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“Turn on-off” fluorescent sensor for protamine and heparin based on label-free silicon quantum dots coupled with gold nanoparticles
Sensors and Actuators B 213 (2015) 131–138
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
“Turn on-off” fluorescent sensor for protamine and heparin based on label-free silicon quantum dots coupled with gold nanoparticles Xue Peng, Qian Long, Haitao Li, Youyu Zhang ∗ , Shouzhuo Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China
a r t i c l e
i n f o
Article history: Received 25 November 2014 Received in revised form 10 February 2015 Accepted 13 February 2015 Available online 23 February 2015 Keywords: Silicon quantum dots Gold nanoparticle Protamine Heparin
a b s t r a c t An ultrasensitive “turn on-off” fluorescent sensor was presented for determination of protamine and heparin based on the high quenching ability of gold nanoparticles (AuNPs) to the fluorescence of silicon quantum dots (SiQDs) as well as high binding affinity of protamine with heparin. The fluorescence of SiQDs was quenched significantly by adding AuNPs into SiQDs solution. Upon addition of protamine, the AuNPs aggregated and the fluorescence of SiQDs was recovered due to the competitive adsorption of protamine and SiQDs on AuNPs. Addition of heparin disturbed the interaction between protamine and AuNPs due to its strong affinity to protamine, resulting in an adsorption of SiQDs on the surface of AuNPs. Thus the fluorescence of SiQDs was quenched. The strategy was simply achieved by measuring the changes in the fluorescence of SiQDs. The research results suggested that the developed method has several advantages such as label-free, high sensitivity, low cost, ease of operation. The linear response range was obtained over the range 0–1.2 g/mL and 0.002–1.4 g/mL with the low detection limit of 6.7 ng/mL and 0.67 ng/mL for protamine and heparin, respectively. The sensing platform was successfully applied to determination of heparin and protamine in serum samples. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The polyionic drugs heparin and protamine are widely used in clinical procedures [1]. Heparin, a negatively charged linear polysaccharide, has been extensively used in medicine as an anticoagulant during clinical procedures for the prevention of blood clotting [2]. However, the higher dose and prolonged use of heparin often induce some adverse effects such as thrombocytopenia and hemorrhage [3,4]. Protamine is a low molecular weight protein with a positive charge of 20 in physiological condition. And it is usually administered following surgery to reverse the anticoagulant activity of heparin [5,6]. The polycationic protamine is rich in basic arginine residues, and the characteristic guanidinium groups of protamine were easy to react with acid to form a stable ion pair complex that is devoid of anticoagulation activity [7]. Based on the clinical importance of these two polyionic drugs and their toxic effects, it is vital to develop a simple, rapid, and cost-effective method for the quantification of both heparin and protamine. To date, various analytical methodologies have been developed for the sensitive assay of heparin and protamine. Several
∗ Corresponding author. Tel.: +86 731 8865515; fax: +86 731 8865515. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.snb.2015.02.070 0925-4005/© 2015 Elsevier B.V. All rights reserved.
conventional methods such as activated clotting time [8], activated thromboplastin time [9], ion chromatography [10], and ion pair high-performance liquid chromatography [11] have been used for the estimation of heparin or protamine. These methods are often not sufficiently sensitive, and the results are affected by many variables. Accordingly, a number of advanced methods have recently been developed to identify and quantitate heparin and protamine, such as electrochemistry [12], light scattering [13], nuclear magnetic resonance, colorimetry [14]. Although these methods have high selectivity and adequate reliability, most of these means suffer from the disadvantages of high costs, sophisticated instruments, and generally limited to the detection limit. Therefore, it is urgently required to develop simple, sensitive and cost-effective methods. Fluorescent methods, in particular, are extremely attractive because they offer advantages of low-cost portable instruments as well as easy-to-operation. Quantum dots (QDs) are versatile inorganic nanoparticles with unique photophysical and chemical properties [15,16] such as broad excitation spectra [17], photobleaching resistance [18], narrow and tunable emission spectra, simultaneous excitation of multiple fluorescence colors [19], and a larger Stokes shift over conventional organic fluorophores [20]. Recently, polyethyleneimine-capped Mn-doped ZnS quantum dots [12], CdTe quantum dots [21] and CuInS2 quantum dots [22] have been
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used as probes for detection of heparin. These methods were successfully applied to detect heparin, however, synthesis procedures of these probes are complicated, and only one analyte could be tested. And the applications to the clinical field have been hampered owing to the high toxicity of the CdTe quantum dots and eventually would cause serious environmental problems due to the leakage of cadmium. Therefore, it is also crucial to develop excellent nanomaterials which are easily obtained for fabricating highly sensitive and selective biosensors to quantify both heparin and protamine. As inert, nontoxic, abundant and low-cost nanomaterials, SiQDs have been demonstrated to be environmentally friendly photoluminescence probes and have attracted much interest. In comparison to other QDs, SiQDs have excellent biocompatibility and noncytotoxic properties. These advantages enable them to play a great role in a variety of applications. Recently, our group have developed fluorescent and colorimetric methods for the detection of glucose [23,24] and organophosphorus pesticides [25] based on the high fluorescence quenching ability of H2 O2 to the fluorescence of SiQDs and the peroxidase mimetics of SiQDs. Herein, we established a SiQDs-based sensing platform which coupled with AuNPs for ultrasensitive detection of heparin and protamine. AuNPs are unique quenchers for organic dyes or QDs through both energy-transfer and electron-transfer processes. It was found that AuNPs can quench fluorescence of SiQDs. Polycationic protamine was used as a medium for inducing the aggregation of citrate-capped AuNPs through electrostatic interaction, resulting in the detaching of the SiQDs and the fluorescence of the SiQDs being recovered. Addition of polyanionic heparin disturbed the interaction of protamine and AuNPs due to the strong affinity of heparin to protamine. Scheme 1 illustrates the proposed mechanism for the sensing of heparin and protamine. The proposed biosensor developed a simple, sensitive method for monitoring of both heparin and protamine. 2. Experimental 2.1. Materials Chloroauric acid (HAuCl4 ), protamine sulfate salt and heparin sodium salt were purchased from Sigma (Shanghai, China). Silicon wafers (phosphorus-doped (p-type), 8 resistivity) and phosphomolybdic acid (POM) were purchased from Sigma–Aldrich. Anhydrous ethanol (analytical grade), hydrofluoric acid (HF), and H2 O2 were purchased from Shanghai Chemical Reagent. All chemicals from commercial sources were of analytical grade and used without further purification. PBS buffer (pH 7.4, 10 mM sodium phosphate) and Milli-Q ultrapure water (Millipore, ≥18 M cm) were used throughout the experiments. The reconstituted serum sample was prepared by dissolving 20 mg of KCl, 800 mg of NaCl, 20 mg of KH2 PO4 , 115 mg of Na2 HPO4 and 4 g of bovine albumin in 100 mL of water.
