ARTICLE pubs.acs.org/Langmuir
Iminodiacetic Acid-Functionalized Gold Nanoparticles for Optical Sensing of Myoglobin via Cu2þ Coordination Xianfeng Zhang, Xianming Kong, Wenjuan Fan, and Xuezhong Du* Key Laboratory of Mesoscopic Chemistry (Ministry of Education), State Key Laboratory of Coordination Chemistry, and School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China
bS Supporting Information ABSTRACT: A novel gold nanoparticle (AuNP)-based optical sensing system has been developed for the detection of myoglobin (Mb), which is of significant importance for early disease diagnosis. Two thiol molecules containing an iminodiacetic acid moiety (IDA) were synthesized. This detection is based on the Mb-induced aggregation of IDA-functionalized AuNPs resulting from the structures of Mb sandwiched between the functionalized AuNPs via Cu2þ bridges in the coordination interactions of IDACu2þhistidine residues available on the Mb surface, which was confirmed by UVvis spectroscopy, transmission electron microscopy, dynamic light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The induction aggregation resulted in a red shift in plasmon resonance band of the AuNPs concomitant with a change in solution color from red to purple. The qualitative and quantitative detections of Mb can be achieved by colorimetric observations and UVvis spectral measurements, respectively. The selectivity of protein assay with the functionalized AuNPs was further investigated, and it is found that the optical sensing of histidine-rich proteins is closely related to number and distribution of surface histidine residues as well as size of proteins.
’ INTRODUCTION Myoglobin (Mb) is mainly from muscle tissues of vertebrates and mammals, and its main function is to bind oxygen reversibly for transport and storage as well as cytoprotection against reactive oxygen species and NO scavenging.1 When muscle tissues are damaged, Mb will be released and filtered by kidneys but is toxic to the renal tubular epithelium, which may cause acute renal harm.2 Mb is absent or present in very low concentrations (below 0.01 μg/mL) in the urine of healthy people and appears in the urine for patients with severe muscle damage sometimes at extremely high levels to reach 750 μg/mL.3 Mb in serum of healthy people is 0.0080.098 μg/mL and is increased to 79.9 μg/mL when damaged.3 Mb plays an important role in disease diagnosis because it is a sensitive marker for muscle injury and a potential code for heart attack in patients with chest pain.4 There is ample literature suggesting that Mb is associated with acute myocardial infarction and is the best candidate marker for early diagnosis.5 Obviously, both qualitative and quantitative detections of Mb are of significant importance for early disease diagnosis. At present, the main method for detection of Mb in urine or serum is immunoassay,6 however, antibodies used in immunoassay are usually very expensive. Chen and co-workers synthesized uniformly sized coreshell superparamagnetic silica-coated magnetite particles immobilized with metal-affinity ligands, iminodiacetic acid (IDA), for the adsorption of bovine hemoglobin (BHb) containing histidine (His) residues via Cu2þ coordination.7 Their results showed that r 2011 American Chemical Society
the submicrospheres had a high adsorption capacity for BHb, low nonspecific adsorption, and good removal of BHb from bovine blood.7 Gold nanoparticles (AuNPs) have received extensive attention because of distinct physical and chemical properties, such as high extinction coefficients, facile functionalization with organic ligands, photostability, and colorimetric readout.810 The colorimetric behaviors of AuNPs are related to localized surface plasmon resonance (LSPR) originating from the coupling of conduction electrons with incident electromagnetic waves.11 The AuNPs smaller than 60 nm in diameter exhibit a red color when well dispersed in aqueous solutions due to the plasmon resonance (PR) absorption. When appropriately functionalized ligands on the AuNP surface are in the presence of analytes, the color turns purple or blue because of near-field PR coupling as a result of the decrease of interparticle distances.1215 Examples of AuNP colorimetric sensors include the detections of DNA,16 proteins,17 amino acids,18 anions,19 saccharides,20 and metal ions.21 To the best of our knowledge, AuNPs functionalized with IDA ligands have not been applied for identification and measurement of Mb. Herein, we designed the two thiol molecules containing an IDA moiety (Chart 1), 6-(iminodiacetic acid)-hexanethiol (IDA6) and 2-(iminodiacetic acid)-ethanethiol (IDA2) (synthesis Received: January 14, 2011 Revised: March 25, 2011 Published: April 13, 2011 6504
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Chart 1. Chemical Structures of 6-(Iminodiacetic Acid)-Hexanethiol (IDA6) and 2-(Iminodiacetic Acid)-Ethanethiol (IDA2)
procedures in the Supporting Information), and used IDA-functionalized AuNPs to recognize and detect Mb. The histidine (His) residues on the surface of Mb bound to the IDA-functionalized AuNPs via Cu2þ coordination, resulting in the PR coupling of the AuNPs concomitant with a change in solution color from red to purple. Therefore, the qualitative detection of Mb could be achieved by colorimetric assay, and the quantitative analysis could be fulfilled using UVvis spectroscopy.
