Article pubs.acs.org/ac
Fluorescent Gold Nanodots Based Sensor Array for Proteins Discrimination Zhiqin Yuan,†,‡,¶ Yi Du,†,§,¶ Yu-Ting Tseng,‡ Meihua Peng,∥ Na Cai,† Yan He,*,† Huan-Tsung Chang,*,‡ and Edward S. Yeung† †
College of Chemistry and Chemical Engineering, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, Hunan 410082, P. R. China ‡ Department of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan § Inspection and Testing Center for Agro-product Safety and Environment Quality, Institute of Applied Ecology Chinese Academy of Sciences (IAE CAS), 72 Wenhua Road, Shenyang, Liaoning 110016, P. R. China ∥ Metabolic Syndrome Research Center, The Second Xiangya Hospital of Central South University, Changsha 410011, Hunan, P. R. China S Supporting Information *
ABSTRACT: A series of dual-ligand cofunctionalized fluorescent gold nanodots with similar fluorescence and diverse surface properties has been designed and synthesized to build a protein sensing array. Using this “chemical nose/tongue” strategy, fluorescence response patterns can be obtained on the array and identified via linear discriminant analysis (LDA). Eight proteins have been well distinguished at low concentration (A280 = 0.005) based on this sensor array. The practicability of this sensor array was further validated by high accuracy (100%) examination of 48 unknown protein samples.
G
detection of various targets, such as metal ions, anions, biomolecules (e.g., peptide, protein, and DNA), etc.19−21 In view of the fact that surface ligands of Au NDs affect the interaction between analytes and Au NDs and finally influence the efficiency of sensing events, surface properties control becomes a key factor on expanding the application of Au NDs. Unfortunately, not all ligands can facilitate the formation or maintain the fluorescence of Au NDs. Therefore, it would be significant to explore a general method for preparation of fluorescent Au NDs with controllable surface properties. For most Au NDs, the surface ligands cannot be optionally replaced, because these ligands contribute to not only the stability but also the optical properties of Au NDs,2,22 making it difficult to prepare surface property diverse Au NDs using a similar synthetic route. To solve this problem, we developed a one-pot, two-step strategy to prepare dual-ligand cofunctionalized fluorescent Au NDs where one ligand acts as the molecular recognition probe and the other serves as the fluorescence turn-on switch.23,24 We synthesized glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) cofunctionalized Au NDs and demonstrated their capability of highly
old nanodots (Au NDs), composed of a few to hundreds of Au atoms, have been widely studied during the past years.1−4 They bridge the gap between individual atoms and large nanocrystals and display molecule-like properties.5 Because the size of Au NDs is comparable to the Fermi wavelength of electrons, they show discrete electronic energy levels and unique physical and/or chemical properties different from their corresponding large nanocrystals (with sizes > ∼3 nm). Au NDs exhibit strong light absorption through electronic transitions between molecule-like energy levels and usually result in the appearance of fluorescence.6 It is generally accepted that the photophysical properties of Au NDs depend on their size, structure, oxidation state, and surface ligand.7−9 Various types of Au NDs have been synthesized by using thiolates, polymers, and biomolecules as ligands/scaffolds,7,10−12 and the potential chemical and biological applications of Au NDs have attracted much interest due to their photophysical/chemical properties, good stability, and excellent biocompatibility, compared to semiconductor quantum dots or other metal (e.g., silver and copper) NDs.13−15 It is well-known that ligands on the Au NDs surface play an important role for analyte recognition and different target responses can be achieved by controlling the surface environments of Au NDs.16−18 By modulating the surface properties, many Au NDs based nanosensors have been developed for the © XXXX American Chemical Society
Received: December 6, 2014 Accepted: March 31, 2015
A
DOI: 10.1021/ac5045302 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry sensitive and selective lead ions (Pb2+) detection based on Pb2+ induced Au ND aggregation and fluorescence quenching.23 Furthermore, upon replacing GSH with 1-(10-mercaptodecyl)5-methylpyrimidine-2,4-dione (TSH), sulfide specific turn-on Au ND nanosensors have been successfully constructed.24 Notably, regardless of the different surface properties, the as prepared Au NDs show similar fluorescence characteristics. The different analyte response behaviors, as well as similar fluorescence characteristics of these Au NDs, encourages us to design and synthesize a series of dual-ligand cofunctionalized Au NDs for detecting various analytes. Proteins are the fundamental units of life, and they are closely related to various forms of life activities (e.g., immunity and metabolism). The presence of irregular protein concentrations or special proteins may indicate the disease states, thereby precise and rapid protein detection is of great importance in proteomics and medical diagnostics.25,26 However, it is a challenging problem because of the structural