Biosensors and Bioelectronics 57 (2014) 186–191

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A polyadenosine–coralyne complex as a novel fluorescent probe for the sensitive and selective detection of heparin in plasma Szu-Ying Hung a, Wei-Lung Tseng a,b,c,n a

Department of Chemistry, National Sun Yat-sen University, Kaohsiung 804, Taiwan School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan c Center for Nanoscience and Nanotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan b

art ic l e i nf o

a b s t r a c t

Article history: Received 12 December 2013 Received in revised form 27 January 2014 Accepted 5 February 2014 Available online 12 February 2014

This study presents the development of a simple, label-free, sensitive, and selective detection system for heparin based on the use of a complex of 20-repeat adenosine (A20) and coralyne. Coralyne emits relatively weak fluorescence in an aqueous solution. In the presence of A20, coralyne molecules complexed with A20 through A2-coralyne-A2 coordination. An increase in the fluorescence of coralyne was observed because coralyne remained separate from water in the hydrophobic environment of the folded A20. The presence of heparin and the formation of the coralyne–heparin complex caused coralyne to be removed from the A20–corlayne complex. Because heparin promoted coralyne dimerization, the fluorescence of coralyne decreased as a function of the concentration of added heparin. This detection method is effective because the electrostatic attraction between heparin and coralyne is substantially stronger than the coordination between A20 and coralyne in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at pH 7.0. Under optimal conditions (5 μM coralyne, 1 μM poly A20, and 10 mM HEPES), this probe exhibited high selectivity ( 490-fold) toward heparin over hyaluronic acid and chondroitin sulfate. The probe's detection limit for heparin was determined to be 4 nM (75 ng/mL) at a signal-to-noise ratio of 3. This study validates the practicality of using the A20–corlayne complex to determine the concentration of heparin in plasma. & 2014 Elsevier B.V. All rights reserved.

Keywords: Heparin Polyadenosine Coralyne Fluorescent sensor Protamine

1. Introduction Heparin, which consists primarily of trisulfated disaccharide repeating units, is the most highly charged polysaccharide in biological systems. Because of its crucial roles in regulating cell growth and differentiation, immune defense, and blood coagulation, heparin has been used clinically as an anticoagulant drug for over 80 years. Heparin is an effective drug in treating and preventing venous thromboembolism and blood clotting because it is capable of accelerating the inactivation rate of coagulation factors such as fibrinbound thrombin. The recommended therapeutic ranges of heparin levels are 2–8 U/mL (17–67 μM) during cardiovascular surgery and 0.2–1.2 U/mL (1.7–10 μM) during postoperative and long-term care. The accurate quantification of heparin is essential during anticoagulant therapy and surgery because a high heparin dose leads to adverse effects such as hemorrhaging and thrombocytopenia (Girolami and Girolami, 2006; Warkentin et al., 1995). n Corresponding author at: Department of Chemistry, National Sun Yat-sen University, 70, Lien-hai Road, Kaohsiung 804, Taiwan. Tel.: þ886 7 5252000; fax: þ886 7 3684046. E-mail address: [email protected] (W.-L. Tseng).

http://dx.doi.org/10.1016/j.bios.2014.02.010 0956-5663 & 2014 Elsevier B.V. All rights reserved.

Because of its clinical importance, numerous methods have been devised for quantifying heparin levels in the blood. Activated clotting time (ACT) assays, such as the activated partial thromboplastin time and chromogenic antifactor Xa assay, are traditional clinical procedures for quantifying heparin levels (Murray et al., 1997; Raymond et al., 2003). However, the ACT method is influenced by many variables such as hypothermia, hemodilution-induced dilution of clotting factors, and interpatient differences in antithrombin levels (Despotis et al., 1997; Machin and Devine, 2005). The Hepcon HMS assay system, which is based on the titration of heparin with protamine and clot formation in the end point, enables more accurate monitoring of heparin concentrations; however, this method involves indirect sensing, and is time consuming and expensive (Ramamurthy et al., 1998). Researchers have developed various methods for identifying and quantifying heparin, such as anion exchange chromatography (Ander et al., 2001), capillary electrophoresis (Volpi et al., 2012), potentiometric methods (Crespo et al., 2012), and ion mobility mass spectrometry (Seo et al., 2012). Although all of these reported methods have demonstrated high sensitivity to heparin, they are costly, time consuming, and nonportable. In response to these shortcomings, massive sensors have been devised for the simple and rapid sensing of heparin, using

