Article pubs.acs.org/ac
A Visual Sensor Array for Pattern Recognition Analysis of Proteins Using Novel Blue-Emitting Fluorescent Gold Nanoclusters Shenghao Xu,†,# Xin Lu,‡ Chenxi Yao,† Fu Huang,§ Hua Jiang,† Wenhao Hua,∇ Na Na,† Haiyan Liu,† and Jin Ouyang*,† †
Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, People’s Republic of China # Key Laboratory of Sensor Analysis of Tumor Marker Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People’s Republic of China ‡ National Institutes for Food and Drug Control, Beijing 100050, People’s Republic of China § Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China ∇ Department of Clinical Laboratory, Beijing Ditan Hospital, Capital Medical University, Beijing 100050, People’s Republic of China S Supporting Information *
ABSTRACT: This paper describes a visual sensor array for pattern recognition analysis of proteins based on two different optical signal changes: colorimetric and fluorometric, by using two types of novel blue-emitting collagen protected gold nanoclusters and macerozyme R-10 protected gold nanoclusters with lower synthetic demands. Eight proteins have been welldiscriminated by this visual sensor array, and protein mixtures after one-dimensional polyacrylamide gel electrophoresis also could be well-discriminated. The possible mechanism of this sensor array was illustrated and validated by fluorescence spectra, X-ray photoelectron spectroscopy (XPS), fluorescence lifetime, isothermal titration calorimetry (ITC), and matrixassisted laser desorption/ionization−time-of-flight mass spectrometry (MALDI-TOF MS) experiments. It was attributed to that the adsorption of proteins onto the surface of gold nanoclusters (Au NCs), forming the protein−Au NCs complex. Furthermore, serums from normal and hepatoma patients were also effectively discriminated by this visual sensor array, showing feasible potential for diagnostic applications.
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materials, including dendrimer, DNA, glutathione and proteins, etc.18−21 Among these synthetic methods, because of the threedimensional (3D) complexed structures of proteins which can be easily conjugated with other systems, protein-directed synthesis is especially attractive.22 To date, a variety of different proteins have been developed for the preparation of red fluorescent Au NCs, such as bovine serum albumin (BSA), transferring, insulin, lysozyme, trypsin, horseradish peroxidase, and pepsin.23−28 Nevertheless, to the best of our knowledge, there has been scarce previous studies reporting on proteindirected synthesis of blue-emitting fluorescent Au NCs. Hence, the development of blue-emitting fluorescent Au NCs with lower synthetic demands, which can be used for protein discrimination still remains a challenge. In this work, we report a visual sensor array for pattern recognition analysis of proteins by utilizing two types of novel
rotein analysis plays crucial roles in many areas, such as proteome research, clinical diagnostics, and biomedical research.1−3 Traditional protein detection methods, such as Coomassie Brilliant Blue-R250 (CBB-R250) staining and silver staining, only show the same color for all target proteins and it may be also restricted because of the time-consuming procedures.4,5 Recently, as a rapid, cost-effective, and practical method for protein discrimination, sensor arrays have been successfully applied for the discrimination and recognition of proteins, including using polymers, nanoparticles, graphene oxide, carbon nanotubes, etc.6−9 However, the synthesis or surface modification of these materials are relative complicated or time-consuming. Therefore, it is still very important to develop new materials, which require simple synthetic procedures for protein discrimination. Fluorescent gold nanoclusters (Au NCs) have gained much attention, because of their burgeoning photophysical properties and potential applications in biosensing and bioimaging.10−17 Impelled by their potential applications, fluorescent Au NCs have been synthesized by utilizing many diverse protected © XXXX American Chemical Society
Received: July 17, 2014 Accepted: November 6, 2014
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blue-emitting fluorescent gold nanoclusters. Compared with the traditional protein discrimination sensor arrays, three obvious advantages of this sensor array make it particularly attractive: (1) These Au NCs can be rapidly synthesized within 90 s and can be directly applied as a sensor array for protein discrimination without further surface modification; (2) Protein discrimination can be achieved both by fluorescent intensity and color changes; and (3) Only two types of nanomaterials are needed to distinguish eight proteins, and protein mixtures after polyacrylamide electrophoresis could also be well-discriminated, according to the diverse color response pattern. Thus, a visual sensor array with lower synthetic efforts for pattern recognition analysis of proteins was established.
