Recyclable Decoration of Amine-Functionalized Magnetic

Nov 28, 2013 - It is critically important to accurately determine histidine since it is an indicator for many diseases when at an abnormal level. Here...
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Recyclable Decoration of Amine-Functionalized Magnetic Nanoparticles with Ni2+ for Determination of Histidine by Photochemical Vapor Generation Atomic Spectrometry Yuan Hu,† Qi Wang,† Chengbin Zheng,*,† Li Wu,‡ Xiandeng Hou,†,‡ and Yi Lv*,† †

Key Laboratory of Green Chemistry & Technology of MOE, College of Chemistry, and ‡Analytical & Testing Center, Sichuan University, Chengdu, Sichuan 610064, China S Supporting Information *

ABSTRACT: It is critically important to accurately determine histidine since it is an indicator for many diseases when at an abnormal level. Here, an inexpensive and simple method using an amine-functionalized magnetic nanoparticle-based Ni2+−histidine affinity pair system was developed for highly sensitive and selective detection of histidine in human urine by photochemical vapor generation atomic spectrometry. Ni2+ was first bound to the amine groups of the amine-functionalized magnetic nanoparticles and then liberated to solution via the highly specific interaction between the histidine and Ni2+ in the presence of histidine. The liberated histidine−Ni2+ complex was exposed to UV irradiation in the presence of formic acid to form gaseous nickel tetracarbonyl, which was separated from the sample matrix and determined by atomic absorption/fluorescence spectrometry. Compared to other methods, this approach promises high sensitivity, simplicity in design, and convenient operation. The need for organic solvents, enzymatic reactions, separation processes, chemical modification, expensive instrumentations, and sophisticated and complicated pretreatment is minimized with this strategy. A limit of detection of 1 nM was obtained and provided tens-to-hundreds of fold improvements over that achieved with conventional methods. The protocol was evaluated by analysis of several urine samples with good recoveries and showed great potential for practical application.

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Although the high affinity of Ni2+ to histidine has been long practiced in metal ion affinity chromatography and further shows promising application in purification and tacking of Hiscontaining peptides or histidine-tagged proteins in modern biotechnology and nanotechnology, only a few reports have used the Ni2+−histidine affinity pair to couple with optically active nanomaterials or organic dyes for development of histidine sensors.21−23 To our best knowledge, the utilization of this affinity pair in conjunction with atomic spectrometry for quantification of histidine has not been reported. Photochemical vapor generation (photo-CVG) is a new and promising gaseous sample introduction method for analytical atomic spectrometry, which reduces nonvolatile analytes to their volatile species through the generation of reducing radicals from low molecular weight organic compounds under UV irradiation.24−26 Several advantages arise, including efficient matrix separation, high analyte transport efficiency, high selectivity, simple instrumentation, and ease of automation. Recently, we have successfully accomplished generation of volatile nickel tetracarbonyl (Ni(CO)4) by exposing Ni2+ to UV

t is well-known that histidine (2-amino-3-(4-imidazolyl)propanoic acid, His) is an essential amino acid for human growth and repair of tissues and acts as a neurotransmitter in the central nervous system of mammals.1,2 Because of a common coordinating ligand (imidazole side chain), histidine also controls the transmission of metal ions in biological bases3 and was detected in the active sites of certain enzymes.4 Therefore, an abnormal level of histidine/histidine-rich proteins has been considered as an indicator for many diseases including histidinemia, chronic kidney disease, AIDS, and thrombotic disorders.1,2,5,6 Therefore, accurate determination of histidine in biological fluids is very important in clinical analysis. However, the sensitive and selective determination of histidine in many biological samples is challenging because of both low content and the similar properties and structure of amino acids. Great progress has been made in the development of histidine determination, such as capillary electrophoresis,7,8 highperformance liquid chromatography,9 colorimetry,10,11 fluorometry,12−17 surface-enhanced Raman scattering (SERS),18 and electrochemistry methods.19,20 Some of these approaches involve cumbersome operating procedures, suffer from lowthroughput, require complicated instrumentation, sophisticated analysis, and expensive reagents, which limit the scope of their practical applications. © XXXX American Chemical Society