2.3. Preparation of SiQDs Photoluminescence SiQDs were synthesized by the POMassisted electrochemical etching of bulk Si [26]. Briefly, Si wafers were cleaned in 20% hydrofluoric acid (HF) for 5 min first to remove surface oxides and impurities. The electrolyte of the electrochemical etching process was prepared by dissolving 0.02 g of POM in 30 mL of anhydrous ethanol, then 25 mL of HF was added under stirring, and with a suitable amount (5–10 mL) of H2 O2 (hydrogen peroxide 30%). The electrochemical system composed of graphite as anode and Si wafer as cathode. Graphite and Si wafer were put into the as-prepared electrolyte and connected to the DC power supply by connecting wires. There was a layer of n-hexane covering the electrolyte solution. The current intensity ranged 4–10 mA/cm2 and the whole etching process was performed for about 2 h. Without complicated centrifugation and filtration, just ultrasonication fracturing the etched silicon wafer in absolute ethanol occurred, and then SiQDs with excellent fluorescence were obtained. 2.4. Synthesis of AuNPs The colloidal solution of AuNPs was synthesized by means of citrate reduction of AuCl3 ·HCl·4H2 O [27]. All glassware and magnetic stirrers used for the synthesis were thoroughly cleaned in aqua regia, rinsed with ultrapure water, and then oven-dried prior to use. Briefly, 100 mL chloroauric acid (HAuCl4 ) solution (containing 0.5 mL 2% HAuCl4 ) was firstly heated to boiling, and then 1.8 mL of 1% sodium citrate solution was rapidly added to the boiled HAuCl4 solution under vigorous stirring resulted in a color change from light yellow to wine red. Boiling was continued for 10 min and stirring was continued for an additional 15 min without heating. After the solution reached room temperature, the prepared AuNPs solution was stored in the 4 ◦ C refrigerator before use. The concentration of the AuNPs was determined according to the Beer’s law by using UV–vis spectroscopy [28]. 2.5. Fluorescence assay of protamine and heparin The protamine-induced fluorescence recovery of SiQDs was monitored by observing the spectral change during the addition of different concentrations of protamine to the mixture of SiQDs–AuNPs. In a typical procedure, the mixed solution of SiQDs–AuNPs was prepared using 80 L of AuNPs, 80 L of SiQDs solution and PBS buffer solution (10 mM, pH 7.4). Then different concentrations of protamine were incubated with SiQDs–AuNPs mixture, the final volume is 600 L. Heparin was detected in the following way: first protamine (1.2 g/mL) was added to SiQDs–AuNPs mixture. A sudden color change from red to blue was noticed after the addition of protamine. Then different concentrations of heparin were added to the resulted solution and heparin-driven de-aggregation of the AuNPs occurred resulted in a reversible color change. The spectra were recorded with incubation for 50 min.
2.2. Instrumentation 2.6. Selectivity experiments toward protamine and heparin SiQDs synthesis was conducted on a CHI660A electrochemical workstation (CHI Instrument Inc., USA). UV–vis spectra were measured on a UV2450 spectrophotometer (Shimadzu). The F4500 fluorescence spectrophotometer (Hitachi Co., Japan) was used to collect the fluorescence spectra operating at the excitation wavelength of 360 nm, with both excitation and emission slit widths of 10 nm. Transmission electron microscopy (TEM) images were recorded using a JEOL-1230 model. Fourier transform infrared (FTIR) spectra were performed on a Nicolet Nexus 670 FTIR spectrometer (Nicolet Instrument Co., U.S.A.). The dynamic light scattering (DLS) was measured by Zetasizer3000HS.
The solutions of other potentially competing substances were prepared in PBS buffer solution for the experiments. The fluorescent responses of the proposed sensor to the competing substances were examined by a similar procedure mentioned above. The selectivity experiments for protamine was achieved with the addition of selected competing substances (K+ , collagen, lysozyme, Ca2+ , IgG, Na+ , bovine serum albumin (BSA), Mg2+ and ConA) (0.2 mg/mL) to the mixture SiQDs–AuNPs in PBS buffer solution (10 mM, pH 7.4), respectively. The selectivity procedure of heparin was detected with the following steps: protamine (1.2 g/mL) was added to
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Scheme 1. Schematic illustration of the design rationale for the fluorescence detection of protamine and heparin based on SiQDs coupled with AuNPs.
SiQDs–AuNPs mixture and then interferents (SO4 2− , BSA, sodium citrate, glucose, Cl− ) at 0.2 mg/mL, (dextran (Dex), hyaluronic acid (HA), chondroitin sulfate (Chs)) at 1.4 g/mL were added to the resulted solution, respectively. The spectra were measured by a fluorescence spectrophotometer excited at 360 nm. 2.7. Serum samples analysis The standard addition method was used to test the practical application of the proposed sensor. 60 L reconstituted serum was diluted by 540 L SiQDs/AuNPs solution without aggregating. Then, 0.2, 0.4, and 0.6 g/mL protamine was added to the mixture of reconstituted serum and SiQDs–AuNPs, respectively. The mixture was incubated for 50 min under a pH of 7.4 before fluorescence measurement. Heparin was detected with the following steps: protamine (1.2 g/mL) was added to the mixture of reconstituted serum and SiQDs–AuNPs and then 0.4, 0.8, and 1.2 g/mL heparin were added to the resulted solution. Each experiment was repeated 3 times. 3. Results and discussion 3.1. Characterization of the SiQDs The morphology and optical properties of SiQDs were characterized. TEM (Fig. 1A) and the high resolution transmission electron microscopy (HRTEM; inset of Fig. 1A) images show that the SiQDs with low polydispersity were spherical and the average diameter was about 8 nm. The diameter measured by DLS confirms the small size of the SiQDs with a hydrodynamic diameter of ∼9.36 nm in Fig. 1B. The different diameters measured by TEM and DLS are due to different surface states of the same sample under the two measurement conditions. Fig. 1C shows the normalized PL spectra of SiQDs at different excitation wavelength ranging from 340 to 390 nm. And it was found that the maximum emission of SiQDs locates at approximately 440 nm. The absorption, excitation and emission spectra of SiQDs in aqueous solution are presented in Fig. 1D. The absorbance below 290 nm is a characteristic absorbance of SiQDs [29]. In the FTIR spectra of SiQDs (Fig. 1E), a band centered at around 900 cm−1 represents Si–H bonds, and coupled H–Si–Si–H stretch bonds were observed
at 2100 cm−1 . Proved by FTIR measurement, the as-prepared SiQDs are terminated with Si–H bonds. The PL quantum yield of SiQDs was up to ∼9.6% according to the Williams method [30]. Upon further investigation, we found that the PL intensity of SiQDs was not affected by temperature (Fig. S1A) and reached the maximum when the pH of SiQDs in solution was 7.4 (Fig. S1B). Moreover, the photostability of SiQDs was excellent (Fig. S1C). 3.2. SiQDs fluorescence quenching by AuNPs As shown in Fig. S2A, upon addition of AuNPs to the SiQDs solution, the fluorescence intensity of SiQDs evidently decreased with increasing the amount of AuNPs. AuNPs were prepared using trisodium citrate as reducing agent, so citrate-stabilized AuNPs is negatively charged [27]. SiQDs are positively charged, since the asprepared SiQDs are terminated with Si–H bonds [31]. Therefore, SiQDs can bind with AuNPs through electrostatic interaction. The PL quenching mechanism of SiQDs by AuNPs can be explained as follows: AuNPs can quench the fluorescence of SiQDs due to charge transfer. It is widely reported that Ag and AuNPs in particular can store a fraction of electrons captured from photoexcited semiconductor nanostructures [32–36]. AuNPs can capture the electrons at the conduction bands of SiQDs, and the number density of the electrons in the excited state of the QD decreases relative to the number density of the bare SiQD excited state. Subsequently the radiative recombination of the photoinduced electrons and holes is inhibited, which ultimately resulted in PL quenching. In addition, it is possible that the emitted photons from the system are absorbed by the AuNPs causing interband transitions since the absorbance band of the AuNPs (red line) was partly overlapped with the emission spectrum of SiQDs (black line) [37] (Fig. S3). 3.3. The working principle of the proposed sensor The “turn on-off” fluorescent sensor for protamine and heparin is outlined in Scheme 1. AuNPs can quench the fluorescence of SiQDs significantly. In the absence of polyanionic heparin, AuNPs could indirectly bind with polycationic protamine through electrostatic interaction, resulting in the detaching of SiQDs from the surface of AuNPs and the fluorescence of SiQDs turned on. On the contrary, in the existence of heparin, protamine could firstly
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Fig. 1. (A) TEM image of SiQDs. The inset shows the HRTEM image of SiQDs. (B) DLS of SiQDs. (C) The normalized PL spectra of SiQDs at different excitation wavelength. (D) UV–vis absorption and excitation spectra (solid line) and emission spectra (dot line) of SiQDs. (E) FTIR spectra of SiQDs.
