7 (Figure S3 in the Supporting Information). This phenomenon suggests that the neutral or weak acidic solutions are preferred for reducing the reagent blank value because in this case phloxine B primarily exists in the anion species and the amino residues on the surface of PEI are protonated with a large number of positive charges. The influence of temperature in the range of 25−55 °C was then investigated. As shown in Figure S4 in the Supporting Information, the change of RRS intensities of the phloxine B/ PEI complex nearly keep constant at different temperatures, so room temperature was chosen. Subsequently, the reaction time and stability were studied. When PEI was added to the phloxine B solution, the RRS and fluorescence intensities decreased rapidly to reach their minimum values and remained stable for a long time. When heparin was added to the phloxine B/PEI complex, after incubation of 4 min, the intensities remained constant for at least 2 h, so the reaction could be completed in 4 min (Figure S5 in the Supporting Information). This result indicates a promising application in the fast and stable detection of heparin. Interference caused by electrolyte or buffer solution was investigated. The concentration of sodium chloride or Tris-HCl varied from 10 to 80 mM, and the RRS spectrum at 554 nm was recorded after each addition. Data are presented as % interference. As shown in Figure S6 in the Supporting Information, at 10 mM sodium chloride, there is little perturbation, even though Na+ ion is present in a 10 000-fold excess compared to the concentration of heparin itself. In general, the buffer causes greater interference than sodium chloride. As shown in the inset of Figure S7 in the Supporting Information, the RRS intensities are proportional to the concentrations of the probe in the range of 0.1−4 μM. Generally, the sensitivity of the probe may be further enhanced if a lower concentration of probe is used. However, on the other hand, the linear range of the detection would be narrow when too low of a concentration of probe was used.31 Thus, the concentration of 2 μM phloxine B was selected in the
determination of heparin for obtaining the widest linear response and the lowest detection limit. Characterization and Sensing Mechanism of Heparin Sensor. Phloxine B and PEI can form a complex through electrostatic interaction (step I in Scheme 1). Nevertheless, Scheme 1. Schematic Illustration of Heparin Detection Based on Phloxine B and PEI
heparin has stronger interactions with PEI through electrostatic interactions and hydrogen bonding. Addition of heparin to the phloxine B/PEI system induces a binding competition between phloxine B and heparin toward PEI. Phloxine B is then gradually released from the phloxine B/PEI complex, resulting in recovery of the RRS, fluorescence, and absorption signals (step II in Scheme 1). This detection strategy was simply based on the reversible adsorption and desorption and confirmed by RRS, fluorescence, UV−vis absorption, and FT-IR spectra. It can be seen from Figure 1A that the RRS spectrum of phloxine B (curve a) has a broad peak in the short wavelength region and a scattering peak at about 554 nm, whereas those of PEI (curve b) and heparin (curve c) have a broad peak in the short wavelength range. When the PEI was added to the phloxine B solution, the RRS intensity of the phloxine B/PEI system (curve d) increased greatly in the short wavelength region compared to that of PEI. However, its RRS intensity at 554 nm decreased greatly compared to that of phloxine B, which demonstrated that phloxine B could associate with PEI to form a complex. When heparin was added to the phloxine B/PEI solution, the RRS intensity at 554 nm (curve e) increased greatly. This is because strong electrostatic interactions between PEI and heparin result in the formation of the PEI/ heparin complex. The RRS spectrum of PEI/heparin (curve f) has no peak at 554 nm. The RRS intensity of phloxine B/ heparin (curve g) at 554 nm was almost not changed compared to that of phloxine B (curve a) due to the electrostatic repulsion between phloxine B and heparin. In order to explore the mechanism further, the system was characterized by fluorescence, UV−vis absorption, and FT-IR spectroscopy. It can be seen from Figure 1B that the phloxine B had a fluorescence emission at 560 nm in aqueous solution (curve a), whereas the PEI (curve b), heparin (curve c), phloxine B/PEI (curve d), and the PEI/heparin system (curve f) had no fluorescence signals. When PEI was added to the phloxine B solution, the formation of the phloxine B/PEI 1577
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changed, whereas the PEI (curve b), heparin (curve c), phloxine B/PEI (curve d), and the PEI/heparin system (curve f) had no absorption signals at 538 nm. Then, the absorbance was reduced by adding PEI because of the formation of the phloxine B/PEI complex. After that, the absorbance increased with the addition of heparin due to the desorption of phloxine B. It can be seen from Figure 2 that the band at 1176 cm−1 is related to the C−O stretching vibration of phloxine B (curve
Figure 1. (A) Resonance Rayleigh scattering spectra of the phloxine B/PEI/heparin system in the wavelength range of 240−630 nm. (B) Fluorescence spectra of the phloxine B/PEI/heparin system in the range of 540−630 nm when excited at the maximum excitation wavelength (530 nm). (C) Absorption spectra of the phloxine B/PEI/ heparin system in the wavelength range of 200−600 nm. (a) Phloxine B, (b) PEI, (c) heparin, (d) phloxine B/PEI, (e) phloxine B/PEI/ heparin, (f) PEI/heparin, and (g) phloxine B/heparin. Concentrations: phloxine B (2 μM), PEI (0.8 μM), and heparin (10 U mL−1).