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fluorophores (Dai et al., 2011; Wang et al., 2008; Wang and Chang, 2008), chromphores (Zhong and Anslyn, 2002), cationic polymers (Pu and Liu, 2009; Zhan et al., 2010), boronic acidcontaining copolymers (Sun et al., 2007), polyethyleneiminemodified quantum dots (Yan and Wang, 2011), graphene oxide (Fu et al., 2012), positive-charged gold nanoparticles (Cao and Li, 2011), and 4-mercaptopyridine-modified silver nanoparticles (Wang et al., 2013). In general, the mechanism for these sensors relies on electrostatic binding between a cationic sensor and anionic heparin, the ability of the boronic acid group to chelate to sugar diol units, or both. Nevertheless, most of these sensors cannot distinguish heparin from its analogues, such as hyaluronic acid (HA) and chondroitin sulfate (ChS). We recently proposed a molecular beacon (MB) strategy for the fluorescence turn-on detection of heparin in plasma (Kuo and Tseng, 2013), Because coralyne can drive adenosine (A)—A mismatches to form A2-coralyne-A2 coordination (Lin and Tseng, 2011, 2012), the designed MB containing 16-mer A bases formed a hairpin structure in the presence of coralyne. When a hairpin-shaped MB encounters heparin, the electrostatic force between coralyne and heparin splits the stem and switches on the MB fluorescence. Although this MB sensor provides high sensitivity and selectivity for heparin, chemically modifying the MB with fluorophores and quenchers is relatively complex and expensive. Herein, this study presents a convenient, label-free, selective, and sensitive assay for standard (unfractionated) heparin through the competitive binding between heparin and 20 repeat A (A20) to coralyne. We demonstrated that the presence of A20 can efficiently enhance the fluorescence of coralyne and the selectivity of coralyne toward heparin. Scheme 1 illustrates the mechanism for a fluorescence turn-off assay of heaprin using the A20–coralyne complex. Coralyne drives the A–A mismatches to form a stable A2-coralyne-A2 complex in the hydrophobic environment of A20, causing an enhancement in the fluorescence of coralyne at a neutral pH. The presence of heparin promotes the dimerization of coralyne through electrostatic attraction, thereby removing coralyne from the formed A20–coralyne complex. Because of dimer-promoted fluorescence quenching of coralyne, the fluorescence of the A20–coralyne complex was switched off in the presence of heparin. To demonstrate the practicality of this probe, we applied it to quantifying heparin in plasma.

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2. Experimental section 2.1. Chemicals 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride (NaCl), protamine sulfate (from salmon), coralyne sulfoacetate, heparin (sodium salt; MW 18,000) from porcine intestinal mucosa, ChS (sodium salt) from bovine trachea from, and HA (sodium salt) from bovine vitreous humor were purchased from Sigma-Aldrich (St. Louis, MO, USA). All DNA samples were obtained from Neogene Biomedicals Corporation (Taipei, Taiwan). Milli-Q ultrapure water (Milli-Pore, Hamburg, Germany) was used in all of the experiments. 2.2. Apparatus The absorption and fluorescence spectra of coralyne were recorded using JASCO V-670 spectrophotometer (JASCO, Tokyo, Japan) and a Hitachi F-7000 fluorometer (Hitachi, Tokyo, Japan). Fluorescence polarization was recorded using a Hitachi F-7000 fluorometer equipped with an autopolarization measurement. Circular dichroism (CD) was performed on a JASCO model J-815 CD spectropolarimeter (JASCO, Tokyo, Japan). 2.3. Sample preparation All DNA samples and polysaccharides were prepared in a solution containing 10 mM HEPES (pH 3.0–10.0) and 0–160 mM NaCl. Polyadenosine (0–8 μM, 100 μL) was incubated with coralyne (20 μM, 100 μL) at ambient temperature for 30 min. Polysaccharides (0–2 μM, 200 μL), including heparin, Chs, and HA, were added to an equal volume of the resulting solutions (200 μL). After 0–20 min incubation at ambient temperature, the mixed solutions were transferred separately into a 4 mL quartz cuvette. Their fluorescence sepctra were collected using a Hitachi F-7000 fluorometer at an excitation wavelength of 425 nm. For sensing protamine, heparin (50 μL, 0–10 μM) was incubated with protamine (50 μL, 0–70 μM) at ambient temperature for 10 min. The mixture (100 μL) was added to a solution (400 μL) containing 5 μM coralyne, 1 μM A20, 10 mM HEPES (pH 7.0). After 5 min, the fluorescence spectra of the resulting solutions were recorded using a Hitachi F-7000 fluorometer at the same excitation wavelength.