combine with heparin due to their strong affinity, and then the negatively charged AuNPs would interact with SiQDs, displaying a decrease of SiQDs fluorescence. To demonstrate the strategy, the fluorescence spectra of SiQDs at various conditions were measured, the results were shown in Fig. 2. The fluorescent spectra of SiQDs (curve a in Fig. 2) were approximate to that of the mixture of SiQDs and protamine (curve b in Fig. 2), which indicates there was no interaction between protamine and SiQDs. When AuNPs was mixed with SiQDs, the fluorescence of SiQDs was quenched by AuNPs (curve c in Fig. 2). However, the fluorescence of the SiQDs–AuNPs mixture was enhanced upon addition of protamine (curve d in Fig. 2) because the SiQDs was replaced by protamine and departed from the surface of AuNPs. Addition of heparin disturbed the combination of protamine and AuNPs due to its strong affinity to protamine, thus the fluorescence was quenched (curve e in Fig. 2). The phenomenon was further evidenced by TEM (Fig. 3). As shown in Fig. 3A, the free
AuNPs was in a dispersed state. In the presence of SiQDs, the AuNPs were still in a dispersed state (Fig. 3B). However, upon addition of protamine into the solution of SiQDs–AuNPs assembly, AuNPs were obviously aggregated (Fig. 3C). When heparin was further added into the test system, the de-aggregation of assembled AuNPs occurred owing to the interaction between protamine and heparin (Fig. 3D). Furthermore, we have also provided DLS of AuNPs at various conditions. The results indicated that aggregation of AuNPs occurred in the presence of protamine while de-aggregation was observed in the presence of heparin. As shown in Fig. 4A, the average size of free AuNPs was 15.7 nm, which indicated AuNPs was in a dispersed state. In the presence of SiQDs, the average size measured by DLS was 22.95 nm (Fig. 4B). While protamine was added into the solution of SiQDs–AuNPs assembly, AuNPs were obviously aggregated and the average size was 178.9 nm (Fig. 4C). The deaggregation of AuNPs occurred after heparin was added to the test system, owing to the prior interaction of protamine with heparin over AuNPs and the average size was 27.09 nm (Fig. 4D).
3.4. Optimization of the experimental conditions
Fig. 2. Fluorescence emission spectra: SiQDs (a); SiQDs and protamine (b); SiQDs and AuNPs (c); mixture of AuNPs, SiQDs and protamine (d); SiQDs–AuNPs incubated with heparin and protamine (e) were collected at the excitation wavelength of 360 nm. [AuNPs]: 1.32 nmol/L; [Protamine]: 1.2 g/mL; [Heparin]: 1.4 g/mL.
Sensitivity of the detection method strongly depended on the relevant experimental parameters including concentrations of AuNPs and protamine, pH values of solution, and incubation time. Firstly, the effect of the concentration of AuNPs was investigated. Fig. 5A shows the fluorescent enhancement efficiency (F − F0 )/F0 changes with the concentration of the AuNPs, where F0 is the fluorescent intensity of SiQDs in the presence of AuNPs and F is the fluorescent intensity of SiQDs/AuNPs mixture in presence of protamine. It was observed that the (F − F0 )/F0 value increased with the increases of AuNPs concentrations in the range of 0.63–1.32 nmol/L, which resulted from the observed decrease in F0 and the increase in F value while increasing the concentration of AuNPs. However, the fluorescent enhancement efficiency decreased when the concentration of AuNPs exceeded 1.32 nmol/L. Because the excess AuNPs will further quench the fluorescence of SiQDs and result in a decrease in the F value, the value of (F − F0 )/F0 is decreased. Hence, 1.32 nmol/L of AuNPs was chosen as the optimum concentration.
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Fig. 3. TEM images of AuNPs: free AuNPs (A); in the presence of SiQDs (B); in the presence of SiQDs and protamine (C) and in the presence of SiQDs, protamine and heparin (D).
The pH values of the solution would affect the interaction of protamine and AuNPs. It could be seen that the highest enhancement efficiency was obtained in the range of 6.0–7.4 (Fig. 5B). Thus, pH 7.4 was chosen in this work. Fig. 5C shows the time-dependent fluorescent response in the presence of protamine. The fluorescent enhancement efficiency maintained a stable value when the incubation time reached 50 min. On the basis of those observations, the analyte systems were incubated for 50 min under a pH of 7.4 before fluorescence measurement. We further studied the effect of the amount of protamine on heparin quantification. As illustrated in Fig. 5D, the fluorescence response increased with increasing concentration of protamine and became stable when the concentration of protamine was
1.2 g/mL. Thus, 1.2 g/mL of protamine was selected for heparin detection. 3.5. Sensitive detection of protamine and heparin On the basis of the above standard procedures and optimized assay conditions, various concentrations of protamine were introduced to the SiQDs–AuNPs system to evaluate the sensitivity. As shown in Fig. 6A, the fluorescence intensity at 440 nm increases with the protamine concentrations. The fluorescence intensity was found to be linear with the concentration of protamine in the range from 0 to 1.2 g/mL (Fig. 6B) and a detection limit of 6.7 ng/mL was estimated based on S/N = 3. The equation for the resulting
Fig. 4. DLS of AuNPs: free AuNPs (A); in the presence of SiQDs (B); in the presence of SiQDs and protamine (C) and in the presence of SiQDs, protamine and heparin (D).
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Fig. 5. Effects of AuNPs concentration (A), pH (B) and incubation time (C) on the fluorescence responses of SiQDs-based sensor for protamine detection. Effects of protamine concentration on fluorescence responses the SiQDs-based sensor for heparin detection (D).
calibration plot was F = 0.6182C + 0.0124 (F was the measured fluorescence intensity, C was the concentration of protamine) with correlation coefficient of 0.9956. Fig. 7 shows the fluorescence intensity of SiQDs–AuNPs system in the presence of 1.2 g/mL protamine with variable concentrations of heparin. In the range of 0.002–1.4 g/mL of heparin, the quenched fluorescence intensity follows the equation of F = 0.2852C + 0.0367 (F was the measured fluorescence intensity, C was the concentration of heparin) with the correlation coefficient of 0.9965, and a detection limit of 0.67 ng/mL (S/N = 3) was calculated. The detection limit values are lower than that of the reported methods for determination of protamine and heparin and the linear response ranges are wider than reported (Table S1). The proposed strategy was demonstrated to be ultrasensitive and highly reliable for the detection of protamine and heparin levels.