Received: October 19, 2013 Accepted: November 28, 2013

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pressure mercury vapor UV lamp (253.7 nm, 15 W, Philip, Holland). The reaction solution was flushed to the GLS by using DIW as carrier solution to separate the generated Ni(CO)4 from the liquid phase and further sweep it by argon carrier gas to an atomic fluorescence spectrometer for quantification. An additional hydrogen flow was introduced and mixed with the argon carrier gas to form a stable Ar−H2 flame in the quartz atomizer of the AFS for the atomization of nickel tetracarbonyl. A schematic of the instrumentation is presented in Figure S1 (see the Supporting Information). The size and surface morphology of MNP-NH2 was characterized by scanning electron microscopy (SEM, Hitachi, S3400). Wide-angle (10−70°, 40 kV/35 mA) powder X-ray diffraction (XRD) measurements were carried out using a X’Pert Pro X-ray diffractometer (Philips) with Cu Kα radiation (K = 1.5406 Å). Fourier transform infrared spectra (FTIR) were recorded in a KBr disk on a Nicolet IS10 FTIR spectrometer (Thermo Inc., America) from 4000 to 400 cm−1 to evaluate the functional groups of the surface of MNPNH2. An atomic absorption spectrometer (Beijing Haiguang Instrumental Co., Beijing, China) was used to construct a quartz tube atomizer electrothermal atomic absorption spectrometer (QTA-ET-AAS) for evaluation of the reusability of the MNP-NH2. Reagents and Materials. All chemicals were at least of analytical grade. High-purity 18.2 MΩ cm deionized water (DIW) was obtained from a water purification system (Chengdu ULTRAPURE Technology Co. Ltd., Chengdu, China) and used to prepare all solutions. FeCl3·6H2O and anhydrous sodium acetate were supplied by Kermel Chemical Reagent Co. Ltd. (Tianjin, China). Other chemicals were purchased from Kelong Chemical Reagent Company (Chengdu, China). The standard stock solution of 1000 μM Ni2+ was prepared by dissolving 0.2910 g of Ni(NO3)2·6H2O with 2.0 mL of nitric acid and subsequently diluting to a final volume of 100 mL with DIW. The standard solutions were prepared by stepwise dilution of the stock solutions prior to use. A Tris-HCl buffer solution of 10 mM was prepared by dissolving 1.2109 g of tris(hydroxymethyl)aminomethane in 1 L of DIW, adjusting to the required pH values with HCl or NaOH solution (0.1 M). Urine Sample Procedures. Human urine samples were collected from healthy adult volunteers and diluted 200-fold with Tris-HCl buffer solution prior to analysis. No additional preconcentration or matrix separation is required for sample preparation. Synthesis and Characterization of Amine-Functionalized Magnetic Nanoparticles. Amine-functionalized magnetic nanoparticles (MNP-NH2) were synthesized according to a reported method25 (see section 1 of the Supporting Information). Figure 2A displays a representative SEM image of the prepared MNP-NH2, showing the spherical MNP-NH2 with an average diameter below 60 nm. These nanosized particles had large specific surface area and were qualified for binding abundant Ni2+, which was beneficial for subsequent applications. The crystalline structure and phase purity of the prepared MNP-NH2 were examined by powder X-ray diffraction, as exhibited in Figure 2B. The positions and relative intensities of diffraction peaks appeared at 30.2, 35.5, 43.3, 53.6, 57.3, and 63.0°, corresponding to the (220), (311), (400), (422), (511), and (440) crystal planes in the pure Fe3O4 crystals, respectively, and this matched well with the standard pattern of Fe3O4 indexed from the JCPDS card. The sharp and intense peaks

irradiation in the presence of formic acid and then developed a highly sensitive method for determination of nickel by atomic spectrometry using this species.27−29 Here we describe a completely different but simple approach for the highly sensitive and selective determination of histidine based on combining an amine-functionalized magnetic nanoparticle-based Ni2+−histidine affinity pair system with photoCVG atomic spectrometry. As illustrated in Figure 1, Ni2+ in

Figure 1. Schematic illustration of the principle of the histidine determination method using MNP-NH2-based histidine−Ni2+ affinity pair/FI/photo-CVG-AFS.

aqueous solution was first bound to the amine groups of the amine-functionalized magnetic nanoparticles (MNP-NH2), which were synthesized according to a facile one-pot template-free method reported by Li et al.,30 and then free Ni2+ could be easily separated with a permanent hand-held magnet to reduce its interferences in the subsequent experiments. Upon addition of histidine, the immobilized Ni2+ could be released from the particles to form a His−Ni2+ complex in the aqueous phase due to the higher affinity of Ni2+ for histidine over that of Ni2+ for NH2. By taking advantage of simple magnetic separation of MNP-NH2, the released His− Ni2+ complex in the supernatant was pumped to a photoreactor to generate Ni(CO)4, which was further separated from the liquid phase and subsequently transported to an atomic spectrometer detector for accomplishment of highly sensitive and selective determination of histidine.