Bruke Autoflex mass spectrometer (Burker Daltonics, Bremen, Germany). All the mass spectra were collected in the reflectron and positive ion mode with an accelerating voltage of 20 kV and proceeded by Flex Analysis 3.4 software (Bruker Daltonics). Synthesis of Col-Au NCs and Mac-Au NCs. All glass bottles were washed with aqua regia and rinsed with ultrapure water. In a typical MW-assisted synthesis experiment, a solution of 40 mg/mL collagen was added to an equal volume of 2.5 mM HAuCl4, and the pH of the resulting solutions was adjusted to 12 by adding NaOH solution. The mixture then was subjected to microwave treatment at 300 W for 90 s. Thus, blue-emitting fluorescent Col-Au NCs were achieved. For the Mac-Au NCs synthesis, a solution of 40 mg/mL Macerozyme R-10 was added to an equal volume of 1.25 mM HAuCl4. The mixture then was subjected to direct microwave treatment at 300 W for 90 s. Protein Discrimination. Twenty microliters (20 μL) of 30 mg/mL target proteins were added to 2 mL of Col-Au NCs and Mac-Au NCs, respectively. They then were incubated at 37 °C for 3 h. Subsequently, the images of the different fluorescent colors were obtained by the bioimaging system at the excitation wavelength of 365 nm. At last, the fluorescent data were processed using linear discriminant analysis (LDA) in SPSS v16.0.
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EXPERIMENTAL SECTION Chemicals. All reagents were of analytical reagent grade. Collagen (Col), Macerozyme R-10 (Mac), lysozyme (Lys), human serum albumin (HSA), egg white albumin (EA), pepsin (Pep), hemoglobin (Hb), trypsin (Try), catalse (CAT), trandferrin (Tf), and tetramethylethylenediamine (TEMED) were obtained from Sigma (St. Louis, MO, USA). Tris(hydroxymethyl)aminomethane), aminoacetic acid (glycine), Bis(N,N′-methylenebisacrylamide), ammonium persulfate, acrylamide, 3′,3″,5′,5″-tetra bromophenol sulfone phthalein (Bromophenol Blue), and hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O) were purchased from Sino-American Biotechnology Co. (Beijing, China). Sodium hydroxide (NaOH) and glycerine were purchased from Beijing Fine Chemical Factory (Beijing, China). Protein markers (myoglobin, catalase, aldolase, and albumin) were supplied by GE Healthcare (Piscataway, NJ, USA). All solutions were prepared using ultrapure water from a Millipore Simplicity 185 water purification system (Millipore, Bedford, MA, USA). Serum samples of healthy people were obtained from the Affiliated Hospital of the Beijing Normal University. Serum samples of hepatoma patients were obtained from the Beijing Ditan Hospital. Serum samples from thalassemia patients were acquired from Guangzhou Children’s Hospital (Guangzhou, China). These samples were obtained with informed consent from the human subjects. In addition, this sample collection was approved by the Institutional Review Board of Beijing Ditan Hospital. Instruments. An advanced microwave digestion system was used for the microwave pyrolysis (Milestone, USA). Fluorescence spectra was recorded on a fluorescence spectrometer (Perkin−Elmer, Model LS55). High-resolution transmission electron microscopy (HRTEM) images were taken on a JEOL TEM 2100 microscope operating at 200 kV. X-ray photoelectron spectroscopy (XPS) was carried out on a thermoelectron instrument (Thermo-VG Scientific ESCALAB 250). Time-resolved fluorescence measurements were performed using an OB920 single-photon counting fluorometer (Edinburgh Analytical Instruments) with a pulsed nanosecond nitrogen lamp as excitation source. All spectra were referenced to the C 1s peak at 285.0 eV. Dynamic light scattering (DLS) experiment was performed on a Zetasizer Nano-ZS (Malvern Instruments, Malvern, U.K.). The electrophoresis systems consisted of DYCZ-24D and DYCZ-21 vertical electrophoresis tanks (Liuyi Instrument Factory, Beijing, China). The fluorescence images were recorded by a bioimaging system (UVP EC3 Imaging System, UVP, Inc., USA) at the wavelength of 365 nm. MALDI-TOF MS analysis was performed on the
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RESULTS AND DISCUSSION Characterization of Col-Au NCs and Mac-Au NCs. Figures 1A-a and 1B-a present typical transmission electron microscopy (TEM) images of the as-prepared Col-Au NCs and Mac-Au NCs, appearing as spherical particles with good monodispersity. Moreover, the well-resolved lattice planes with a spacing of ∼0.23 nm in the HRTEM image (inset in Figure 1A-a and B-a) demonstrates the excellent crystalline