diversity and complexity of proteins. Considerable efforts have been devoted to develop protein sensing methods,27−29 and the most generally used detection method is the enzyme-linked immunosorbent assay (ELISA).30 Despite the high sensitivity of ELISA, high costs and instability restrict its application and simultaneous multiple proteins discrimination is still difficult using ELISA. On the other hand, many proteins are important intracellular or extracellular biomarkers of cancerous cells. Onsite, high-throughput protein discrimination would be very important for effective differentiation of normal and cancerous cells. Using electrophoresis based technology, hundreds of proteins could be separated and visualized;31,32 however, the inherent disadvantages of being biased toward certain proteins and being labor intensive limit its application in fast, multiple protein differentiation. Thus, developing efficient protein discrimination methods is still appealing. Array based sensing approaches, also called “chemical nose/ tongue” strategies, are complementary to some traditional immunosensing strategies (e.g., ELISA).33,34 Compared to ELISA, array based sensing utilizes the selective rather than specific interactions between receptors and analytes,35 and different analytes generate unique response patterns that can be differentiated using linear discriminant analysis (LDA), providing versatile systems that can be “trained” to identify various analytes. For instance, Rotello and co-workers fabricated a sensor array to identify proteins by integrating an anionic fluorescent polymer and six cationic gold nanoparticles (Au NPs).36 On the basis of electrostatic attraction, the anionic polymer adsorbs onto the surface of Au NPs and its fluorescence is quenched by Au NPs. Due to the protein-Au NP affinity, the presence of protein disrupts the interaction of polymer with Au NPs and generates a distinct fluorescence response pattern. Using an aptamer functionalized Au NPs based colorimetric sensor array, Zhang and co-workers successfully achieved protein discrimination with the naked eye at 50 nM levels.37 Because of the unnecessariness of specific recognition, NP array based sensing systems have been widely used for bioanalysis. Sugars, cells, and bacteria with extremely high similarity have been discriminated using NP based sensor arrays.34,38,39 Since the size, shape, composition, and surface properties of NPs show a marked effect to the interaction between NPs and analytes,40 controlling these parameters would be an effective strategy for versatile array development. Therefore, exploring a simple method that prepares NPs with
similar optical but diverse chemical/physical properties is valuable for constructing sensor arrays for multiple bioanalyses. In this study, we synthesized eight dual-ligand cofunctionalized Au NDs by using the one-pot and two-step strategy. These eight Au NDs showed similar fluorescence characteristics but different surface properties, as supported by fluorescence, UV−vis absorption, and Fourier transform infrared spectroscopy and zeta potential measurements. We constructed an array based protein discrimination system by using these eight Au NDs as efficient protein receptors and competent signal transducers. On the basis of this sensor array, eight proteins were well distinguished at low concentration (A280 = 0.005) by analyzing the fluorescence response patterns via LDA. Furthermore, this method was successfully used to identify 48 unknown protein (eight different proteins) samples with an accuracy of 100%. Since our Au ND synthetic strategy efficiently simplifies the process of array-receptor preparation, we expect that the application of Au NDs could be significantly expanded by simply varying the molecular recognition probe and versatile Au NDs based sensor arrays could be constructed.
■
EXPERIMENTAL SECTION Chemicals. Bovine serum albumin (BSA), cytochrome c (CytC, from equine heart), histone (His, from calf thymus, type III-S), human serum albumin (HSA), lysozyme (Lys, from chicken egg white), myoglobin (Myo, from equine heart), streptavidin (SA, from Streptomyces avidinii), Trypsin (Try, 1:250), 11-mercaptoundecanoic acid (MUA), 3-mercaptopropionic acid (MPA), mercaptosuccinic acid (MSA), 6-mercapto1-hexanol (MH), (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide (NSH), 4-mercaptobenzoic acid (MBA), glutathione (GSH), (2-mercaptoethyl)amine (MEN), tetrakis (hydroxymethyl) phosphonium chloride (THPC), quinine sulfate, and chloroauric acid (HAuCl4) were purchased from Sigma-Aldrich (Milwaukee, USA). 