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EXPERIMENTAL SECTION Instrumentation. A commercial hydride generation nondispersive atomic fluorescence spectrometer (AFS-9600, Beijing Haiguang Instrumental Co., Beijing, China) fitted with a quartz gas−liquid separator (GLS), a quartz atomizer, and a coded high-intensity nickel hollow cathode lamp was used for quantification. The instrument has earlier been described in detail.31−33 The volatile nickel carbonyl was generated using a homemade flow injection photochemical vapor generation system, which consists of two three-channel peristaltic pumps (BT100-02, Baoding Qili Precision Pump Co., Ltd., Baoding, China), a six-port injection valve, and a photochemical reactor. The six-port injection valve, fitted with a 2 mL polytetrafluoroethylene (PTFE) sample loop, was used to transport the Ni2+− histidine complex solution to mix with a solution containing 80% (v/v) formic acid prior to generation of Ni(CO)4 in the photochemical reactor. The latter consisted of a coiled quartz tube (100 cm × 4 mm i.d. × 6 mm o.d.) wrapped around a lowB

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Figure 2. Characterization of MNP-NH2: (A) SEM image; (B) XRD; (C) FTIR spectrum; and (D) well dispersion and magnetic separation of MNP-NH2.

buffer solution (10 mM, pH 9.0) containing 8 μM Ni2+. After standing for 30 min, the MNP-NH2 with fully bound Ni2+ (MNP-NH2/Ni2+) was separated from the mixture with the aid of a permanent hand-held magnet and rinsed with Tris-HCl buffer solution (10 mM, pH 9.0) for several times to remove the unbound Ni2+. Finally, the MNP-NH2/Ni2+ was stored in the dark at 4 °C. Its lifetime was determined to be longer than 12 months. To realize selective elution of Ni2+ from the decorated MNPNH2 by histidine, 10 mL of standard solution or sample solution containing 10 mM Tris-HCl (pH 9.0) and various concentrations of histidine were added to the MNP-NH2/Ni2+. After standing for 15 min, quantitative and selective dissociation of Ni2+ from the MNP-NH2 can be obtained by formation of a stable histidine−Ni2+ affinity pair. The supernatant containing the His−Ni2+ affinity pair was separated with the aid of a magnet. Quantification of histidine via determination of nickel containing the histidine−Ni2+ affinity pair with flow injection photo-CVG-AFS (FI/photo-CVG-AFS) was computer-programmable, and the optimum instrumental parameters are described in detail in Table S1 (see the Supporting Information). Typically, 2 mL of the obtained supernatant from the standard solution or sample was initially directed to the sample loop through a six-port valve with the aid of a peristaltic pump. The valve was activated to pass the carrier solution (DIW) to flush the analyte solution to mix with

also confirmed good crystallinity and pure spinel structure of the MNP-NH2, indicating that the phase of nanoparticles was not influenced by its amine functionalization. The FTIR spectrum of MNP-NH2 is shown in Figure 2C. The IR band at 576 cm−1 is characteristic of Fe−O vibrations. In contrast to the nonfunctionalized MNP synthesized by the same method, only in the absence of 1,6-hexadiamine (Figure 2C,a), the bands around 1628, 1322, 1090, and 877 cm−1 from the MNP-NH2 (Figure 2C,b) are evident, indicating that the MNP-NH2 had been functionalized with amino function groups, which were responsible for decoration of Ni2+. In order to visually demonstrate the good water dispersion and strong magnetic property of as-synthesized MNP-NH2, the same amount of its powder was weighed into two glass test tubes filled with DIW and vigorously shaken by hand. As expected, the MNP-NH2 could be well-dispersed in water because of the hydrophilic character of the reactive amine functional groups, which is beneficial to the subsequent experiments for fast decoration and dissociation of Ni2+. Importantly, this well-dispersed MNP-NH2 can be quickly and completely collected from the aqueous phase with the aid of an external magnetic field, as shown in Figure 2D. This fast magnetic separation arising from the strong magnetic response of the MNP-NH2 significantly simplifies the procedure and reduces the analytical time. Procedure. For decoration of MNP-NH2 with Ni2+, 0.01 g of the prepared MNP-NH2 was dispersed in 10 mL of Tris-HCl C

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Figure 3. Effect of pH and time on decoration and dissociation of Ni2+. (A) Effect of pH on the binding efficiency of Ni2+ onto MNP-NH2 (red) and effect of pH on dissociation of Ni2+ from MNP-NH2 (black). (B) Effect of binding time on the response from the solution containing 8 μM Ni2+ and 0.01 g of MNP-NH2 under optimum pH (black) and effect of dissociation time on the response from the solution containing 2 μM histidine and 0.01 g of decorated MNP-NH2 under optimum pH (red).