Figure 1. (A-a) Typical TEM image of Col-Au NCs. The top and bottom insets display size distribution and the HRTEM image of ColAu NCs, respectively. (A-b) Fluorescence excitation (λex = 330 nm, blue line) and emission (λem= 420 nm, red line) spectra of the Col-Au NCs; inset shows a photograph of the Col-Au NCs under sunlight (left) and 365-nm UV light illumination (right). (A-c) XPS spectrum of Au 4f for Mac-Au NCs. (B-a) Typical TEM image of Mac-Au NCs; the top and bottom insets display size distribution and the HRTEM image of Mac-Au NCs, respectively. (B-b) Fluorescence excitation (λex = 366 nm, blue line) and emission (λem = 440 nm, red line) spectra of the Mac-Au NCs, the inset is the photograph of the Mac-Au NCs under sunlight (left) and 365 nm UV light illumination (right). (B-c) XPS spectrum of Au 4f for Mac-Au NCs. B
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structure of the as-prepared Au NCs, which was consistent with the previous report.29 The diameter histograms in Figure 1A-a and 1B-a, calculated by measuring more than 100 particles in the TEM image, reveal that the Col-Au NCs and Mac-Au NCs have average sizes of 1.4 and 2.0 nm, respectively, which was in accordance with the DLS data (see Figure S1 in the Supporting Information). The as-prepared two Au NCs showed a broad absorption band and the absence of localized surface plasmon resonance bands, suggesting the formation of the Au NCs (see Figure S2 in the Supporting Information). In addition, the fluorescence emission were also investigated to confirm the formation of Au NCs. As shown in Figures 1A-b and 1B-b, the emission peak of the as-prepared Col-Au NCs and Mac-Au NCs was located at 420 and 440 nm, respectively. This suggests the presence of Au8 clusters according to the spherical Jellium model,30 which are consistent with those of Au8 nanoclusters reported in the previous studies.31 Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was performed to confirm the production of Au8 clusters (see Figure S3 in the Supporting Information). The quantum yield (QY) was 3.1% and 4.2% (calibrated with quinoline as the reference), and the fluorescence lifetime was 2.65 and 5.82 ns for Col-Au NCs and Mac-Au NCs, respectively (see Figure S4 in the Supporting Information). In order to obtain additional information about the as-prepared Au NCs, X-ray photoelectron spectroscopy (XPS) was conducted and the results are shown in Figures 1A-c and 1Bc. The binding energies (BEs) of the Au 4f5/2 and Au 4f7/2 peaks are 85.18 and 88.58 eV for Col-Au NCs and 84.48 and 87.93 eV for Mac-Au NCs, inferring that both Au0 and Au+ exist in the Au NCs.32 Furthermore, these Au NCs are relatively stable and the fluorescence quenching only occurred slightly after one month of storage in darkness at 4 °C (see Figure S5 in the Supporting Information). In addition, a systematic study was performed to explore the optimal conditions (e.g., reaction time and the concentrations of Col, Mac, HAuCl4, and NaOH) to prepare the fluorescent Col-Au NCs and Mac-Au NCs (see Figures S6 and S7 in the Supporting Information). Meanwhile, we also investigated whether red-shifted Au NCs could be synthesized by changing five different molar ratio of Au/protein (0.02, 0.04, 0.08, 0.16, and 0.33), using Col-Au NCs as an example. However, as shown in Figure S8 in the Supporting Information, red-shifting still could not be observed. We could only observe the change of the fluorescence intensity. Protein Discrimination by Linear Discriminant Analysis (LDA). As a proof-of-concept system, we select eight proteins that have diverse isoelectric points and molecular weights as the sensing targets (see Table S1 in the Supporting Information). As shown in Figure 2A, after adding different target proteins, the as-prepared blue-emitting Col-Au NCs and Mac-Au NCs gave diverse responses to the target proteins and caused a various fluorescent color change, which could be observed by the naked eye. These color differences could provide rough discriminations among the target proteins. For example, HSA, Hb, and Tf caused visible color changes of ColAu NCs from blue to red, green, and pink, respectively. Similarly, these three target proteins caused visible color changes of Mac-Au NCs from blue to pink, yellow, and purple, respectively. In other words, after adding different target proteins, it will also cause different wavelength shifts. These different wavelength shifts, which act as “fingerprints”, encouraged us to exploit a pattern recognition approach for protein discrimination. Therefore, linear discriminate analysis