1-(10-Mercaptodecyl)-5methylpyrimidine-2,4-dione) (TSH) was synthesized according to our previous report. Anhydrous ethanol, sodium hydroxide (NaOH), and phosphate buffered saline (PBS, pH 7.4, 1X), were obtained from Sinophrarm Chemical Reagent Corporation (Shanghai, China). All chemicals were used without further purification. Ultrapure water (18.2 MΩ) was obtained from a Millipore system. All glassware was cleaned by fresh aqua regia. Characterization. The fluorescence spectra of Au NDs were recorded using a F-7000 fluorescence spectrophotometer (Hitachi, Japan). The UV absorption spectra of the Au NDs were obtained through a UV-1800 spectrophtometer (Shimadzu, Japan). High resolution transmission electron microscopy (HRTEM) was performed on a Tecnai F20 high resolution transmission electron microscope (FEI, USA). Infrared spectra (FT-IR) were recorded by using a FT/IR-410 Fourier transform infrared spectrophotometer (JASCO, Japan). Zeta potential measurements were performed with a Zetasizer Nano ZS (Malvern, U.K.). Static Rayleigh scattering intensities of Au NDs solutions were obtained using a F-7000 fluorescence spectrophotometer by setting the emission wavelength equal to excitation wavelength. Mass spectrometry (MS) experiments were performed in the reflectron negative- and positive-ion modes using an AutoflexIII LDI time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany). The samples were irradiated using pulsed laser irradiation (355 nm Nd:YAG, 100 Hz; pulse width: 6 ns). A total of 500 pulsed B
DOI: 10.1021/ac5045302 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry laser shots at a laser density of 3.19 × 104 W·cm−2 was applied to five random positions on the LDI target. Synthesis of Dual-Ligand Cofunctionalized Au NDs. Dual-ligand cofunctionalized Au NDs were synthesized according to our previous reports with slight modification,23 that is, first, introducing an independent functional probe during the synthesis of the nonfluorescent small gold nanoparticles (Au NPs) or the “off-state” Au NDs and, second, using MUA to “turn on” the Au NDs fluorescence. Such a protocol was illustrated in Scheme 1. Typically, 100 μL of
Array Based Sensing of Proteins. To facilitate accurate discrimination of proteins, we generated patterns with proteins at the absorbance level of A280 = 0.005 (Table S1, Supporting Information, also shown are their corresponding molar and mass concentrations), the lowest concentration that can be detected using ultraviolet absorption spectroscopy. At such a low concentration, the interference from other proteins could be minimized. In the protein sensing study, eight Au NDs were first diluted in 1X PBS to get solutions with Au ND concentrations of 25 nM, and eight proteins were dissolved with 1X PBS (A280 = 0.1). Each Au ND solution (190 μL) was added into a well on a 96-well plate (300 μL Whatman Glass Bottom microplate), respectively; then, 10 μL of protein solution or 1X PBS (control) was added to each well, and the fluorescence spectrum was recorded with excitation at 387 nm. The relative fluorescence variation ((I − I0)/I0) was used as the fluorescence response, where I0 and I are the fluorescence intensities of the Au ND solution in the absence and presence of the proteins. Since the maximum emission wavelengths of all Au NDs were around 500 nm, to minimize the data analysis error, we chose the integrated fluorescence intensity between 480 and 520 nm as the raw data during all LDA treatments. This process was repeated for eight proteins to generate five replicates of each. Thus, the eight proteins were tested against the eight Au NDs array five times, to afford an 8 × 8 × 5 training data matrix. The raw data matrix was processed using classical LDA treatment. To test the unknown proteins, stock solutions of eight proteins in 1X PBS (A280 = 0.1) were first prepared. Then, the Au ND solution (190 μL, 25 nM) was added into a well, and 10 μL of randomly selected protein solution was added. The fluorescence spectrum was recorded with excitation at 387 nm, and the relative integrated fluorescence intensity variation ((I − I0)/I0) during the range from 480 to 520 nm was used to generate the fluorescence response pattern. The resulting fluorescence response was analyzed with LDA to identify the tested protein by comparing the results with the training matrix.
Scheme 1. Synthetic Strategy of Dual-Ligand Functionalized Au NDs
NaOH (1 M) solution and 24 μL of THPC (8%, wt) solution were first mixed with 8 mL of ultrapure water under violent stirring for 5 min, and then, 400 μL of HAuCl4 (Au3+, 24 mM) solution was added rapidly. The color of the solution turns from light-yellow to brown in 1 min, indicating the formation of small Au NPs. At this point, 100 μL of thiolate (R-SH, 10 mM) solutions was added to obtain different thiolate-protected Au NPs. After stirring for another 15 min at room temperature, the solution was cooled to 4 °C for further use. Notice that the thiolate needs to be introduced into the reactant mixture a little later than THPC. This is because thiolates can reduce HAuCl4 directly to the Au(I)-SR complex which is difficult to be further reduced to AuNPs with a mild reducing agent like THPC. After aging for 1 day, 1 mL of thiolate-protected Au NP stock solution was mixed with 200 μL of carbonate buffer (0.1 M, pH 9.0) and 75 μL of MUA (0.1 M) ethanol solution in a thermomixer. The solution turns to light yellow (from brown) in color within 1 h and emits green light under UV (365 nm) light illumination, indicating the formation of fluorescent Au NDs. At the 2 h point, the reaction was stopped. The resulting Au ND solution was purified by centrifugation at 13 000 rpm for 20 min to eliminate large aggregates and, then, was filtered with 10 kDa cutoff ultrafiltration centrifuge tubes to remove excess reactants (MUA, thiolates, salts, etc.). The purified Au NDs were stored under a dark condition for further use. The final concentration of the Au ND solution was calculated to be 0.94 μM.