Scheme 1. Turn-off fluorescence detection of heparin based on competitive binding between heparin and the A20–coralyne complex to coralyne.

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2.4. Analysis of heparin in plasma Blood samples were collected from a healthy adult female with the age of 23 years. To collect plasma samples, the whole blood samples were immediately centrifuged at 3000 rpm for 10 min at 4 1C. The obtained plasma samples were spiked with standard solutions of heparin (0–40 μM). The spiked samples were diluted to 40-fold with a solution containing 10 mM HEPES (pH 7.0). We incubated the resulting solutions with an equal volume of the A20– coralyne complex (2 μM A20 and 10 μM coralyne) at ambient temperature for 5 min and collected their fluorescence spectra at an excitation wavelength of 425 nm.

3. Result and discussion 3.1. Optical properties of coralyne Although previous studies have demonstrated that coralyne can specifically and strongly bind to polyadensoine through the formation of one coralyne per four A bases (Persil et al., 2004; Polak and Hud, 2002), few studies have investigated the effect of polyadenosine on the optical properties of coralyne. Therefore, we first compared the optical properties of coralyne alone and in the presence of A20. Fig. 1A shows that the absorption spectrum of 5 μM coralyne exhibited maximum absorption at 420 nm. The equilibrium constant for the dimerization of coralyne was 1.1  105 M  1 (Gough et al., 1979), implying that 73% of coralyne molecules were present as monomers at this concentration. Adding 1 μM A20 to a solution of 5 μM coralyne resulted in two new bands centered at 412 and 435 nm. Because positively charged coralyne has a planar hydrophobic moiety, it facilitates movement into the relatively nonpolar interior of A20 through A2-coralyne-A2 coordination. Consequently, the effective overlapping of the π electron cloud of adenosine bases with that of coralyne caused the change in the fluorescence of coralyne. The spectrum

change of coralyne induced by A20 is consistent with that caused by polyadenosine (Xing et al., 2005). Upon excitation at 425 nm, the emission band of coralyne was centered at approximately 475 nm (Fig. 1B). The presence of A20 induced a remarkable increase in the fluorescence of coralyne. This result might arise from the fact that coralyne molecules are separated and arrayed in folded A20. Another reason is that the hydrophobic environment of the folded A20 prevents coralyne from contacting water. To prove this hypothesis, other control DNA samples, including 20-repeat thymines (T20), cytosines (C20), and guanines (G20), were used in place of A20 under the same conditions (Fig. 1C). Upon the addition of T20, we observed a reduction in the fluorescence intensity of coralyne, mainly because of the lack of coordination between T20 and coralyne. In addition, the electrostatic force between cationic coralyne and anionic T20 facilitates the dimerization of coralyne, resulting in the fluorescence quenching of coralyne. Similar results were observed in the study of C20 and G20. Our results are in agreement with those of previous studies showing that anionic polysaccharides promote the dimerization of coralyne and quench its fluorescence (Megyesi et al., 2009). The quantum yield of coralyne was determined to be 36% by using quinine sulfate as the reference (Fig. S1, Supplementary materials). This value was improved to 55% by introducing A20 to a solution of coralyne. The interaction between A20 and coralyne was examined using CD spectroscopy and fluorescence polarization. Compared with the CD spectrum of A20, a mixture of coralyne and A20 generated a new band between 320 and 340 nm, signifying that coralyne triggers a dramatic change in the conformation of A20 (Fig. 1D). Similarly, Xing et al. (2005) demonstrated that the addition of coralyne to a solution of polyadenosine caused the appearance of a new band between 320 and 340 nm. In addition, the fluorescence polarization value P of 5 μM coralyne was examined with and without 1 μM A20. Fluorescence polarization is a highly sensitive means for probing the interaction between a small-molecule ligand and a macromolecule (Smith and Eremin, 2008). In contrast to coralyne, a considerable increase in the value of P was observed in