3.6. Selectivity of the method To determine the selectivity of the developed method, the interference from other potentially competing substances, including structural analogues and commonly coexistent physiological level species was investigated. Potentially competing substances including K+ , collagen, lysozyme, Ca2+ , IgG, Na+ , bovine serum albumin (BSA), Mg2+ , ConA were added to the SiQDs–AuNPs system, and then the fluorescence intensity was recorded. As indicated in Fig. 8A, the FL recovery by protamine was larger than the other ions and compounds, which indicated that the potential interfering substances could hardly produce distinct interference for the detection of protamine. Fig. 8B shows the changes in the fluorescence intensity of the SiQDs–AuNPs/protamine mixed solution after the addition of Dex, HA, Chs, SO4 2− , BSA, sodium citrate,
Fig. 6. (A) Fluorescent spectra of SiQDs–AuNPs mixed solution upon addition of different concentrations of protamine. The concentrations of protamine (from bottom to top) are 0, 2 × 10−8 , 4 × 10−8 , 8 × 10−8 , 1 × 10−7 , 2 × 10−7 , 3 × 10−7 , 4 × 10−7 , 6 × 10−7 , 8 × 10−7 , 1 × 10−6 and 1.2 × 10−6 g/mL respectively. (B) The linear fitting of the relative fluorescent intensity versus concentrations of protamine.
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Fig. 7. (A) Fluorescent spectra of SiQDs–AuNPs/protamine mixed solution with various concentration of heparin. The concentrations of heparin (from top to bottom) are 0, 2 × 10−9 , 2 × 10−8 , 1 × 10−7 , 2 × 10−7 , 4 × 10−7 , 6 × 10−7 , 8 × 10−7 , 1 × 10−6 , 1.2 × 10−6 and 1.4 × 10−6 g/mL respectively. [Protamine]: 1.2 g/mL. (B) The linear fitting of the relative fluorescent intensity versus concentrations of heparin.
Fig. 8. (A) Fluorescent enhancement efficiency of SiQDs–AuNPs mixture upon the addition of protamine (0.8 g/mL) or other substances (0.2 mg/mL): (a) blank; (b) K+ ; (c) collagen; (d) lysozyme; (e) Ca2+ ; (f) IgG; (g) Na+ ; (h) BSA; (i) Mg2+ ; (j) ConA; (k) protamine. (B) Fluorescent quenching efficiency of SiQDs–AuNPs/protamine mixed solution upon addition of heparin or other substances: (a) blank; (b) Dex (1.4 g/mL); (c) HA (1.4 g/mL); (d) Chs (1.4 g/mL); (e) SO4 2− (0.2 mg/mL); (f) BSA (0.2 mg/mL); (g) sodium citrate (0.2 mg/mL); (h) Glucose (0.2 mg/mL); (i) Cl− (0.2 mg/mL); (j) heparin (1.4 g/mL).
glucose, Cl− and heparin. The fluorescence response of the sensor to heparin is obviously higher than other samples. Other polysaccharides such as HA and Chs bear low charge density per disaccharide unit: one is formed by a 1/3 linked N-acetylglucosamine and glucuronic acid, which has only one carboxyl group; the other is formed by 1/3 linked N-acetylgalactosamine and glucuronic acid and modified by sulfation in position 4, which possesses one sulfate and one carboxylate moieties. In contrast, interestingly, heparin is composed of a 1/4 linked iduronic acid and glucosamine per repeating disaccharide unit, which carries three sulfate groups and one carboxylate per repeat unit. Therefore, the electrostatic attraction between HA or Chs and protamine is significantly weaker than that between heparin and protamine [38]. The binding constant for the protamine toward heparin was high, while the reaction of the protamine toward other substances has a somewhat lower affinity constant [39], which indicated the developed sensor possessed good selectivity toward heparin. All these results clearly indicated that the approach possessed good selectivity. 3.7. Application to the serum samples With excellent sensitivity and selectivity in buffer solution, the proposed sensor was further tested with serum samples to demonstrate its practical application. The records of fluorescence response for the spiked serum sample with protamine and heparin separately are shown in Tables 1 and 2. The experimental
Table 1 Detection of protamine in 10% serum samples. Sample
Added (g/mL)
Calculated (g/mL)
Recovery (%)
RSD (%)
1 2 3
0.20 0.40 0.60
0.21 0.38 0.59
105.00 95.00 98.33
0.58 0.67 0.85
Table 2 Detection of heparin in 10% serum samples. Sample
Added (g/mL)
Calculated (g/mL)
Recovery (%)
RSD (%)
1 2 3
0.40 0.80 1.20
0.42 0.82 1.23
105.00 102.50 102.50
0.38 0.51 0.36
results exhibited that recoveries of protamine and heparin reached to 98.33–105.00% and 102.50–105.00%, with the relative standard deviation (RSD) of 0.58–0.85% and 0.36–0.51% respectively. The results indicated that the proposed method can be successfully applied for detecting protamine and heparin in real samples. 4. Conclusion In conclusion, we have successfully developed a label-free fluorescent nanosensor for dual detection and quantification of heparin and protamine by taking advantage of the fluorescence
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“turn-off” and “turn-on” feature. The fluorescent sensor showed good selectivity for protamine and heparin against other ions and their analogs. The sensor also can be used for the detection of protamine and heparin in real serum samples. We believe that the fluorescent sensor provides a versatile method for the determination of heparin and protamine in other applications. Acknowledgments This work was supported by the National Natural Science Foundation of China (21375037, 21275051 and 21475043), Scientific Research Fund of Hunan Provincial Science and Technology Department and Education Department (13JJ2020, 12A084), and Doctoral Fund of Ministry of Education of China (no.: 20134306110006). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2015.02.070. References [1] B.F. Akl, G.M. Vargas, J. Neal, J. Robillard, P. Kelly, Clinical experience with the activated clotting time for the control of heparin and protamine therapy during cardiopulmonary bypass, J. Thorac. Cardiovasc. Surg. 79 (1980) 97–102. [2] K.L. Gemene, M.E. Meyerhoff, Reversible detection of heparin and other polyanions by pulsed chronopotentiometric polymer membrane electrode, Anal. Chem. 82 (2010) 1612–1615. [3] M. Wang, D. Zhang, G. Zhang, D. Zhu, The convenient fluorescence turn-on detection of heparin with a silole derivative featuring an ammonium group, Chem. Commun. (2008) 4469–4471. [4] R. Zhan, Z. Fang, B. Liu, Naked-eye detection and quantification of heparin in serum with a cationic polythiophene, Anal. Chem. 82 (2009) 1326–1333. [5] A. Shvarev, E. Bakker, Reversible electrochemical detection of nonelectroactive polyions, J. Am. Chem. Soc. 125 (2003) 11192–11193. [6] L.B. Jaques, Heparins—anionic polyelectrolyte drugs, Pharmacol. Rev. 31 (1979) 99–166. [7] J.C. Horrow, Protamine: a review of its toxicity, Anesth. Analg. 64 (1985) 348–361. [8] D.J. Murray, W.J. Brosnahan, B. Pennell, D. Kapalanski, J.M. Weiler, J. Olson, Heparin detection by the activated coagulation time: a comparison of the sensitivity of coagulation tests and heparin assays, J. Cardiothorac. Vasc. Anesth. 11 (1997) 24–28. [9] P.G. Hattersley, ACtivated coagulation time of whole blood, J. Am. Med. Assoc. 196 (1966) 436–440. [10] B. Ander, A. Karlsson, Å. Öhrlund, Determination of heparin on intraocular lens surfaces by ion chromatography, J. Chromatogr. A 917 (2001) 105–110. [11] R.P. Patel, C. Narkowicz, G.A. Jacobson, Effective reversed-phase ion pair high-performance liquid chromatography method for the separation and characterization of intact low-molecular-weight heparins, Anal. Biochem. 387 (2009) 113–121. [12] H. Yan, H.-F. Wang, Turn-on room temperature phosphorescence assay of heparin with tunable sensitivity and detection window based on target-induced self-assembly of polyethyleneimine capped Mn-doped ZnS quantum dots, Anal. Chem. 83 (2011) 8589–8595. [13] Z. Zhang, B. Li, J. Suwan, F. Zhang, Z. Wang, H. Liu, B. Mulloy, R.J. Linhardt, Analysis of pharmaceutical heparins and potential contaminants using 1 H-NMR and PAGE, J. Pharm. Sci. 98 (2009) 4017–4026. [14] R. Cao, B. Li, A simple and sensitive method for visual detection of heparin using positively-charged gold nanoparticles as colorimetric probes, Chem. Commun. 47 (2011) 2865–2867. [15] J. Li, X. Hong, D. Li, K. Zhao, L. Wang, H. Wang, Z. Du, J. Li, Y. Bai, T. Li, Mixed ligand system of cysteine and thioglycolic acid assisting in the synthesis of highly luminescent water-soluble CdTe nanorods, Chem. Commun. (2004) 1740–1741. [16] X. Hong, J. Li, M. Wang, J. Xu, W. Guo, J. Li, Y. Bai, T. Li, Fabrication of magnetic luminescent nanocomposites by a layer-by-layer self-assembly approach, Chem. Mater. 16 (2004) 4022–4027. [17] P. Wu, Y. Li, X.-P. Yan, CdTe quantum dots (QDs) based kinetic discrimination of Fe2+ and Fe3+ , and CdTe QDs-fenton hybrid system for sensitive photoluminescent detection of Fe2+ , Anal. Chem. 81 (2009) 6252–6257. [18] S. Gosso, D. Gavello, C.N.G. Giachello, C. Franchino, E. Carbone, V. Carabelli, The effect of CdSe–ZnS quantum dots on calcium currents and catecholamine secretion in mouse chromaffin cells, Biomaterials 32 (2011) 9040–9050. [19] C. Ratanatawanate, A. Chyao, K.J. Balkus, S-Nitrosocysteine-decorated PbS QDs/TiO2 nanotubes for enhanced production of singlet oxygen, J. Am. Chem. Soc. 133 (2011) 3492–3497.