’ EXPERIMENTAL SECTION Chemicals and Materials. Chloroauric acid (99.99%, HAuCl4), trisodium citrate dihydrate (99.9%), Mb (pI 7.0, 17 kDa) from equine skeletal muscle (95100%), ovalbumin (pI 4.6, 43.5 kDa), and human serum albumin (HSA, pI 4.8, 66.5 kDa) were purchased from SigmaAldrich. BHb (pI 7.1, 64.5 kDa), bovine pepsin (pI 1.0, 34.5 kDa), and bovine serum albumin (BSA, pI 4.7, 67 kDa) were provided by Amresco. Ethyl bromoacetate (99%) and triphenylphosphine (99%) were of chemical grade, and other chemicals (99%, Sinopharm Chemical Reagent Co., Ltd.) were of analytical grade. Semipermeable membrane (MW: 800014400) were purchased from Nanjing Superbio Biotechnology Co., Ltd. All aqueous protein solutions were prepared with 10 mM phosphate buffer (PB, pH 7.0) without NaCl, and other aqueous solutions were prepared with double-distilled water. Preparation of AuNPs. Colloidal AuNPs of approximately 13 nm in diameter were synthesized by the citrate reduction of HAuCl4.22 The glassware used was thoroughly cleaned with aqua regia followed by rinsing with copious double-distilled water and drying for use. HAuCl4 (100 mL, 1 mM) was heated to reflux under vigorous stirring, and then 10 mL of 1% trisodium citrate was rapidly added, and the color of the mixture changed from pale yellow to wine red within several minutes. The solution was allowed to reflux for 30 min to ensure complete reduction, then cooled to room temperature under continuous stirring. The gold colloids were stored in a refrigerator at 4 °C prior to use. Functionalization of AuNPs. Ligand-exchange reactions were performed overnight at room temperature by mixing a 10 mL of the asprepared gold colloids with 60 μL of aqueous IDA solution (2 mM) under stirring, and then the mixture was subjected to centrifugation at 12 000 rpm for 12 min. The supernatant was removed, and the precipitate was resuspended in 10 mL of 10 mM PB solution. The concentration of functionalized AuNPs was measured using UVvis spectroscopy with a molar extinction coefficient of 2.7 108 M1cm1 according to the literature23 to be approximately 5.8 109 M. Instruments and Assay. UVvis spectra were recorded on a LAMBDA-35 spectrophotometer (PerkinElmer Corporation). The aggregation of IDA-functionalized AuNPs was monitored by UVvis spectral changes upon addition of Mb of different amounts in the presence of Cu2þ ions. 100 μL of 1 mM Cu2þ ions was first added to 950 μL of IDAfunctionalized AuNPs, then aqueous Mb solutions of different volumes were added, and finally PB solutions of varied volumes were added so that the total volumes of all samples were identical.