Figure 2. FT-IR spectra of phloxine B (a), PEI (b), heparin (c), phloxine B/PEI (d), PEI/heparin (e), phloxine B/heparin (f), and phloxine B/PEI/heparin (g).
a), while for the phloxine B/PEI complex, this peak is blueshifted to 1240 cm−1 (curve d). All the data of the wavelength (marked in Figure 2) were read out by the FT-IR spectrometer precisely. Meanwhile, the characteristic peak at 1123 cm−1 which corresponds to the stretching vibration of the C−N bonds of PEI (curve b) is red-shifted to 1079 cm−1 (curve d). These spectral changes indicate that electrostatic interaction causes complexation between the phloxine B and PEI. In addition, the bands assigned to the stretching vibration of SO, O−H, and C−O for heparin which are at 1423, 3385, and 1237 cm−1, respectively (curve c), shift to 1456, 3422, and 1249 cm−1 for the PEI/heparin complex (curve e). Meanwhile, the characteristic peaks of phloxine B and heparin do not change when they are mixed together (curve f). To further confirm the tighter complexation between heparin and PEI than that between phloxine B and PEI, we added the heparin to the phloxine B/PEI system (curve g). As can be seen from curve g, the typical peak for the heparin/PEI complex at 1457 cm−1 is observed, but the characteristic peak of the phloxine B/ PEI complex at 1240 cm−1 is not observed. Thus, both phloxine B and heparin can bind to PEI to form the complex, respectively, but the hydrogen bonding might cause a stronger interaction of heparin with PEI as compared with phloxine B. Selective Detection of Heparin. Selectivity has been considered to be one of the greatest challenges for heparin
complex resulted in the quenching of phloxine B fluorescence (curve d). The absorption spectrum of phloxine B dye changed obviously in the presence of PEI, which suggests the formation of the ground-state complex and a static quenching mechanism.32 In addition, phloxine B can act as an electron acceptor, whereas PEI has abundant nitrogen atoms and would be an electron donator. Hence, photoinduced electron transfer between phloxine B and PEI may be another possible reason for fluorescence quenching.33 The binding constant (Kb) between phloxine B and PEI was calculated to be 9.12 × 104 M−1 by the Job’s plot (Figure S8 in the Supporting Information).34 However, when heparin was added to the phloxine B/PEI complex, the fluorescence recovery of phloxine B at 560 nm occurred (curve e) because of the replacement of phloxine B by heparin in the phloxine B/PEI complex to yield free phloxine B in the solution. Compared to that of phloxine B, the fluorescence intensity of phloxine B/heparin (curve g) was almost not changed. Similarly, in Figure 1C, the maximum absorption peak of phloxine B (curve a) lied at 538 nm, and the absorbance of phloxine B/heparin (curve g) was almost not 1578
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Figure 3. Resonance Rayleigh scattering spectra (A), fluorescence emission spectra (C), and absorption spectra (E) of the phloxine B/PEI complex upon addition of different concentrations of heparin. B, D, and F show the relationship between different concentrations of heparin and ΔIRRS, ΔIF, and A, respectively. The insets of B, D, and F display that the linear ranges for heparin are 0.001−0.15 U mL−1 for RRS, 0.005−0.3 U mL−1 for fluorescence, and 0.01−0.1 U mL−1 for absorption, respectively.