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Biographies Xue Peng is a graduate student in Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, China. Qian Long is a graduate student in Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, China. Haitao Li is a Professor in Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University. He received his MSc degree from Hunan Normal University and Doctor degree from College of Chemistry, Nankai University, China. His research is focused on organic synthesis and chemical and biological sensing. Youyu Zhang is a Professor in Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University. She received her MSc degree from Hunan Normal University and Doctor degree from State Key Laboratory of Chemo/Biological Sensing and Chemometrics, Hunan University, China. Her research is focused on chemical and biological sensing. Her current research interests are in the areas nanomaterials and nanoanalysis. Shuozhuo Yao is a Professor in Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University and he is an academician of Chinese Academy of Science as well.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
“Turn on-off” fluorescent sensor for protamine and heparin based on label-free silicon quantum dots coupled with gold nanoparticles Xue Peng, Qian Long, Haitao Li, Youyu Zhang ∗ , Shouzhuo Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China
a r t i c l e
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Article history: Received 25 November 2014 Received in revised form 10 February 2015 Accepted 13 February 2015 Available online 23 February 2015 Keywords: Silicon quantum dots Gold nanoparticle Protamine Heparin
a b s t r a c t An ultrasensitive “turn on-off” fluorescent sensor was presented for determination of protamine and heparin based on the high quenching ability of gold nanoparticles (AuNPs) to the fluorescence of silicon quantum dots (SiQDs) as well as high binding affinity of protamine with heparin. The fluorescence of SiQDs was quenched significantly by adding AuNPs into SiQDs solution. Upon addition of protamine, the AuNPs aggregated and the fluorescence of SiQDs was recovered due to the competitive adsorption of protamine and SiQDs on AuNPs. Addition of heparin disturbed the interaction between protamine and AuNPs due to its strong affinity to protamine, resulting in an adsorption of SiQDs on the surface of AuNPs. Thus the fluorescence of SiQDs was quenched. The strategy was simply achieved by measuring the changes in the fluorescence of SiQDs. The research results suggested that the developed method has several advantages such as label-free, high sensitivity, low cost, ease of operation. The linear response range was obtained over the range 0–1.2 g/mL and 0.002–1.4 g/mL with the low detection limit of 6.7 ng/mL and 0.67 ng/mL for protamine and heparin, respectively. The sensing platform was successfully applied to determination of heparin and protamine in serum samples. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The polyionic drugs heparin and protamine are widely used in clinical procedures [1]. Heparin, a negatively charged linear polysaccharide, has been extensively used in medicine as an anticoagulant during clinical procedures for the prevention of blood clotting [2]. However, the higher dose and prolonged use of heparin often induce some adverse effects such as thrombocytopenia and hemorrhage [3,4]. Protamine is a low molecular weight protein with a positive charge of 20 in physiological condition. And it is usually administered following surgery to reverse the anticoagulant activity of heparin [5,6]. The polycationic protamine is rich in basic arginine residues, and the characteristic guanidinium groups of protamine were easy to react with acid to form a stable ion pair complex that is devoid of anticoagulation activity [7]. Based on the clinical importance of these two polyionic drugs and their toxic effects, it is vital to develop a simple, rapid, and cost-effective method for the quantification of both heparin and protamine. To date, various analytical methodologies have been developed for the sensitive assay of heparin and protamine. Several
∗ Corresponding author. Tel.: +86 731 8865515; fax: +86 731 8865515. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.snb.2015.02.070 0925-4005/© 2015 Elsevier B.V. All rights reserved.
conventional methods such as activated clotting time [8], activated thromboplastin time [9], ion chromatography [10], and ion pair high-performance liquid chromatography [11] have been used for the estimation of heparin or protamine. These methods are often not sufficiently sensitive, and the results are affected by many variables. Accordingly, a number of advanced methods have recently been developed to identify and quantitate heparin and protamine, such as electrochemistry [12], light scattering [13], nuclear magnetic resonance, colorimetry [14]. Although these methods have high selectivity and adequate reliability, most of these means suffer from the disadvantages of high costs, sophisticated instruments, and generally limited to the detection limit. Therefore, it is urgently required to develop simple, sensitive and cost-effective methods. Fluorescent methods, in particular, are extremely attractive because they offer advantages of low-cost portable instruments as well as easy-to-operation. Quantum dots (QDs) are versatile inorganic nanoparticles with unique photophysical and chemical properties [15,16] such as broad excitation spectra [17], photobleaching resistance [18], narrow and tunable emission spectra, simultaneous excitation of multiple fluorescence colors [19], and a larger Stokes shift over conventional organic fluorophores [20]. Recently, polyethyleneimine-capped Mn-doped ZnS quantum dots [12], CdTe quantum dots [21] and CuInS2 quantum dots [22] have been
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used as probes for detection of heparin. These methods were successfully applied to detect heparin, however, synthesis procedures of these probes are complicated, and only one analyte could be tested. And the applications to the clinical field have been hampered owing to the high toxicity of the CdTe quantum dots and eventually would cause serious environmental problems due to the leakage of cadmium. Therefore, it is also crucial to develop excellent nanomaterials which are easily obtained for fabricating highly sensitive and selective biosensors to quantify both heparin and protamine. As inert, nontoxic, abundant and low-cost nanomaterials, SiQDs have been demonstrated to be environmentally friendly photoluminescence probes and have attracted much interest. In comparison to other QDs, SiQDs have excellent biocompatibility and noncytotoxic properties. These advantages enable them to play a great role in a variety of applications. Recently, our group have developed fluorescent and colorimetric methods for the detection of glucose [23,24] and organophosphorus pesticides [25] based on the high fluorescence quenching ability of H2 O2 to the fluorescence of SiQDs and the peroxidase mimetics of SiQDs. Herein, we established a SiQDs-based sensing platform which coupled with AuNPs for ultrasensitive detection of heparin and protamine. AuNPs are unique quenchers for organic dyes or QDs through both energy-transfer and electron-transfer processes. It was found that AuNPs can quench fluorescence of SiQDs. Polycationic protamine was used as a medium for inducing the aggregation of citrate-capped AuNPs through electrostatic interaction, resulting in the detaching of the SiQDs and the fluorescence of the SiQDs being recovered. Addition of polyanionic heparin disturbed the interaction of protamine and AuNPs due to the strong affinity of heparin to protamine. Scheme 1 illustrates the proposed mechanism for the sensing of heparin and protamine. The proposed biosensor developed a simple, sensitive method for monitoring of both heparin and protamine. 2. Experimental 2.1. Materials Chloroauric acid (HAuCl4 ), protamine sulfate salt and heparin sodium salt were purchased from Sigma (Shanghai, China). Silicon wafers (phosphorus-doped (p-type), 8 resistivity) and phosphomolybdic acid (POM) were purchased from Sigma–Aldrich. Anhydrous ethanol (analytical grade), hydrofluoric acid (HF), and H2 O2 were purchased from Shanghai Chemical Reagent. All chemicals from commercial sources were of analytical grade and used without further purification. PBS buffer (pH 7.4, 10 mM sodium phosphate) and Milli-Q ultrapure water (Millipore, ≥18 M cm) were used throughout the experiments. The reconstituted serum sample was prepared by dissolving 20 mg of KCl, 800 mg of NaCl, 20 mg of KH2 PO4 , 115 mg of Na2 HPO4 and 4 g of bovine albumin in 100 mL of water.