Figure 1. UVvis spectra of IDA6-functionalized AuNPs upon addition of (a) none, (b) Cu2þ ions (90.9 μM), (c) Mb (45.5 μg/mL), and (d) Cu2þ ions (90.9 μM) and Mb (45.5 μg/mL) in the PB solution. Insert shows the corresponding colorimetric responses. The shapes and sizes of the AuNPs were examined using a transmission electron microscope (TEM, JEOL JEM-2100) operating at an acceleration voltage of voltage of 200 KV. Cu2þ ions (100 μL, 1 mM) were added to 950 μL of IDA-functionalized AuNPs, then 50 μL of double-distilled water or 50 μL of Mb (1 mg/mL) was added followed by incubating for 15 min. Ten microliters of colloidal AuNPs were dropped onto carbon-coated copper grids to leave in air for drying followed by TEM observations. Dynamic light scattering (DLS) measurements were performed on a BI200SM apparatus (Brookhaven Instruments) at 25 °C with a scattering angle of 90°. Cu2þ ions (100 μL, 1 mM) were added to 950 μL of IDAfunctionalized AuNPs, and then 50 μL of 1 mg/mL Mb was added followed by incubating for 15 min. The mixture was finally diluted 3 times for the DLS assay. Matrix-assisted laser desorption/ionization time-of-flight mass spectra were carried out on an Auto Flex MALDI-TOF-MS (Bruker Daltonics). IDA-functionalized AuNPs were subjected to centrifugation, and then the precipitate was resuspended in double-distilled water. The concentrations of IDA-functionalized AuNPs were identical to those used in the UVvis spectral measurements. Cu2þ ions (100 μL, 1 mM) were added to 950 μL of IDA-functionalized AuNPs, and then 50 μL of 1 mg/mL Mb was added. The mixture was incubated for 1 h and then subjected to centrifugation at 12 000 rpm for 12 min. The supernatant was removed, the precipitate was resuspended in 1 mL of doubledistilled water followed by recentrifugation at 12 000 rpm for 12 min, and finally the precipitate was resuspended in 10 μL of double-distilled water. One microliter of the sample solution was spotted onto a MALDI plate, and then another 1 μL of saturated sinapic acid (SA) matrix solution was introduced. After evaporation of the solvent, the sample was ready for the mass spectral measurement. All mass spectra were generated at 300 pulsed laser shots.
’ RESULTS AND DISCUSSION Optical Sensing of Mb with IDA-Functionalized AuNPs. Figure 1 shows UVvis spectral changes of the IDA6-functionalized AuNPs in the presence of Cu2þ ions and/or Mb. The functionalized AuNPs showed an intense PR band at 518 nm. When Cu2þ ions or Mb was individually added to the functionalized AuNPs, the spectra remained unchanged. The peak at 408 nm is due to the Soret absorption band of Mb associated with a six-coordinated high-spin ferric heme with covalently bound water and a highly polar distal pocket.24 However, a red shift of 6505
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Figure 2. TEM images of IDA6-funtctionalized AuNPs: (a) alone, (b) after addition of Cu2þ ions (90.9 μM), (c) after addition of Cu2þ ions (90.9 μM) and Mb (45.4 μg/mL); (d) schematic illustration of Mb-induced aggregation of functionalized AuNPs via Cu2þ coordination.