Figure 2. Array-based sensing of eight proteins: (A) photographs of the fluorescence color change upon addition of protein solutions at 300 μg/mL; (B) canonical score plot for the wavelength shift as obtained from LDA with 95% confidence ellipses; and (C) canonical score plot for the FL response patterns as obtained from LDA with 95% confidence ellipses.
(LDA), which is a statistic approach,33 was used to differentiate the wavelength shift of the Col-Au NCs and Mac-Au NCs with target proteins. For each protein, we tested its wavelength shift against the two Au NCs three times, generating a matrix of 2 Au NCs × 8 proteins × 3 replicates. LDA analysis transforms the matrix into canonical factors and the two factors can be visualized in a two-dimensional (2D) plot shown in Figure 2B under the 95% confidence ellipses. As shown in Figure 2B, all target proteins were separated from each other, demonstrating that they were effectively discriminated by LDA, based on wavelength shift. Moreover, adding different target proteins to the Col-Au NCs and Mac-Au NCs could also result in diversity of fluorescent intensity (FL) responses (see Figure S9 in the Supporting Information). (Here, the change of the fluorescence intensity refers to the fluorescent intensity change before and after adding the target proteins to the Au NCs. Before adding target proteins to the Au NCs, the fluorescence intensity was the intensity of the Au NCs. After adding target proteins to the Au NCs, the target proteins adsorbed onto the surface of Au NCs, forming the target protein−Au NCs complexes. The fluorescence intensity was the intensity of the target protein− Au NCs complex. For example, the emission peak of Au NCs used in Figure2B was 630 nm.) Similarly, these different FL responses could also be a “fingerprint” map for the protein discrimination. As shown in Figure 2C, all target proteins were separated from each other demonstrating that they were effectively discriminated according to the diverse fluorescent intensity (FL) responses. It is noteworthy that the quantum yield of Mac-Au NCs (4.2%) is higher than that of Col-Au NCs (3.1%). However, in Figure 2A, the emission intensity of MacAu NCs is weaker than that of Col-Au NCs. We presented the relevant explanation in the Supporting Information (Figure S10). In addition, target proteins could still be discriminated with a low protein concentration (10 μg/mL) (see Figure S11 in the Supporting Information). Moreover, protein mixtures after polyacrylamide electrophoresis could also be welldiscriminated, according to the diverse color response pattern (see Figure S12 in the Supporting Information). Discussion of the Possible Mechanism. Subsequently, we studied the possible mechanism, taking Col-Au NCs as an C
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example. First, in order to understand the interaction between Au NCs with target proteins, an isothermal titration calorimetry (ITC) experiment using Col-Au NCs and HSA was conducted as an example. As we know, the thermodynamic parameters ΔH and ΔS could be used to describe the binding affinity between Au NCs and target proteins, which can be classified into different models.34 In detail, the interaction is mainly attributed to hydrophobic bonds when ΔH > 0 and ΔS > 0, while the interaction is mainly attributed to van der Waals forces and hydrogen bonding when ΔH < 0 and ΔS < 0. Furthermore, the interaction is mainly attributed to electrostatic interactions when ΔH ≈ 0 and ΔS > 0.34 In this experiment, as shown in Figure 3, ΔH1 < 0, ΔS1 < 0, K1 = 7.24
Figure 4. (A) Fluorescence emission spectra of the Col-Au NCs after adding different concentrations of HSA; inset shows the corresponding photos under UV light. (B) Fluorescence emission spectra of ColAu NCs (spectrum a), Col-Au NCs + CAT (spectrum b), Col-Au NCs + Hb (spectrum c), and Col-Au NCs + HSA (spectrum d). (C) XPS spectrum of Au 4f for Col-Au NCs (spectrum a), Col-Au NCs + CAT (spectrum b), Col-Au NCs + Hb (spectrum c), and Col-Au NCs + HSA (spectrum d). (D) Fluorescence lifetime of the Col-Au NCs (spectrum a), Col-Au NCs + CAT (spectrum b), Col-Au NCs + Hb (spectrum c), and Col-Au NCs + HSA (spectrum d), respectively.