Fig. 1. Absorption (A) and fluorescence (B) spectra of 5 μM coralyne in the (a) absence and (b) presence of 1 μM A20. (C) Fluorescence intensity at 475 nm of 5 μM coralyne (a) before and ((b)–(e)) after the addition of 1 μM (b) A20, (c) T20, (d) C20, and (e) G20. (D) CD spectra of solutions of (a) 50 μM A20 and (b) 50 μM A20 and 50 μM coralyne. ((A)–(D)) Coralyne was incubated with polyadenosine in 10 mM HEPES (pH 7.0) for 30 min at ambient temperature.

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the presence of A20, indicating the formation of the A20–coralyne complex (Fig. S2, Supplementary materials). This study subsequently investigated the effects of the polyadenosine length on the fluorescence of coralyne in a 10 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.0). A series of polyadenosine, including A8, A12, A20, A28, A36, and A44, was used to test this effect. Fig. 2A shows A8- and A12-induced fluorescence quenching of coralyne, which occurred because coralyne is almost impossible to cause short DNA strands to form a folded structure through A2-coralyne-A2 coordination. When the number of bases of polyadenosine exceeded 12, the fluorescence intensity of coralyne at 475 nm progressively increased as the number of bases of polyadenosine increased and reached a saturation level at A36. Obviously, long DNA strands enable easier formation of a folded structure than short DNA strands do. The stability of the A20–coralyne complex was then tested by varying the NaCl concentration and solution pH. Fig. 2B shows that the fluorescence intensity of the A20–coralyne complex at 475 nm remained nearly constant in the presence of 0–160 mM NaCl. This result revealed that the A20–coralyne complex was stable under physiological conditions. Fig. 2C shows that the fluorescence intensity of the A20–coralyne complex at 475 nm reached a maximum plateau at pH 7, indicating that coralyne binds to polyadenosine more tightly at pH 7 than at other pH values. Similarly, Lim et al. reported that the coordination of A2-coralyneA2 was stable at a neutral pH (Kim et al., 2011). We also optimized the concentration of A20 in the presence of 5 μM coralyne. As indicated in Fig. 2D, the fluorescence intensity of coralyne at 475 nm achieved a plateau after 1 μM A20 was added, which is reasonable because one molecule of A20 can bind to five molecules of coralyne. According to the information in Fig. 2D and a Scatchard plot fitting McGhee and von Hippel (1974) analysis (Fig. S3, Supplementary materials), the binding constant between coralyne and A20 was calculated to be 4.1  104 M  1.

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3.2. Sensing of heparin A previous study demonstrated that chondroitin-6-sulfate and dextran sulfate were capable of promoting the dimerization of coralyne through electrostatic attraction, leading to a remarkable fluorescence quenching of coralyne (Megyesi et al., 2009). This result suggested that negatively charged heparin can quench the fluorescence of cationic coralyne. Fig. S4 (Supplementary materials) shows that the fluorescence of coralyne gradually diminished with an increase in the heparin concentration. Based on a Scatchard plot fitting McGhee and von Hippel (1974) analysis (inset in Fig. S4, Supplementary materials), the binding constant of heparin and coralyne was estimated to be 2.0  107 M  1. Apparently, the binding of coralyne to heparin is stronger than that to A20, implying that the A20–coralyne complex might have an ability to detect heparin based on competitive binding between heparin and A20 to coralyne. Following the addition of an equal volume of 0.2 μM heparin to a solution consisting of 10 mM HEPES (pH 7.0), 10 μM coralyne, and 2 μM A20, we observed a remarkable reduction in the fluorescence intensity at 475 nm (Fig. 3A). The heparin-induced fluorescence quenching of the A20–coralyne complex was nearly complete after 5 min (inset in Fig. 3A). This study subsequently tested whether the length of polyadenosine influences the sensitivity of the proposed probe to heparin. Fig. 3B shows that four types of single-stranded DNA (namely, A20, A28, A36, and A44) complexed with coralyne were all effective in detecting heparin. After increasing the number of A bases, the difference in fluorescence intensity of the formed probe at 475 nm between the absence and presence of heparin decreased. Because longer poladenosine interacts more strongly with coralyne, heparin exhibits more difficulty in removing coralyne from the polyadenosine–coralyne complex. Thus, A20 was chosen to complex with coralyne in the following study.