2.3. Preparation of SiQDs Photoluminescence SiQDs were synthesized by the POMassisted electrochemical etching of bulk Si [26]. Briefly, Si wafers were cleaned in 20% hydrofluoric acid (HF) for 5 min first to remove surface oxides and impurities. The electrolyte of the electrochemical etching process was prepared by dissolving 0.02 g of POM in 30 mL of anhydrous ethanol, then 25 mL of HF was added under stirring, and with a suitable amount (5–10 mL) of H2 O2 (hydrogen peroxide 30%). The electrochemical system composed of graphite as anode and Si wafer as cathode. Graphite and Si wafer were put into the as-prepared electrolyte and connected to the DC power supply by connecting wires. There was a layer of n-hexane covering the electrolyte solution. The current intensity ranged 4–10 mA/cm2 and the whole etching process was performed for about 2 h. Without complicated centrifugation and filtration, just ultrasonication fracturing the etched silicon wafer in absolute ethanol occurred, and then SiQDs with excellent fluorescence were obtained. 2.4. Synthesis of AuNPs The colloidal solution of AuNPs was synthesized by means of citrate reduction of AuCl3 ·HCl·4H2 O [27]. All glassware and magnetic stirrers used for the synthesis were thoroughly cleaned in aqua regia, rinsed with ultrapure water, and then oven-dried prior to use. Briefly, 100 mL chloroauric acid (HAuCl4 ) solution (containing 0.5 mL 2% HAuCl4 ) was firstly heated to boiling, and then 1.8 mL of 1% sodium citrate solution was rapidly added to the boiled HAuCl4 solution under vigorous stirring resulted in a color change from light yellow to wine red. Boiling was continued for 10 min and stirring was continued for an additional 15 min without heating. After the solution reached room temperature, the prepared AuNPs solution was stored in the 4 ◦ C refrigerator before use. The concentration of the AuNPs was determined according to the Beer’s law by using UV–vis spectroscopy [28]. 2.5. Fluorescence assay of protamine and heparin The protamine-induced fluorescence recovery of SiQDs was monitored by observing the spectral change during the addition of different concentrations of protamine to the mixture of SiQDs–AuNPs. In a typical procedure, the mixed solution of SiQDs–AuNPs was prepared using 80 L of AuNPs, 80 L of SiQDs solution and PBS buffer solution (10 mM, pH 7.4). Then different concentrations of protamine were incubated with SiQDs–AuNPs mixture, the final volume is 600 L. Heparin was detected in the following way: first protamine (1.2 g/mL) was added to SiQDs–AuNPs mixture. A sudden color change from red to blue was noticed after the addition of protamine. Then different concentrations of heparin were added to the resulted solution and heparin-driven de-aggregation of the AuNPs occurred resulted in a reversible color change. The spectra were recorded with incubation for 50 min.
2.2. Instrumentation 2.6. Selectivity experiments toward protamine and heparin SiQDs synthesis was conducted on a CHI660A electrochemical workstation (CHI Instrument Inc., USA). UV–vis spectra were measured on a UV2450 spectrophotometer (Shimadzu). The F4500 fluorescence spectrophotometer (Hitachi Co., Japan) was used to collect the fluorescence spectra operating at the excitation wavelength of 360 nm, with both excitation and emission slit widths of 10 nm. Transmission electron microscopy (TEM) images were recorded using a JEOL-1230 model. Fourier transform infrared (FTIR) spectra were performed on a Nicolet Nexus 670 FTIR spectrometer (Nicolet Instrument Co., U.S.A.). The dynamic light scattering (DLS) was measured by Zetasizer3000HS.
The solutions of other potentially competing substances were prepared in PBS buffer solution for the experiments. The fluorescent responses of the proposed sensor to the competing substances were examined by a similar procedure mentioned above. The selectivity experiments for protamine was achieved with the addition of selected competing substances (K+ , collagen, lysozyme, Ca2+ , IgG, Na+ , bovine serum albumin (BSA), Mg2+ and ConA) (0.2 mg/mL) to the mixture SiQDs–AuNPs in PBS buffer solution (10 mM, pH 7.4), respectively. The selectivity procedure of heparin was detected with the following steps: protamine (1.2 g/mL) was added to
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Scheme 1. Schematic illustration of the design rationale for the fluorescence detection of protamine and heparin based on SiQDs coupled with AuNPs.