solution (1.25 mM, 600 μL), the emission peak of Col-Au NCs at 420 nm was gradually decreased while a new emission peak at 630 nm appeared and gradually increased with increasing HSA concentration. We noticed that the emission peak of ColAu NCs at 420 nm was almost disappeared when the concentration of HSA was higher than 2.2 mg/mL (0.032 mM). Meanwhile, the intensity of the new emission peak at 630 nm was almost no longer increased. It was concluded that the adsorption process had a tendency to be saturated when the concentration of HSA was higher than 2.2 mg/mL (0.032 mM). That is to say, 0.16 × 10−9 mol HSA absorbs on the surface of 750 × 10−9 mol Au NCs, forming protein−Au NC complexes. In other words, 0.0002 mol HSA absorbs on the surface of 1 mol Au NCs forming protein−Au NC complexes. In addition, the HSA-induced fluorescence color change (from blue to red) was so obvious that it could be easily observed by the naked eye under an ultraviolet (UV) lamp (see the inset in Figure 4A). Since HSA is nonfluorescent in this spectral region, we speculated that the generation of the new fluorescent peak resulted from the adsorption of HSA onto the surface of ColAu NCs, forming the HSA-Au NCs complex. Similarly, new emission peaks at 450 and 510 nm also appeared after adding CAT and Hb, respectively, to the Col-Au NCs (see Figure 4B). To support this result, first, XPS experiment was carried out to verify this speculation. As shown in Figure 4C, after adding the same concentrations of CAT, Hb, and HSA, respectively, the binding energy of the Au 4f5/2 and Au 4f7/2 decreased by varying degrees. According to the previous reports, a red shift would happen when the energy decreases.38,39 This might be the reason for the resulting fluorescence red shift from 420 nm to 455 nm (blue color), 520 nm (green color), and 630 nm (red color), leading to the fluorescence color changes. The
Figure 3. Isothermal titration calorimetry (ITC) profile for Col-Au NCs titration into HSA at 310 K. The concentration of Col-Au NCs is 0.5 mM and the concentration of HSA is 0.03 mM.