Fig. 2. (A) Fluorescence intensity at 475 nm of coralyne (a) before and ((b)–(g)) after the addition of (b) A8, (c) A12, (d) A20, (e) A28, (f) A36, and (g) A44. Effects of (B) the NaCl concentration, (C) the solution pH, and (D) the A20 concentration on the fluorescence intensity at 475 nm of the A20–coralyne complex. 5 μM coralyne was incubated with ((A)–(C)) 1 μM and (D) 0–2 μM polyadenosine in 10 mM HEPES (pH 7.0) for 30 min at ambient temperature.

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Fig. 3. (A) Fluorescence spectra of the A20–coralyne complex in the (a) absence and (b) presence of 0.1 μM heparin. Inset: time-course measurements of fluorescence intensity (475 nm) of the A20–coralyne complex upon the addition of 0.1 μM heparin. (B) Fluorescence intensity (475 nm) of the polyadenosine–coralyne complex (a) before and (b) after the addition of 0.1 μM heparin. ((A) and (B)) 5 μM coralyne was incubated with 1 μM polyadenosine in 10 mM HEPES (pH 7.0) for 30 min at ambient temperature. The incubation time between the A20–coralyne complex and heparin was 5 min.

Fig. 4. (A) Fluorescence spectra of the A20–coralyne complex in the presence of increasing concentration of heparin. The arrow indicates the signal changes as increases in heparin concentration (0, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800, and 100 nM) Inset: a plot of the (IF0–IF)/IF0 value at 475 nm versus the heparin concentration. The incubation time between the A20–coralyne complex and heparin was 5 min. The error bars represent standard deviations based on three independent measurements. (B) The (IF0–IF)/IF0 value obtained from the addition of polysaccharide (0.1–10 μM) to a solution of the A20–coralyne complex.

3.2.1. Quantification, selectivity, and application Under optimal conditions (10 mM HEPES, 5 μM coralyne, and 1 μM A20; pH 7.0), this study tested the sensitivity of the proposed probe to heparin in a 5-min incubation time. When the concentration of heparin changed from 10 to 1000 nM (0.18 to 18 μg/mL), the fluorescence spectra of the A20–coralyne complex revealed a progressive decrease in the fluorescence intensity at 475 nm and an increase in the (IF0–IF)/IF0 value with an increase in the concentration of heparin (Fig. 4A). IF0 and IF corresponded to the fluorescence intensity of the A20–coralyne complex at 475 nm in the absence and presence of heparin, respectively. Plotting the (IF0–IF)/IF0 value versus the heparin concentration produced a linear calibration curve (R2 ¼0.9813) over the range of 10– 100 nM (inset in Fig. 4A). The relative standard deviation of the (IF0–IF)/IF0 value at three concentration levels (10, 100, and 1000 nM) was less than 3%. The detection limit (LOD) at a signal-to-noise ratio of 3 for heparin was calculated to be 4 nM (approximately 75 ng/mL). The LOD of heparin obtained by this probe is substantially lower than the therapeutic level of heparin required for cardiovascular surgery and postoperative and longterm care. In addition, the sensitivity of this probe for heparin is comparable to that of graphene oxide (Fu et al., 2012), positivecharged gold nanoparticles (Cao and Li, 2011), and MBs (Kuo and Tseng, 2013). Compared with fluorophores (Dai et al., 2011; Wang et al., 2008; Wang and Chang, 2008), chromphores (Zhong and Anslyn, 2002), cationic polymers (Pu and Liu, 2009; Zhan et al.,