our method shows lower detection limit than many other methods (Table S2 in the Supporting Information). Meanwhile, previous research failed to consider that fluorescein derivatives have the special RRS peak in the long wavelength region. Although the RRS peak in the short wavelength region is stronger than that in the long wavelength region, the peak in the long wavelength is located near the absorption band, which is a key factor for producing the RRS spectrum. In addition, the noise level in the long wavelength region is much weaker and the reproducibility is much better; in this way, the sensitivity and selectivity can be improved. Furthermore, the method also exhibits good repeatability (RSD = 3.2%, n = 6) when 0.1 U mL−1 heparin was investigated. Fluorescent and Colorimetric Detection of Heparin. The change of fluorescence intensities at 560 nm (ΔIF) was also proportional to the concentration of heparin; the linear range was 0.005−0.3 U mL−1, and good linear correlation (R2 = 0.9972) was obtained (Figure 3D). The absorbance (A) at 538 nm was used as a function of heparin concentrations and exhibited a linear relationship for 0.01−0.1 U mL−1, and good linear correlation (R2 = 0.9934) was obtained (Figure 3F). The detection limits of fluorescent and colorimetric methods based on 3δ/s were 0.003 and 0.005 U mL−1, respectively. Although the detection limit of the colorimetric response of the sensing detection of heparin is higher than that of RRS detection, the
sensor operating in biological media. Besides heparin, there are five other structurally related glycosaminoglycans: hyaluronic acid (HA), chondroitin sulfate (ChS), heparan sulfate (HS), dermatan sulfate (DS), and keratin sulfate (KS). In this work, two heparin analogues, ChS and HA, are selected as the potentially interfering substances, which are often found as contaminants in heparin samples. As heparin has a much higher charge density than ChS or HA does, it has stronger electrostatic attractions with PEI and induces a much larger response. In any case, this study signifies that the phloxine B/ PEI system can serve as a selective sensor for 1 μM heparin over 1 μM ChS and HA. The signal obtained from 1 μM heparin is lower than that obtained from 10 μM ChS and HA. However, the signal obtained from 0.01 μM heparin is higher than that obtained from 0.1 μM ChS and HA (Figure S9 in the Supporting Information). Therefore, our method shows better selectivity when the concentration of analyte is lower. Sensitivity for Heparin Detection. Under optimum conditions, the linear range for heparin was 0.001−0.15 U mL−1 (8.3 × 10−3 to 1.25 μM), and good linear correlation (R2 = 0.9948) was obtained (Figure 3). The detection limit for heparin based on 3δ/s was approximately 5.0 × 10−4 U mL−1 (4.2 × 10−3 μM), which is much lower than the therapeutic level of heparin during cardiovascular surgery (17−67 mM) and postoperative and long-term care (1.7−10 mM). Moreover, 1579
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Figure 4. Truth table (A), symbol (B), and reversible switching (C) of the IMPLICATION logic gate.
outputs. Only with the input of PEI and without heparin (1/0), the RRS, fluorescence, and absorption signals of phloxine B are low, and the output is “0” (off state). In all other cases, the outputs are “1” (on state). This IMPLICATION logic gate can be used to reflect the relative concentrations of heparin in solutions. We also studied the reversibility of this logic gate operation. Figure 4C shows the repeated switching behavior with alternating addition of PEI and heparin. The outputs gradually decreased after cyclic treatment with PEI and heparin. The observed loss can be attributed to the dilution effect, owing to the increase in the solution volume with the alternating addition of PEI and heparin. The truth table (A), symbol (B), and the repeated switching behavior (C) of the IMPLICATION logic gate are shown in Figure 4.