■
RESULTS AND DISCUSSION Synthesis and Characterization of Dual-Ligand Cofunctionalized Au NDs. As a starting point for our studies, we synthesized eight dual-ligand cofunctionalized Au NDs according to our previously reported one-pot and two-step strategy, as shown in Scheme 1. These eight Au NDs were characterized by UV−vis spectrometry, steady-state fluorescence spectrometry, HRTEM, FT-IR spectrometry, and zeta
Figure 1. Characterization of eight dual-ligand cofunctionalized Au NDs. Photographs of the eight Au NDs solution under room light and 365 nm UV light (a), 1−8 and Au ND1−Au ND8 are GSH/MUA, TSH/MUA, MH/MUA, MPA/MUA, MSA/MUA, MEN/MUA, MBA/MUA, and NSH/ MUA cofunctionalized Au NDs, respectively. Normalized absorption (b) and fluorescence emission (c) spectra of the eight Au NDs. C
DOI: 10.1021/ac5045302 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
Information, showed the FT-IR spectra of dual-ligand stabilized Au NDs. It was seen that the C−S stretch band (610−630 cm−1) can be observed in all Au NDs, suggesting the presence of thiolate ligands on the surface. All Au NDs exhibited two CO stretch bands and a C−O stretch band of the −COO− group centered at ∼1637, ∼1400, and ∼1093 cm −1 , respectively. The two strong CO stretch bands and one C−O stretch band suggested the presence of MUA on the Au ND surface. The broad −OH stretch band appeared around ∼3420 cm−1 maybe due to the water vapor. Several Au NDs possess a small split peak around ∼1617 cm−1; this could be attributed to the pronation of the CO bond, which may be due to the arrangement of multilayered MUA that increases the CO stretch frequency. Since MPA, MSA, and MH have similar IR spectra compared with MUA, no more characteristic IR peaks could be found in these three Au NDs. Both GSH and MEN have the primary amine group, the GSH/MUA and MEN/MUA cofunctionalized Au NDs showed a weak peak around ∼1150 cm−1 (C−N stretch bond). As can be seen in Figure S4h, Supporting Information, an extra C−H stretch bond located at ∼2875 cm−1 that was assigned to −CH3 revealed the existence of NSH in NSH/MUA cofunctionalized Au NDs. On the other hand, the zeta potentials were measured to determine the surface charge (25 nM Au NDs in 1× PBS). It can be seen that the surface zeta potentials of all Au NDs were negatively charged because of the anion form of MUA on the Au ND surface (Figure S5, Supporting Information). However, the zeta potentials of all the eight dual-ligand cofunctionalized Au NDs were smaller than the one of MUA-Au NDs, further suggesting the existence of other thiolate ligands other than MUA. Despite the less negative charge, the difference between those Au NDs were not extremely large; this may due to the small percentage of other thiolate ligands. Such a hypothesis was first verified by the UV−visible spectra variation. As an example, according to the absorbance change at 295 nm, almost 90% MBA was substituted by MUA (data not shown). Further evidence from the MALDI-MS measurement also suggests the low percentage of other ligands (106 M−1·cm−1).42 It has been reported previously that thiolate ligand functionalized Au NDs have an Au0−AuI core−shell nanostructure and their fluorescence is majorly originated from ligand to metal charge transfer (LMCT; S → AuI) mixed with ligand to metal−metal charge transfer (LMMCT; S → AuI···AuI) of the polynuclear AuI− thiolate shell.6,43 Our previous study also proved that the reduction of AuI shell to Au0 would make the fluorescence vanish.23 According to the fluorescence emission spectra under different excitation wavelengths, no emission shift was observed except fluorescence intensity variation (Figure S1, Supporting Information). Thus, the maximum emission wavelength is independent of the excitation wavelength, suggesting the uniform optical properties of these Au NDs in spite of the different chemical structures of the eight thiolate ligands (Figure S1i, Supporting Information). As can be seen from the HRTEM images (Figure S2, Supporting Information), these Au NDs had spherical shapes and the average diameters of the eight Au NDs are all less than 2.2 nm (Figure S2 insets, Supporting Information). When the above data is considered, it can be concluded that the recognition thiolate ligands do not strongly affect the size or optical properties of the resulting Au NDs using our synthetic strategy. Since the surface properties play an important role on analyte recognition, the surface characterization is essential to predict the sensing performance. As can be seen from the absorption spectra of TSH/MUA-Au NDs solution, the absorption peak around 275 nm was assigned to the heterocyclic ring of TSH (Figure S3a, Supporting Information).24 The distinct 275 nm absorption peak, as well as the strong fluorescence suggests the coexistence of TSH and MUA. An evident absorption peak around 295 nm (Figure S3b, Supporting Information) was assigned to the benzene ring of benzoic acid, indicating the successful preparation of MBA/MUA cofunctionalized Au NDs. From the absorption spectra, TSH/MUA and MBA/ MUA cofunctionalized Au NDs can be readily realized. However, the other six thiolates have no characteristic absorption peak in neither visible nor UV range, making their surface characterization difficult using the absorption spectrum. To further investigate the surface properties of these Au NDs, FT-IR and zeta potential measurements were performed. The evidence for the existence of surface thiolate ligands of Au NDs was first provided by FT-IR analysis. Figure S4, Supporting D