the absorption band from 518 to 552 nm was observed upon simultaneous addition of Cu2þ ions and Mb concomitant with a change in solution color from red to purple. The red shift in the PR band is ascribed to the near-field coupling due to the decrease of interparticle distances.25 The observed changes arose from the aggregation of the functionalized AuNPs, which was supported by the increase of average hydrodynamic diameters of the AuNPs from 27.1 to 831.4 nm (Figure S1 of the Supporting Information). Note that hydrodaynamic diameters are generally larger than real ones due to hydration shell layers. Furthermore, the formation of the AuNPs aggregates was determined by TEM observations (Figure 2). The IDA6-functionalized AuNPs were spherical with an average diameter of 13 nm and nearly monodispersed or oligoaggregated (part a of Figure 2). The functionalized AuNPs remained well dispersed only in the presence of Cu2þ ions (part b of Figure 2). However, a great amount of AuNP aggregates were observed in the presence of Cu2þ and Mb (part c of Figure 2), in accordance with the results of the UVvis spectra and DLS measurements. It is known that Cu2þ ions can coordinate strongly with IDA with an association constant of about 1011 M1,26 which is widely used in metal-affinity chromatography.2729 Mb contains 11 His residues, at least four of which are exposed on the surface in the native state.30 The imidazole moieties of the surface His residues can readily coordinate with Cu2þ ions.31 Therefore, the aggregates of Mb sandwiched between the IDA-functionalized AuNPs were formed through the coordination interactions of IDACu2þHis (part d of Figure 2) concomitant with a change in solution color from red (individual AuNPs) to purple (AuNP aggregates). Optimization of molecular structure of the coordination segment of IDA6Cu2þ His was presented in Figure S2 of the Supporting Information. MALDI-TOF mass spectrometry has been widely applied for analysis of biomacromolecules, such as proteins32 and nucleic
acids.33 There are many reports about functionalized nanoparticles as probes for the analyses of peptides and proteins in biological samples using this technique.34 In the MALDI-TOF mass spectral measurements, salts in biological samples can cause suppression of signal intensities,35 so that no m/z peak of Mb could be available in the presence of abundant salts. Herein double-distilled water was used instead of PB solution. The MALDI-TOF mass spectra of Mb binding to the IDA6-functionalized AuNPs in the absence and presence of Cu2þ ions were compared under the same conditions (Figure 3). Although a small m/z peak of Mb was detected in the absence of Cu2þ, the peak in the presence of Cu2þ was about 10-fold stronger than that in the absence of Cu2þ. Mb (pI 7.0) was positive-charged in the case of pure water and could nonspecifically bind to the functionalized AuNPs through the electrostatic interaction. However, the highly specific protein binding to the functionalized AuNPs via Cu2þ coordination was obviously observed. Parameter Optimization. The red colors of the IDA6- and IDA2-functionalized AuNP solutions remained unchanged in a refrigerator at 4 °C after 2 months, indicating the high stability of the functionalized AuNPs. No significant change in color was observed even if the IDA6-functionalized AuNP solutions were centrifugalized once, twice, and thrice, respectively. No shift in maximum absorption wavelength of the PR band for different times was observed but a decrease in absorbance with number of centrifugalization (Figure S3 of the Supporting Information). The decrease of absorbance was due to a spot of the functionalized AuNPs remained in the supernatant after centrifugalization. The UVvis spectra of IDA6-functionalized AuNPs remained almost unchanged in the absence of Mb upon addition of Cu2þ ions of different concentrations up to 136.4 μM (Figure S4 of the Supporting Information). In the presence of Cu2þ (90.9 μM), the UVvis spectra of the IDA6-functionalized AuNPs hardly 6506
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Figure 3. MALDI-TOF mass spectra of IDA6-functionalized AuNPs in the presence of Mb: (a) with Cu2þ ions (90.9 μM), (b) without Cu2þ ions.
change within 24 h (Figure S5 of the Supporting Information), indicating highly stability of the IDA6-functionalized AuNPs. However, the UVvis spectra of IDA2-functionalized AuNPs underwent a change after 1 h (Figure S6 of the Supporting Information), and the solution turned to blue after 3 h, which indicates that Cu2þ ions could induce the aggregation of the IDA2-functionalized AuNPs and that the AuNPs modified with thin organic layers were not stable and could generate PR coupling in the presence of Cu2þ. Upon addition of Mb of various concentrations to the IDA6-functionalized AuNPs in the absence of Cu2þ, no or very subtle shift (