color reactions are easy and rapid: upon addition of different targets (10 μM each), a color change would be observed less than 1 min under a UV lamp. Accordingly, the corresponding solutions emit yellow (heparin), khaki (HA), and dark yellow (ChS), respectively (inset of Scheme 1). Thus, the phloxine B/ PEI system is attractive and promising for visual detection of heparin. Sensing Performances in Real Samples. In practical application, heparin was measured in a heparin sodium injection sample and human serum sample. Moreover, to evaluate the validity of our method, the standard heparin solution was spiked into the human serum sample and then detected by our method. The results, recoveries, and relative standard deviations (RSDs) of two real samples are listed in Tables S3 and S4, respectively, in the Supporting Information, indicating that our method is accurate and reliable. To further evaluate the validity of this work, a standard heparin sodium sample (197 U mg−1, used for determination of anticoagulant potency, National Institutes for Food and Drug Control, China) was detected by our method. Moreover, the t-test has been used to justify whether a significant difference exists. The values of the calculated t for the heparin sodium injection sample and standard heparin sodium sample are 0.48 and 0.16, respectively, and both of them are less than the critical t value for the significance level (α) of 0.05 (t0.05, 4 = 2.78). The results (Table S3 in the Supporting Information) show that no significant difference existed. As shown in Figure S10 in the Supporting Information, the linear ranges of heparin in the serum sample are 0.08−0.4 U mL−1 for RRS, 0.08−0.5 U mL−1 for fluorescence, and 0.2−0.5 U mL−1 for absorption, respectively. The detection limits of heparin for RRS, fluorescent, and colorimetric methods based on 3δ/s are 0.04, 0.06, and 0.15 U mL−1, respectively. Molecular Logic Gates. For input, we defined the presence of PEI or heparin as 1 and their absence as 0. Figure 4B outlines the working principle of the IMPLICATION logic gate by employing PEI and heparin as inputs and the RRS, fluorescence, and absorption signals of phloxine B as the
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CONCLUSIONS
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ASSOCIATED CONTENT
In summary, this paper reports a simple, stable, and commercially available heparin sensor which utilized the special RRS peak in the long wavelength region. Our method can improve the sensitivity and selectivity. Moreover, the detection of heparin can also be performed by the fluorescent and colorimetric methods, which are also attractive and promising. The recognition mechanism is based on the reversible interaction (adsorption and desorption) of phloxine B and PEI. Especially, the detection was applied in heparin sodium injection and human serum samples with satisfactory results. This study not only extends the biological application of RRS but also provides an excellent assay for rapid detection of heparin in real samples. This sensing system has great potential for the detection of heparin in clinical and real analysis/ monitoring in the field.
* 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. 1580
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(30) Liu, S. P.; Luo, H. Q.; Li, N. B.; Liu, Z. F.; Zheng, W. X. Anal. Chem. 2001, 73, 3907−3914. (31) Qu, F.; Li, N. B.; Luo, H. Q. Anal. Chem. 2012, 84, 10373− 10379. (32) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (33) Sadamoto, R.; Tomioka, N.; Aida, T. J. Am. Chem. Soc. 1996, 118, 3978−3979. (34) Connors, K. A. Binding constants: The measurement of molecular complex stability; Wiley: New York, 1987.
AUTHOR INFORMATION
Corresponding Authors
*Tel./Fax: +86 23 6825 3237. E-mail:
[email protected]. *Tel./Fax: +86 23 6825 3237. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge the financial support from the National Natural Science Foundation of China (No. 21273174), the Municipal Science Foundation of Chongqing City (No. CSTC−2013jjB00002), and the Fundamental Research Funds for the Central Universities of China (No. XDJK2014D033).