SiQDs–AuNPs mixture and then interferents (SO4 2− , BSA, sodium citrate, glucose, Cl− ) at 0.2 mg/mL, (dextran (Dex), hyaluronic acid (HA), chondroitin sulfate (Chs)) at 1.4 g/mL were added to the resulted solution, respectively. The spectra were measured by a fluorescence spectrophotometer excited at 360 nm. 2.7. Serum samples analysis The standard addition method was used to test the practical application of the proposed sensor. 60 L reconstituted serum was diluted by 540 L SiQDs/AuNPs solution without aggregating. Then, 0.2, 0.4, and 0.6 g/mL protamine was added to the mixture of reconstituted serum and SiQDs–AuNPs, respectively. The mixture was incubated for 50 min under a pH of 7.4 before fluorescence measurement. Heparin was detected with the following steps: protamine (1.2 g/mL) was added to the mixture of reconstituted serum and SiQDs–AuNPs and then 0.4, 0.8, and 1.2 g/mL heparin were added to the resulted solution. Each experiment was repeated 3 times. 3. Results and discussion 3.1. Characterization of the SiQDs The morphology and optical properties of SiQDs were characterized. TEM (Fig. 1A) and the high resolution transmission electron microscopy (HRTEM; inset of Fig. 1A) images show that the SiQDs with low polydispersity were spherical and the average diameter was about 8 nm. The diameter measured by DLS confirms the small size of the SiQDs with a hydrodynamic diameter of ∼9.36 nm in Fig. 1B. The different diameters measured by TEM and DLS are due to different surface states of the same sample under the two measurement conditions. Fig. 1C shows the normalized PL spectra of SiQDs at different excitation wavelength ranging from 340 to 390 nm. And it was found that the maximum emission of SiQDs locates at approximately 440 nm. The absorption, excitation and emission spectra of SiQDs in aqueous solution are presented in Fig. 1D. The absorbance below 290 nm is a characteristic absorbance of SiQDs [29]. In the FTIR spectra of SiQDs (Fig. 1E), a band centered at around 900 cm−1 represents Si–H bonds, and coupled H–Si–Si–H stretch bonds were observed
at 2100 cm−1 . Proved by FTIR measurement, the as-prepared SiQDs are terminated with Si–H bonds. The PL quantum yield of SiQDs was up to ∼9.6% according to the Williams method [30]. Upon further investigation, we found that the PL intensity of SiQDs was not affected by temperature (Fig. S1A) and reached the maximum when the pH of SiQDs in solution was 7.4 (Fig. S1B). Moreover, the photostability of SiQDs was excellent (Fig. S1C). 3.2. SiQDs fluorescence quenching by AuNPs As shown in Fig. S2A, upon addition of AuNPs to the SiQDs solution, the fluorescence intensity of SiQDs evidently decreased with increasing the amount of AuNPs. AuNPs were prepared using trisodium citrate as reducing agent, so citrate-stabilized AuNPs is negatively charged [27]. SiQDs are positively charged, since the asprepared SiQDs are terminated with Si–H bonds [31]. Therefore, SiQDs can bind with AuNPs through electrostatic interaction. The PL quenching mechanism of SiQDs by AuNPs can be explained as follows: AuNPs can quench the fluorescence of SiQDs due to charge transfer. It is widely reported that Ag and AuNPs in particular can store a fraction of electrons captured from photoexcited semiconductor nanostructures [32–36]. AuNPs can capture the electrons at the conduction bands of SiQDs, and the number density of the electrons in the excited state of the QD decreases relative to the number density of the bare SiQD excited state. Subsequently the radiative recombination of the photoinduced electrons and holes is inhibited, which ultimately resulted in PL quenching. In addition, it is possible that the emitted photons from the system are absorbed by the AuNPs causing interband transitions since the absorbance band of the AuNPs (red line) was partly overlapped with the emission spectrum of SiQDs (black line) [37] (Fig. S3). 3.3. The working principle of the proposed sensor The “turn on-off” fluorescent sensor for protamine and heparin is outlined in Scheme 1. AuNPs can quench the fluorescence of SiQDs significantly. In the absence of polyanionic heparin, AuNPs could indirectly bind with polycationic protamine through electrostatic interaction, resulting in the detaching of SiQDs from the surface of AuNPs and the fluorescence of SiQDs turned on. On the contrary, in the existence of heparin, protamine could firstly
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Fig. 1. (A) TEM image of SiQDs. The inset shows the HRTEM image of SiQDs. (B) DLS of SiQDs. (C) The normalized PL spectra of SiQDs at different excitation wavelength. (D) UV–vis absorption and excitation spectra (solid line) and emission spectra (dot line) of SiQDs. (E) FTIR spectra of SiQDs.
combine with heparin due to their strong affinity, and then the negatively charged AuNPs would interact with SiQDs, displaying a decrease of SiQDs fluorescence. To demonstrate the strategy, the fluorescence spectra of SiQDs at various conditions were measured, the results were shown in Fig. 2. The fluorescent spectra of SiQDs (curve a in Fig. 2) were approximate to that of the mixture of SiQDs and protamine (curve b in Fig. 2), which indicates there was no interaction between protamine and SiQDs. When AuNPs was mixed with SiQDs, the fluorescence of SiQDs was quenched by AuNPs (curve c in Fig. 2). However, the fluorescence of the SiQDs–AuNPs mixture was enhanced upon addition of protamine (curve d in Fig. 2) because the SiQDs was replaced by protamine and departed from the surface of AuNPs. Addition of heparin disturbed the combination of protamine and AuNPs due to its strong affinity to protamine, thus the fluorescence was quenched (curve e in Fig. 2). The phenomenon was further evidenced by TEM (Fig. 3). As shown in Fig. 3A, the free
AuNPs was in a dispersed state. In the presence of SiQDs, the AuNPs were still in a dispersed state (Fig. 3B). However, upon addition of protamine into the solution of SiQDs–AuNPs assembly, AuNPs were obviously aggregated (Fig. 3C). When heparin was further added into the test system, the de-aggregation of assembled AuNPs occurred owing to the interaction between protamine and heparin (Fig. 3D). Furthermore, we have also provided DLS of AuNPs at various conditions. The results indicated that aggregation of AuNPs occurred in the presence of protamine while de-aggregation was observed in the presence of heparin. As shown in Fig. 4A, the average size of free AuNPs was 15.7 nm, which indicated AuNPs was in a dispersed state. In the presence of SiQDs, the average size measured by DLS was 22.95 nm (Fig. 4B). While protamine was added into the solution of SiQDs–AuNPs assembly, AuNPs were obviously aggregated and the average size was 178.9 nm (Fig. 4C). The deaggregation of AuNPs occurred after heparin was added to the test system, owing to the prior interaction of protamine with heparin over AuNPs and the average size was 27.09 nm (Fig. 4D).
3.4. Optimization of the experimental conditions
Fig. 2. Fluorescence emission spectra: SiQDs (a); SiQDs and protamine (b); SiQDs and AuNPs (c); mixture of AuNPs, SiQDs and protamine (d); SiQDs–AuNPs incubated with heparin and protamine (e) were collected at the excitation wavelength of 360 nm. [AuNPs]: 1.32 nmol/L; [Protamine]: 1.2 g/mL; [Heparin]: 1.4 g/mL.
Sensitivity of the detection method strongly depended on the relevant experimental parameters including concentrations of AuNPs and protamine, pH values of solution, and incubation time. Firstly, the effect of the concentration of AuNPs was investigated. Fig. 5A shows the fluorescent enhancement efficiency (F − F0 )/F0 changes with the concentration of the AuNPs, where F0 is the fluorescent intensity of SiQDs in the presence of AuNPs and F is the fluorescent intensity of SiQDs/AuNPs mixture in presence of protamine. It was observed that the (F − F0 )/F0 value increased with the increases of AuNPs concentrations in the range of 0.63–1.32 nmol/L, which resulted from the observed decrease in F0 and the increase in F value while increasing the concentration of AuNPs. However, the fluorescent enhancement efficiency decreased when the concentration of AuNPs exceeded 1.32 nmol/L. Because the excess AuNPs will further quench the fluorescence of SiQDs and result in a decrease in the F value, the value of (F − F0 )/F0 is decreased. Hence, 1.32 nmol/L of AuNPs was chosen as the optimum concentration.
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Fig. 3. TEM images of AuNPs: free AuNPs (A); in the presence of SiQDs (B); in the presence of SiQDs and protamine (C) and in the presence of SiQDs, protamine and heparin (D).