× 105 M−1, and ΔH2 > 0, ΔS2 > 0, K2 = 3.62 × 103 M−1, The spontaneity for ΔH1 < 0 and ΔS1 < 0 might be that when Au NCs insert into the binding sites of hydrophobic part of target proteins, the hydrophobic interactions could lead to exothermic phenomenon. In addition, the rigid structure of target proteins may also be destroyed leading to the exothermic phenomenon.34 At the same time, The spontaneity for ΔH1 > 0 and ΔS1 > 0 might be that when Au NCs close to the binding sites of target protein, the hydration layer of the target protein around the binding sites are partly damaged, resulting in heat absorption phenomenon.35 This indicates that Col-Au NCs and HSA interacted with a hydrophobic force mainly via hydrogen bonding and van der Waals interactions. Therefore, we speculated that the target proteins are adsorbed onto the surface of Au NCs, forming the target protein−Au NCs complexes. In addition, previous works have been reported that the photophysical properties of the Au NCs could be altered when proteins adsorbed to its surface, forming the target protein− NCs complexes.36,37 As shown in Figure 4A, after adding different concentrations of HSA (5 μL) to Col-Au NCs D
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binding energy shift strongly support our claim that the photophysical properties of the Au NCs are directly altered by proteins binding to the Au NCs surface,40 forming the protein− Au NCs complexes. Furthermore, fluorescence lifetime measurement was also performed to further illustrate the underlying mechanism. As shown in Figure 4D, the average lifetime of the Col-Au NCs increased from 2.65 ns to 3.85, 4.17, and 1.22 μs, respectively. This change might originate from the longest-lifetime component, associated with the Au(I)−thiol complex on the particle surface.41 Moreover, the interactions between the target protein and the Au NCs surface could affect the luminescence process of the Au(I)−thiol complex.41,42 Furthermore, upon target protein adsorption onto the Au NCs, the binding energy shift of Au 4f 5/2 and Au 4f7/2 may be another reason for the resulting variation in the lifetimes of Au NCs.40 This result also indicated that the photophysical properties of the Au NCs was altered by the interactions between target proteins and the Au NCs surface,43 forming the target protein− Au NCs complexes. Furthermore, according to the spherical Jellium model, the wavelength of fluorescent AuNCs is also dependent on the size of the Au NC cores.30 In detail, smaller Au NCs emit at shorter wavelengths, while larger Au NCs emit at longer wavelengths (e.g., blue for Au8, green for Au13, and red for Au25 emission). As shown in Figure S13 in the Supporting Information, the average particle size of the protein−Au NCs complex gradually increased. According to the previous report, compared with other target proteins, HSA possesses a relatively large internal space at alkaline pH values.24,44−46 Therefore, it could provide larger internal space to assemble Au8 clusters, eventually forming Au25 clusters.24,26 This may be the reason for the relatively large size distribution of Col-Au NCs + HSA. Meanwhile, this phenomenon was also consistent with the previous research that the binding energy of Au NCs decreases with increasing cluster size.47 We also have used MALDI-TOF MS to characterize the atomic composition of the Au NC cores before and after adding Hb to the Au NCs solution. As shown in Figure S14 in the Supporting Information, after adding Hb to the Col-Au NCs solutions, blue-emitting Au8 clusters have changed to green-emitting Au13 clusters. The results demonstrated that the fluorescence color change was really dependent on the size of the Au NC cores, that is to say, aggregation of Au NCs might also be another reason leading to the change of fluorescence color of Au NCs (from blue to green), which was in accordance with the previous report.26 Discrimination between Serums from Hepatoma Patients and Healthy People. In order to further explore the application of this sensor array to real samples, it was used to distinguish between serums from hepatoma patients and healthy people. Here, serums from hepatoma patients have already been diagnosed by the standard method of chemiluminescent microparticle immunoassay. When serums from hepatoma patients and healthy people were added to this sensor array, different FL responses will be generated. As shown in Figure 5A, the as-prepared blue-emitting Col-Au NCs and Mac-Au NCs generated different responses to the serums from normal and hepatoma patients, respectively, which could be observed by the naked eye under a UV lamp. Clinically, the content of some proteins in the serum of hepatoma patients will be altered, to a certain degree (such as Hb,48 haptoglobin (Hp),49 alpha fetoprotein,50 etc.), compared to those of the healthy people.51,52 Therefore, these different FL responses
Figure 5. Array-based sensing of human serums from five normal people, five hepatoma patients, and five thalassemia patients: (A) Photographs of the fluorescence color change upon the addition of 10 μL of human serums from five normal people (panels a, b, c, d, and e), five hepatoma patients (panels f, g, h, i, and j), and five thalassemia patients (panels k, l, m, n, and o) to the 600 μL pure Au NCs. (B) Canonical score plot for the FL response patterns, as obtained from LDA, with 95% confidence ellipses.
may result from the alteration of certain protein contents in hepatoma patients’ serums.53,54 LDA analysis revealed that serums from normal and hepatoma patients were effectively discriminated (see Figure 5B). This result, which was provided by our sensor, was consistent with those obtained by the standard method. Consequently, this sensor array could provide an auxiliary method for the clinical standard method for the distinction between hepatoma patients and healthy people, which should be beneficial for the clinical diagnosis of liver cancer. Furthermore, in order to find other applications for real clinical diagnosis with this sensor array, we use this sensor to distinguish between serums from thalassemia patients and healthy people. As shown in Figure 5B, serums from thalassemia patients and healthy people could also be discriminated. Different protein contents will change for different diseases, so this may cause the different FL responses of the sensor array. Therefore, this sensor array has the potential to discriminate serums from different diseases.