DOI: 10.1021/ac5045302 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry molecules can be affected by many factors such as hydrogen bonding, electrostatic attraction, hydrophobic interaction, etc.44 Taking this into consideration, eight thiolate ligands with different functional groups, isoelectric points, and hydrophobicity were chosen to provide different binding capabilities toward proteins, and eight proteins were chosen to have a variety of sizes and charges, with molecular weights of the eight proteins ranging from 12.3 to 69.4 kDa and isoelectric points varying from 4.8 to 11.0. Table 1 shows the basic properties of Table 1. Basic Properties of the Eight Proteins Used as Sensing Targets protein
Mw (kDa)
pI
metal contained
bovine serum albumin (BSA) human serum albumin (HSA) lysozyme (Lys) trypsin (Try) 1:250 myoglobin (Myo) cytochrome C (CytC) histone (His) streptavidin (SA)
66.3 69.4 14.4 24.0 17.0 12.3 21.5 65.0
4.8 5.2 11.0 10.5 7.2 10.7 10.8 6.0
N N N Y Y Y N N
Figure 2. Relative fluorescence increase (I − I0)/I0 patterns of the Au NDs array against proteins as an average of five parallel measurements.
differentiate two or more classes of events or objects, it can maximize the variance ratio of events and enable maximal data separation. The LDA reduced the size of the training matrix (8 Au NDs × 8 proteins × 5 replicates) and transformed them into canonical factors. The canonical patterns were clustered into eight different groups, which were visualized as a wellclustered three-dimensional plot (Figure 3a). The similarity of proteins was analyzed using hierarchical cluster analysis (HCA), a statistical classification method based on Euclidean distance. As shown in Figure 3b, all of the 40 cases (8 proteins × 5 replicates) were correctly assigned to their respective groups. Significantly, the fluorescence response patterns were characteristic and highly reproducible for particular proteins, indicating the excellent reproducibility of protein identification. With less Au NDs (e.g., six and three), the canonical patterns can also be clustered into eight different groups through LDA (Figures S7a and S8b, Supporting Information), and the 40 cases (8 proteins × 5 replicates) were also correctly assigned to their respective groups (Figure S9a,b, Supporting Information), suggesting the excellent separating capability of the dual-ligand cofunctionalized Au NDs based sensor array. However, the eight Au NDs based sensor array showed more concentrated canonical patterns than the six or three Au NDs based sensor array, suggesting better resolution capability. On the other hand, the throughput of a sensor array is proportional to the dimension. Thus, the eight Au NDs based sensor array should have higher throughput in theory, which means it has the potential to discriminate more proteins. On the basis of these results, we choose the eight Au NDs based sensor array as the working array. The fluorescence of polymer or quantum dots might be quenched by adsorbed proteins due to energy or electron transfer.45,46 Therefore, one of the possible mechanisms of the protein induced fluorescence response may be attributed to the energy or electron transfer between Au NDs and adsorbed proteins. The clue of energy or electron transfer between proteins and Au NDs was supported by the absorption spectra of protein solution. It can be seen that both Cytochrome C (CytC) and Myoglobin (Myo) solution exhibit red color and have two absorption peaks close to the excitation and emission centers of the Au NDs (Figure S10a, Supporting Information). Thus, the reduction of CytC/Au ND and Myo/Au ND fluorescence can be attributed to fluorescence quenching through energy or electron transfer upon protein adsorption onto surfaces of Au NDs. Since both surface oxidation state and dispersion state of Au NDs play important roles to the fluorescence, any change in these parameters may also lead to