dependent binding efficiency probably results from the pKa (about 8.87) of the amine groups.37 The primary amine group is partially protonated at pH lower than the pKa, thereby inhibiting its coordination with Ni2+, resulting in low binding efficiency. However, Ni2+ would start to precipitate with further increase of pH beyond 9.0, resulting in lower binding efficiency. Therefore, pH 9.0 was chosen for subsequent experiments to ensure high binding efficiency of Ni2+ and a low blank value. The binding kinetics of Ni2+ onto MNP-NH2 were examined by stirring 10 mL of Tris-HCl buffer solution (pH 9.0) containing 8 μM Ni2+ and 0.01 g of MNP-NH2 in a time range of 0−60 min, as shown in Figure 3B. The results show that the response from the supernatant sharply decreased and reached about 80% binding efficiency after 5 min. This reveals that binding Ni2+ onto MNP-NH2 is favorable because of the modified amine group and good dispersion of the prepared MNP-NH2. Therefore, 30 min was selected as the equilibration time for subsequent experiments to ensure sufficient binding reaction. To investigate the binding capacity of MNP-NH2 for Ni2+, 0.01 g of MNP-NH2 was added to a series of 10 mL solutions containing various concentrations of Ni2+. The mixture was shaken vigorously and then allowed to stand for 30 min prior to determination of the residual Ni2+ in the supernatant by FI/ photo-CVG-AFS. The results are summarized in Figure S2 (see the Supporting Information) and show that the binding efficiency remains over 90% in the range of 2−8 μM and then decreases at higher concentration of Ni2+. Therefore, an 8 μmol g−1 of binding capacity was obtained for the prepared MNP-NH2, which is enough for routine analysis of many types of samples. The excessive introduction of Ni2+ might cause trouble in removing the free Ni2+ and result in false-positive results. Consequently, 8 μM of Ni2+ was selected to decorate 0.01 g of MNP-NH2 for the following experiments. Effects of Experimental Conditions on Dissociation of Ni2+ with Histidine. The high-affinity interaction of histidine to Ni2+ was accomplished through the coordination of Ni2+ and the imidazole residue as well as the primary amine and/or carboxylic groups of histidine. Thus, the pH of the solution not only influences the binding efficiency of Ni2+ but also affects the subsequent dissociation of Ni2+ from MNP-NH2 by histidine (Figure 3A). As expected, the response from the dissociated Ni2+ increased significantly with pH from 6.0 to 9.0, followed

HCOOH, with the mixture then being transported through the photochemical reactor for UV irradiation for 160 s to generate Ni(CO)4. Finally, the reaction solution was flushed into the GLS by the carrier solution and carrier gas, wherein the resultant nickel carbonyl was separated from liquid phase and then introduced into the AFS. Peak area measurement of the nickel atomic fluorescence intensity was monitored.

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RESULTS AND DISCUSSION

Methodology Design. MNP-NH2 was validated to be capable of immobilizing metal ions on its surface.34 Therefore, an initial experiment added MNP-NH2 to a 4 μM Ni2+ standard solution and was allowed to stand for 30 min. After magnetic separation, the AFS signal from the supernatant decreased by 95%, but no obvious decrease on the response was observed when the unmodified MNP was used as an alternative to MNPNH2. These confirmed the response decrease attributed to the amine function groups modified on the surface of MNP-NH2. Previous studies21−23,35,36 found that histidine has a high affinity for Ni2+ because it contains imidazole, primary amine, and carboxyl groups, permitting various methods for determination or separation of histidine/histidine-containing peptides from complex matrices to be developed. The mechanism provides the potential to dissociate Ni2+ from the surface of MNP-NH2, thereby permitting a novel method for quantification of histidine via determination of nickel using atomic spectrometry (Figure 1). To demonstrate the feasibility of the proposed strategy, 2 μM histidine was initially added to 0.01 g of MNP-NH2/Ni2+. A significant response from the supernatant was observed by detecting through FI/photo-CVG-AFS. However, no obvious signal from the Tris-HCl buffer solution was detected in the absence of histidine, indicating that the dissociation of Ni2+ by the buffer solution was negligible. These observations support the potential of this method for determination of histidine. Effects of Experimental Conditions on Binding Ni2+ on MNP-NH2. First, we investigated the effect of pH on the binding efficiency in the range of 6.0−9.5, as shown in Figure 3A. The binding efficiency, defined as the variation rate of nickel concentration in the solution before and after adding MNP-NH2, increases significantly with increasing pH in the range of 6.0−9.0 and decreases beyond 9.0. Such a pHD

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used for subsequent experiments. It was worth noting that a large blank was obtained when reagent grade formic acid was used because commercial formic acid usually contains several to tens of μg L−1 nickel as a contaminant, necessitating use of subboil distilled formic acid for this work. The effect of irradiation time is described in Figure 4. No signal was detected in the absence of UV irradiation. The optimum irradiation time is 160 s, which is longer than that required for photo-CVG of Ni2+ (typically