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REFERENCES
(1) Wu, P.; Miao, L. N.; Wang, H. F.; Shao, X. G.; Yan, X. P. Angew. Chem., Int. Ed. 2011, 50, 8118−8121. (2) Xie, W. Y.; Huang, W. T.; Zhang, J. R.; Luo, H. Q.; Li, N. B. J. Mater. Chem. 2012, 22, 11479−11482. (3) Alcantara-Licudine, J. P.; Bui, N. L.; Kawate, M. K.; Li, Q. X. J. Agric. Food Chem. 1998, 46, 1005−1011. (4) Rasooly, R. FEMS Immunol. Med. Microbiol. 2007, 49, 261−265. (5) Xia, T.; Kovochich, M.; Liong, M.; Meng, H.; Kabehie, S.; George, S.; Zink, J. I.; Nel, A. E. ACS Nano 2009, 3, 3273−3286. (6) Wen, T.; Li, N. B.; Luo, H. Q. Anal. Chem. 2013, 85, 10863− 10868. (7) Lei, Y. G.; Segura, T. Biomaterials 2009, 30, 254−265. (8) Glodde, M.; Sirsi, S. R.; Lutz, G. J. Biomacromolecules 2006, 7, 347−356. (9) Shi, Y.; Luo, H. Q.; Li, N. B. Chem. Commun. 2013, 49, 6209− 6211. (10) Kuo, C. Y.; Tseng, W. L. Chem. Commun. 2013, 49, 4607−4609. (11) Bromfield, S. M.; Barnard, A.; Posocco, P.; Fermeglia, M.; Pricl, S.; Smith, D. K. J. Am. Chem. Soc. 2013, 135, 2911−2914. (12) Girolami, B.; Girolami, A. S. Thromb. Hemostasis 2006, 32, 803− 809. (13) Hattersley, P. G. J. Am. Med. Assoc. 1966, 196, 436−440. (14) Basu, D.; Gallus, A.; Hirsh, J.; Cade, J. N. Engl. J. Med. 1972, 287, 324−327. (15) Levine, M. N.; Hirsh, J.; Gent, M.; Turpie, A. G.; Cruickshank, M.; Weitz, J.; Anderson, D.; Johnson, M. Arch. Int. Med. 1994, 154, 49−56. (16) Bromfield, S. M.; Wilde, E.; Smith, D. K. Chem. Soc. Rev. 2013, 42, 9184−9195. (17) Beni, S.; Limtiaco, J. F.; Larive, C. K. Anal. Bioanal. Chem. 2011, 399, 527−539. (18) Qi, H. T.; Li, Z.; Yang, L. F.; Yu, P.; Mao, L. Q. Anal. Chem. 2013, 85, 3439−3445. (19) Zhong, A. L.; Anslyn, E. V. J. Am. Chem. Soc. 2002, 124, 9014− 9015. (20) Dai, Q.; Liu, W. M.; Zhuang, X. Q.; Wu, J. S.; Zhang, H. Y.; Wang, P. F. Anal. Chem. 2011, 83, 6559−6564. (21) Cai, L. P.; Zhan, R. Y.; Pu, K. Y.; Qi, X. Y.; Zhang, H.; Huang, W.; Liu, B. Anal. Chem. 2011, 83, 7849−7855. (22) Zhan, R. Y.; Fang, Z.; Liu, B. Anal. Chem. 2010, 82, 1326−1333. (23) Cao, R.; Li, B. X. Chem. Commun. 2011, 47, 2865−2867. (24) Wang, S. L.; Chang, Y. T. Chem. Commun. 2008, 10, 1173− 1175. (25) Yan, H.; Wang, H. F. Anal. Chem. 2011, 83, 8589−8595. (26) Hung, S. Y.; Tseng, W. L. Biosens. Bioelectron. 2014, 57, 186− 191. (27) Li, T.; Wang, E. K.; Dong, S. J. J. Am. Chem. Soc. 2009, 131, 15082−15083. (28) Freeman, R.; Finder, T.; Willner, I. Angew. Chem., Int. Ed. 2009, 48, 7818−7821. (29) Park, K. S.; Jung, C.; Park, H. G. Angew. Chem., Int. Ed. 2010, 49, 9757−9760. 1581
DOI: 10.1021/ac504023b Anal. Chem. 2015, 87, 1575−1581