the 8 proteins used in sensing experiments. We investigated the fluorescence profile change of the Au NDs in the presence of these proteins. Sensing of individual analytes in isolation provides a test for array based sensors, giving insight into their ability to detect small changes in analyte structure. To ensure the detection of a low concentration of protein, we tested the fluorescence responses using protein samples with a UV adsorption value of 0.005 at 280 nm. Upon adding protein to the Au ND solution, the fluorescence profiles of the Au NDs showed distinct changes in intensity but only small shifts in the maximum emission wavelength. For example, as shown in Figure S6, Supporting Information, the presence of the same absorbance level of different proteins resulted in large variations of the fluorescence intensity but only a small wavelength shift (∼7 nm) of the GSH/MUA cofunctionalized Au NDs, and with increasing protein concentration, the fluorescence intensities of the Au NDs showed a continuous decrease (Figure S7, Supporting Information). These results suggested that the interaction between certain protein and dual-ligand cofunctionalized Au NDs would affect the excitation/emission processes and produce distinct fluorescence profiles. Subsequently, for each protein, we tested its fluorescence response against the Au NDs array five times, generating an 8 × 8 × 5 matrix. As shown in Figure 2, the relative fluorescence increase pattern (I − I0)/I0 induced by different proteins is distinct, suggesting the feasibility of protein identification using such a sensor array. Sensor arrays are made of many sensor elements, and each of them may show a different response toward even the same analyte.38 In general, the response pattern of each analyte generated by a sensor array is unique. Compared to traditional methods, array based sensing strategies avoid the use of specific acceptors, greatly expanding the range of detectable analytes. On the basis of the nonspecific interaction between receptor-protein and unique response pattern, different proteins with similar structure could be effectively discriminated. To further generate the fluorescence response pattern of the Au ND array against the eight proteins, the raw data obtained were subjected to LDA using SPSS v16.0. Since LDA can recognize the linear combination of features that E
DOI: 10.1021/ac5045302 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
decrease when they aggregate.23 The static Rayleigh scattering intensities of Au ND solutions in the absence and presence of His were recorded (Figure S10b, Supporting Information). It was found that the scattering intensities of Au NDs increased after adding His to the Au ND probe solutions, indicating the increased degree of aggregation. Therefore, the fluorescence quenching induced by His might be attributed to the aggregation. In contrast, the disaggregation of Au ND aggregates increased the fluorescence. When all the data are combined, the protein induced fluorescence response of the Au NDs may involve electrostatic as well as hydrophobic interactions between the surface thiolate ligands and protein amino acid residues and appears to be a result of the combination of Au NDs to protein energy or electron transfer, Au NDs surface charge state, and dispersion state variation. To validate the detection efficiency of our array based sensing strategy, a series of protein solutions (A280 = 0.005) was chosen as the unknown samples. The response patterns and LDA analysis were performed by the same approach. Notably, of the 48 unknown protein samples, none of them was incorrectly identified, affording an identification accuracy of 100% (Table S3, Supporting Information, for original data and the result of identification). However, the accuracy becomes 97.9% and 95.8% when using the six or three Au NDs based sensor array (data not shown), suggesting the better discrimination capability of the eight Au NDs based sensor array. Such good results demonstrated that our Au NDs sensing array holds substantial promise for the discrimination of proteins. Since this system shows satisfactory single protein discrimination capability, complicated analysis with real samples (e.g., blood plasma and cell culture) should also be able to be performed according to Rotello and others’ related works. To address the sample matrix effect in practical analysis, a different training set would be designed, though we expect the sensitivity and efficiency in protein discrimination may not be as good as the ideal cases. However, a combination of the “chemical nose/ tongue” strategy with mature protein separation technologies may facilitate analysis of complex protein samples. Future works focusing on selectivity verification and algorithm improvement may play important roles to achieve this goal.
■
Figure 3. Canonical score plots for the first three factors of relative fluorescence increase (I − I0)/I0 pattern analyzed by LDA (a). HCA analysis of protein samples with five parallel measurements (b).