2010), boronic acid-containing copolymers (Sun et al., 2007), and polyethyleneimine-modified quantum dots (Yan and Wang, 2011), this probe provides greater sensitivity to heparin. Additionally, the proposed probe can be used to quantify protamine. When high-pI protamine neutralizes negative charges of heparin through electrostatic attraction between them, the formed complex is unable to remove coralyne from the proposed probe. Therefore, we initially mixed 0–7000 nM protamine with 0.2 μM heparin. Following the addition of the resulting solutions to the A20–coralyne complex, the fluorescence intensity at 475 nm gradually increased as the concentration of protamine increased (Fig. S5, Supplementary materials). Evidently, this probe is capable of sensing protamine. The selectivity of the A20–coralyne complex to heparin was then tested, and Fig. 4B and Fig. S6 (Supplementary materials) shows that changes in the (IF0–IF)/IF0 value occurred within 5 min after separately adding anionic polysaccharide, metal ions, anions, bovine serum albumin, adenosine triphosphate. Evidently, this probe can provide greater (490-fold) selectivity for heparin than Chs, HA, metal ions, anions, bovine serum albumin, and triphosphate. This is mainly because heparin possesses more negative charge sites for complexing coralyne than Chs and HA do. Under the condition in which Chs generated an equivalent signal for comparison with heparin, the ratio of the concentrations of heparin to Chs was 91. The value of this discrimination ratio obtained from the A20–coralyne complex was superior to that obtained from the reported heparin sensor (Table S1, Supplementary

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4. Conclusions This study demonstrated that the A20–coralyne complex is a simple, cost-effective, sensitive, and selective probe for sensing heparin in plasma. We showed polyadenosine (n 412) to be a highly efficient fluorescence enhancer for coralyne through A2-coralyne-A2 coordination. By contrast, heparin was capable of efficiently quenching coralyne through electrostatic attraction. The sensing specificity relied on competition between A20 and heparin for interacting with coralyne. This probe provided numerous distinctive advantages, including high sensitivity (LOD ¼4 nM), short analysis time (approximately 5 min), and high selectivity (discrimination ratio ¼ 91). In contrast to adenosine-based MBs (Kuo and Tseng, 2013), there is no need to modify the probe with fluorophores and quenchers. Our study results indicated that the proposed probe has great potential for use in routine assays of heparin in clinical samples.

Acknowledgment We would like to thank National Science Council (NSC 1002628-M-110-001-MY 4) for the financial support of this study.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.02.010. References Fig. 5. (A) Fluorescence spectra obtained from the addition of heparin-spiked plasma sample to a solution containing 1 μM A20 and 5 μM coralyne. The arrow indicates the signal changes as increases in analyte concentration (0, 0.4, 0.8, 1.6, 2.4, 3.2, 4, 8, 16, 24, 32, and 40 μM). Inset: a plot of the (IF0–IF)/IF0 value at 475 nm versus the spiked concentration of heparin. A mixture of A20 and coralyne was incubated in 10 mM HEPES (pH 7.0) for 30 min. The incubation time between the A20–coralyne complex and heparin was 5 min. The error bars represent standard deviations based on three independent measurements. (B) Three linear calibration curves for the quantification of heparin in human plasma Samples 1–3 based on the use of the A20–coralyne complex. The error bars represent standard deviations based on three independent measurements.

materials). Additionally, in the absence of A20, coralyne exhibited poor selectivity toward heparin (Fig. S7, Supplementary materials), demonstrating that A20 is effective to improve the selectivity of coralyne. The feasibility of the proposed probe for sensing heparin in plasma was, therefore, validated. Progressive decreases in the fluorescence intensity at 527 nm and the (IF0–IF)/IF0 value were observed after three individual plasma samples (denoted as Samples 1–3) were spiked with standard solutions containing 0.4–40 μM heparin (Fig. 5A; Figs. S8 and S9, Supplementary materials). Three linear calibration curves for determining the concentration of heparin in human plasma Samples 1–3 were constructed by plotting the (IF0–IF)/IF0 values versus the spiked concentrations of heparin over a range of 0.4–4 μM (Fig. 5B, Supplementary materials). Because the plasma samples were diluted 40-fold throughout the sample preparation, the method detection limit of heparin in plasma was determined to be 0.2 μM (approximately 4 μg/mL). The slope of the calibration curve obtained from Sample 1 resembled those obtained from Samples 2 and 3, suggesting that the proposed probe is largely free from matrix effect of the plasma sample.

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

This study presents the development of a simple, label-free, sensitive, and selective detection system for heparin based on the use of a complex of 20...
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