The pH values of the solution would affect the interaction of protamine and AuNPs. It could be seen that the highest enhancement efficiency was obtained in the range of 6.0–7.4 (Fig. 5B). Thus, pH 7.4 was chosen in this work. Fig. 5C shows the time-dependent fluorescent response in the presence of protamine. The fluorescent enhancement efficiency maintained a stable value when the incubation time reached 50 min. On the basis of those observations, the analyte systems were incubated for 50 min under a pH of 7.4 before fluorescence measurement. We further studied the effect of the amount of protamine on heparin quantification. As illustrated in Fig. 5D, the fluorescence response increased with increasing concentration of protamine and became stable when the concentration of protamine was
1.2 g/mL. Thus, 1.2 g/mL of protamine was selected for heparin detection. 3.5. Sensitive detection of protamine and heparin On the basis of the above standard procedures and optimized assay conditions, various concentrations of protamine were introduced to the SiQDs–AuNPs system to evaluate the sensitivity. As shown in Fig. 6A, the fluorescence intensity at 440 nm increases with the protamine concentrations. The fluorescence intensity was found to be linear with the concentration of protamine in the range from 0 to 1.2 g/mL (Fig. 6B) and a detection limit of 6.7 ng/mL was estimated based on S/N = 3. The equation for the resulting
Fig. 4. DLS of AuNPs: free AuNPs (A); in the presence of SiQDs (B); in the presence of SiQDs and protamine (C) and in the presence of SiQDs, protamine and heparin (D).
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Fig. 5. Effects of AuNPs concentration (A), pH (B) and incubation time (C) on the fluorescence responses of SiQDs-based sensor for protamine detection. Effects of protamine concentration on fluorescence responses the SiQDs-based sensor for heparin detection (D).
calibration plot was F = 0.6182C + 0.0124 (F was the measured fluorescence intensity, C was the concentration of protamine) with correlation coefficient of 0.9956. Fig. 7 shows the fluorescence intensity of SiQDs–AuNPs system in the presence of 1.2 g/mL protamine with variable concentrations of heparin. In the range of 0.002–1.4 g/mL of heparin, the quenched fluorescence intensity follows the equation of F = 0.2852C + 0.0367 (F was the measured fluorescence intensity, C was the concentration of heparin) with the correlation coefficient of 0.9965, and a detection limit of 0.67 ng/mL (S/N = 3) was calculated. The detection limit values are lower than that of the reported methods for determination of protamine and heparin and the linear response ranges are wider than reported (Table S1). The proposed strategy was demonstrated to be ultrasensitive and highly reliable for the detection of protamine and heparin levels.
3.6. Selectivity of the method To determine the selectivity of the developed method, the interference from other potentially competing substances, including structural analogues and commonly coexistent physiological level species was investigated. Potentially competing substances including K+ , collagen, lysozyme, Ca2+ , IgG, Na+ , bovine serum albumin (BSA), Mg2+ , ConA were added to the SiQDs–AuNPs system, and then the fluorescence intensity was recorded. As indicated in Fig. 8A, the FL recovery by protamine was larger than the other ions and compounds, which indicated that the potential interfering substances could hardly produce distinct interference for the detection of protamine. Fig. 8B shows the changes in the fluorescence intensity of the SiQDs–AuNPs/protamine mixed solution after the addition of Dex, HA, Chs, SO4 2− , BSA, sodium citrate,
Fig. 6. (A) Fluorescent spectra of SiQDs–AuNPs mixed solution upon addition of different concentrations of protamine. The concentrations of protamine (from bottom to top) are 0, 2 × 10−8 , 4 × 10−8 , 8 × 10−8 , 1 × 10−7 , 2 × 10−7 , 3 × 10−7 , 4 × 10−7 , 6 × 10−7 , 8 × 10−7 , 1 × 10−6 and 1.2 × 10−6 g/mL respectively. (B) The linear fitting of the relative fluorescent intensity versus concentrations of protamine.
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Fig. 7. (A) Fluorescent spectra of SiQDs–AuNPs/protamine mixed solution with various concentration of heparin. The concentrations of heparin (from top to bottom) are 0, 2 × 10−9 , 2 × 10−8 , 1 × 10−7 , 2 × 10−7 , 4 × 10−7 , 6 × 10−7 , 8 × 10−7 , 1 × 10−6 , 1.2 × 10−6 and 1.4 × 10−6 g/mL respectively. [Protamine]: 1.2 g/mL. (B) The linear fitting of the relative fluorescent intensity versus concentrations of heparin.
Fig. 8. (A) Fluorescent enhancement efficiency of SiQDs–AuNPs mixture upon the addition of protamine (0.8 g/mL) or other substances (0.2 mg/mL): (a) blank; (b) K+ ; (c) collagen; (d) lysozyme; (e) Ca2+ ; (f) IgG; (g) Na+ ; (h) BSA; (i) Mg2+ ; (j) ConA; (k) protamine. (B) Fluorescent quenching efficiency of SiQDs–AuNPs/protamine mixed solution upon addition of heparin or other substances: (a) blank; (b) Dex (1.4 g/mL); (c) HA (1.4 g/mL); (d) Chs (1.4 g/mL); (e) SO4 2− (0.2 mg/mL); (f) BSA (0.2 mg/mL); (g) sodium citrate (0.2 mg/mL); (h) Glucose (0.2 mg/mL); (i) Cl− (0.2 mg/mL); (j) heparin (1.4 g/mL).
glucose, Cl− and heparin. The fluorescence response of the sensor to heparin is obviously higher than other samples. Other polysaccharides such as HA and Chs bear low charge density per disaccharide unit: one is formed by a 1/3 linked N-acetylglucosamine and glucuronic acid, which has only one carboxyl group; the other is formed by 1/3 linked N-acetylgalactosamine and glucuronic acid and modified by sulfation in position 4, which possesses one sulfate and one carboxylate moieties. In contrast, interestingly, heparin is composed of a 1/4 linked iduronic acid and glucosamine per repeating disaccharide unit, which carries three sulfate groups and one carboxylate per repeat unit. Therefore, the electrostatic attraction between HA or Chs and protamine is significantly weaker than that between heparin and protamine [38]. The binding constant for the protamine toward heparin was high, while the reaction of the protamine toward other substances has a somewhat lower affinity constant [39], which indicated the developed sensor possessed good selectivity toward heparin. All these results clearly indicated that the approach possessed good selectivity. 3.7. Application to the serum samples With excellent sensitivity and selectivity in buffer solution, the proposed sensor was further tested with serum samples to demonstrate its practical application. The records of fluorescence response for the spiked serum sample with protamine and heparin separately are shown in Tables 1 and 2. The experimental
Table 1 Detection of protamine in 10% serum samples. Sample
Added (g/mL)
Calculated (g/mL)
Recovery (%)
RSD (%)
1 2 3
0.20 0.40 0.60
0.21 0.38 0.59
105.00 95.00 98.33
0.58 0.67 0.85
Table 2 Detection of heparin in 10% serum samples. Sample
Added (g/mL)
Calculated (g/mL)
Recovery (%)
RSD (%)
1 2 3
0.40 0.80 1.20
0.42 0.82 1.23
105.00 102.50 102.50
0.38 0.51 0.36
results exhibited that recoveries of protamine and heparin reached to 98.33–105.00% and 102.50–105.00%, with the relative standard deviation (RSD) of 0.58–0.85% and 0.36–0.51% respectively. The results indicated that the proposed method can be successfully applied for detecting protamine and heparin in real samples. 4. Conclusion In conclusion, we have successfully developed a label-free fluorescent nanosensor for dual detection and quantification of heparin and protamine by taking advantage of the fluorescence
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Biographies Xue Peng is a graduate student in Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, China. Qian Long is a graduate student in Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, China. Haitao Li is a Professor in Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University. He received his MSc degree from Hunan Normal University and Doctor degree from College of Chemistry, Nankai University, China. His research is focused on organic synthesis and chemical and biological sensing. Youyu Zhang is a Professor in Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University. She received her MSc degree from Hunan Normal University and Doctor degree from State Key Laboratory of Chemo/Biological Sensing and Chemometrics, Hunan University, China. Her research is focused on chemical and biological sensing. Her current research interests are in the areas nanomaterials and nanoanalysis. Shuozhuo Yao is a Professor in Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University and he is an academician of Chinese Academy of Science as well.