CONCLUSIONS We have designed and synthesized eight dual-ligand functionalized Au NDs with similar fluorescence profiles and diverse surface properties using a one-pot and two-steps strategy and built a protein sensor array based on these Au NDs. Using this sensor array, eight proteins with a variety of sizes and charges were successfully discriminated at low concentration (A280 = 0.005) through LDA. The high accuracy of blind sample tests further validated the practical application of this sensor array. Taken together, our method was rapid and efficient and can be readily generalized. By varying the surface properties of the Au NDs, the fluorescent Au NDs based array might hold the potential for DNAs, cells, tissues, and heavy metal ions discrimination.
fluorescence variation. It was reported that hemin can quench the fluorescence of MUA capped Au NDs.47 This is because the Fe2+ center can induce surface Au+ reduction that inhibit ligand-to-metal charge transfer processes and subsequently decrease the fluorescence. Both CytC and Myo contain hemin moiety; thus, they may quench the fluorescence of all Au NDs, and hemin contained proteins can also induce the fluorescence quenching of protein stabilized Au NDs.48 Besides CytC and Myo, trypsin (Try) can also quench the fluorescence of all Au NDs. Although Try has no strong absorption near the excitation or emission wavelength range of Au NDs, the metal center of Try may possibly be the reason for the induced fluorescence quenching. On the other hand, histone (His), that without hemin or an absorption peak near the excitation or emission wavelength of Au NDs, can also quench the fluorescence of all Au NDs. Our previous study indicated that the degree of aggregation of Au NDs is very important to the fluorescence intensity, and the fluorescence of Au NDs would
■
ASSOCIATED CONTENT
S Supporting Information *
Additional figures and tables as indicated in the main text. This material is available free of charge via the Internet at http:// pubs.acs.org. F
DOI: 10.1021/ac5045302 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
■
(24) Yuan, Z.; Peng, M.; Shi, L.; Du, Y.; Cai, N.; He, Y.; Chang, H.T.; Yeung, E. S. Nanoscale 2013, 5, 4683−4686. (25) Pepys, M. B.; Hirschfield, G. M.; Tennent, G. A.; Ruth Gallimore, J.; Kahan, M. C.; Bellotti, V.; Hawkins, P. N.; Myers, R. M.; Smith, M. D.; Polara, A.; Cobb, A. J. A.; Ley, S. V.; Andrew Aquilina, J.; Robinson, C. V.; Sharif, I.; Gray, G. A.; Sabin, C. A.; Jenvey, M. C.; Kolstoe, S. E.; Thompson, D.; Wood, S. P. Nature 2006, 440, 1217− 1221. (26) He, G.; Luo, W.; Li, P.; Remmers, C.; Netzer, W. J.; Hendrick, J.; Bettayeb, K.; Flajolet, M.; Gorelick, F.; Wennogle, L. P.; Greengard, P. Nature 2010, 467, 95−98. (27) Ho, Y. Nature 2002, 415, 180−183. (28) Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884−1886. (29) Ho, H.-A.; Najari, A.; Leclerc, M. Acc. Chem. Res. 2008, 41, 168−178. (30) Lequin, R. M. Clin. Chem. 2005, 51, 2415−2418. (31) Mann, M.; Jensen, O. N. Nat. Biotechnol. 2003, 21, 255−261. (32) Görg, A.; Weiss, W.; Dunn, M. J. Proteomics 2004, 4, 3665− 3685. (33) Haab, B. B. Curr. Opin. Biotechnol. 2006, 17, 415−421. (34) Askim, J. R.; Mahmoudi, M.; Suslick, K. S. Chem. Soc. Rev. 2013, 42, 8649−8682. (35) Wright, A. T.; Anslyn, E. V. Chem. Soc. Rev. 2006, 35, 14−28. (36) You, C. C.; Miranda, O. R.; Gider, B.; Ghosh, P. S.; Kim, I. B.; Erdogan, B.; Krovi, S. A.; Bunz, U. H.; Rotello, V. M. Nat. Nanotechnol. 2007, 2, 318−323. (37) Lu, Y.; Liu, Y.; Zhang, S.; Wang, S.; Zhang, S.; Zhang, X. Anal. Chem. 2013, 85, 6571−6574. (38) Miranda, O. R.; Creran, B.; Rotello, V. M. Curr. Opin. Chem. Biol. 2010, 14, 728−736. (39) Bajaj, A.; Miranda, O. R.; Kim, I. B.; Phillips, R. L.; Jerry, D. J.; Bunz, U. H. F.; Rotello, V. M. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 10912−10916. (40) Mahmoudi, M.; Lynch, I.; Ejtehadi, M. R.; Monopoli, M. P.; Bombelli, F. B.; Laurent, S. Chem. Rev. 2011, 111, 5610−5637. (41) Haiss, W.; Thanh, N. T. K.; Aveyard, J.; Fernig, D. G. Anal. Chem. 2007, 79, 4215−4221. (42) Tseng, Y.-T.; Yuan, Z.; Yang, Y.-Y.; Huang, C.-C.; Chang, H.-T. RSC Adv. 2014, 4, 33629−33635. (43) Zhou, C.; Sun, C.; Yu, M.; Qin, Y.; Wang, J.; Kim, M.; Zheng, J. J. Phys. Chem. C 2010, 114, 7727−7732. (44) Berg, T. Angew. Chem., Int. Ed. 2003, 42, 2462−2481. (45) Fan, C.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2002, 124, 5642−5643. (46) Choi, J. H.; Chen, K. H.; Strano, M. S. J. Am. Chem. Soc. 2006, 128, 15584−15585. (47) Chen, L.-Y.; Huang, C.-C.; Chen, W.-Y.; Lin, H.-J.; Chang, H.T. Biosens. Bioelectron. 2013, 43, 38−44. (48) Kong, H.; Lu, Y.; Wang, H.; Wen, F.; Zhang, S.; Zhang, X. Anal. Chem. 2012, 84, 4258−4261.
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Phone: +86 731 88823074. Fax: +86 731 88821904. *E-mail:
[email protected]. Phone/Fax: +011 886 02 33661171. Author Contributions ¶
Z.Y. and Y.D. contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China with grant numbers of 20975036, 91027037, 21127009, and 21221003, Natural Science Foundation of Hunan Province 13JJ1015, and Hunan University 985 fund. This work was also supported by the Ministry of Science and Technology of Taiwan under contracts NSC 101-2113-M002-002-MY3 and 103-2923-M-002-002-MY3. Z.Y. is grateful to the Ministry of Science and Technology of Taiwan for a postdoctoral fellowship under contract NSC 103-2811-M-002169. We would like to thank the Mass Spectrometry-based Proteomics Core Facility in National Taiwan University for assistance with the MALDI-TOF mass spectra measurements.
■
REFERENCES
(1) Li, G.; Jin, R. Acc. Chem. Res. 2013, 46, 1749−1758. (2) Huang, C.-C.; Yang, Z.; Lee, K.-H.; Chang, H.-T. Angew. Chem., Int. Ed. 2007, 46, 6824−6828. (3) Zheng, J.; Nicovich, P. R.; Dickson, R. M. Annu. Rev. Phys. Chem. 2007, 58, 409−431. (4) Shang, L.; Dong, S.; Nienhaus, G. U. Nano Today 2011, 6, 401− 418. (5) Jin, R.; Zhu, Y.; Qian, H. Chem.Eur. J. 2011, 17, 6584−6593. (6) Zheng, J.; Zhou, C.; Yu, M.; Liu, J. Nanoscale 2012, 4, 4073− 4083. (7) Zheng, J.; Zhang, C. W.; Dickson, R. M. Phys. Rev. Lett. 2004, 93, 077402. (8) Duan, H.; Nie, S. J. Am. Chem. Soc. 2007, 129, 2412−2413. (9) Wu, Z.; Jin, R. Nano Lett. 2010, 10, 2568−2573. (10) Jin, R. Nanoscale 2010, 2, 343−362. (11) Xie, J.; Zheng, Y.; Ying, J. Y. J. Am. Chem. Soc. 2009, 131, 888− 889. (12) Kennedy, T. A. C.; MacLean, J. L.; Liu, J. Chem. Commun. 2012, 48, 6845−6847. (13) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2003, 4, 11−18. (14) Yuan, Z.; Chen, Y.-C.; Li, H.-W.; Chang, H.-T. Chem. Commun. 2014, 50, 9800−9815. (15) Ma, J.-Y.; Chen, P.-C.; Chang, H.-T. Nanotechnology 2014, 25, 195502. (16) Huang, C.-C.; Chen, C.-T.; Shiang, Y.-C.; Lin, Z.-H.; Chang, H.T. Anal. Chem. 2009, 81, 875−882. (17) Tseng, Y.-T.; Chang, H.-T.; Chen, C.-T.; Chen, C.-H.; Huang, C.-C. Biosens. Bioelectron. 2011, 27, 95−100. (18) Wang, Y.; Chen, J.; Irudayaraj, J. ACS Nano 2011, 5, 9718− 9725. (19) Liu, J. TrAC, Trends Anal. Chem. 2014, 58, 99−111. (20) Luo, Z.; Zheng, K.; Xie, J. Chem. Commun. 2014, 50, 5143− 5155. (21) Chen, L.-Y.; Wang, C.-W.; Yuan, Z.; Chang, H.-T. Anal. Chem. 2015, 87, 216−229. (22) Jung, J.; Kang, S.; Han, Y.-K. Nanoscale 2012, 4, 4206−4210. (23) Yuan, Z.; Peng, M.; He, Y.; Yeung, E. S. Chem. Commun. 2011, 47, 11981−11983. G
DOI: 10.1021/ac5045302 Anal. Chem. XXXX, XXX, XXX−XXX