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CONCLUSIONS In summary, we have first synthesized novel blue-emitting ColAu NCs and Mac-Au NCs and successfully used them as a visual sensor array to discriminate eight proteins, based on the diverse fluorescence color and intensity responses patterns. Five experiments were performed to illustrate and validate the possible mechanism of this sensor array. Furthermore, it was also successfully applied to distinguish between serums from hepatoma patients and healthy people, showing feasible potential in the clinical diagnosis. The findings demonstrated in this study may be of interest to researchers in fields such as nanochemistry, cluster chemistry, biosensors, and biochemistry. We expect that the present study may contribute to future experiments that are focused on the synthesis of fluorescent nanomaterials with diverse colors and research on the interaction between proteins and nanomaterials. E
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Fax: +86-10-62799838. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the support from the National Nature Science Foundation of China (Nos. 21175014, 21475011) and National Grant of Basic Research Program of China (No. 2011CB915504).
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REFERENCES
(1) Bunz, U. H. F.; Rotello, V. M. Angew. Chem., Int. Ed. 2010, 49, 3268−3279. (2) Na, N.; Liu, L.; Taes, Y. E. C.; Zhang, C. L.; Huang, B. R.; Liu, Y. L.; Ma, L.; Ouyang, J. Small 2010, 6, 1589−1592. (3) Liang, S. S.; Chen, S. L.; Chen, S. H. Chem. Commun. 2011, 47, 8385−8387. (4) Laing, S.; Santana, A. H.; Sassmannshausen, J.; Asquith, D. L.; Mclnnes, I. B.; Faulds, K.; Graham, D. Anal. Chem. 2011, 83, 297−302. (5) Jenner, M.; Ellis, J.; Huang, W. C.; Raven, E. L.; Roberts, G. C. K.; Oldham, N. J. Angew. Chem., Int. Ed. 2011, 50, 8291−8294. (6) Li, X. N.; Wen, F.; Creran, B.; Jeong, Y.; Zhang, X. R.; Rotello, V. M. Small 2012, 8, 3589−3592. (7) Li, H. P.; Bazan, G. C. Adv. Mater. 2009, 21, 964−967. (8) Lu, Y. X.; Kong, H.; Wen, F.; Zhang, S. C.; Zhang, X. R. Chem. Commun. 2013, 49, 81−83. (9) Triulzi, R. C.; Micic, M.; Giordani, S.; Serry, M.; Chiou, W. A.; Leblanc, R. M. Chem. Commun. 2006, 66, 5068−5070. (10) Cai, S.; Lao, K. M.; Lau, C. W.; Lu, J. Z. Anal. Chem. 2011, 83, 9702−9708. (11) Zhang, B. C.; Lan, T.; Huang, X. Y.; Dong, C. Q.; Ren, J. C. Anal. Chem. 2013, 85, 9433−9438. (12) Kwak, K.; Kumar, S. S.; Pyo, K.; Lee, D. ACS Nano 2014, 8, 671−679. (13) Wen, Q.; Gu, Y.; Tang, L. J.; Yu, R. Q.; Jiang, J. H. Anal. Chem. 2013, 85, 11681−11685. (14) Hu, L. Z.; Deng, L.; Alsaiari, s.; Zhang, D. Y.; Khashab, N. M. Anal. Chem. 2014, 86, 4989−4994. (15) Chen, Y. N.; Chen, P. C.; Wang, C. W.; Lin, Y. S.; Ou, C. M.; Ho, L. C.; Chang, H. Y. Chem. Commun. 2014, 50, 8571−8574. (16) Sun, J.; Yang, F.; Zhao, D.; Yang, X. R. Anal. Chem. 2014, 86, 7883−7889. (17) Selvaprakash, K.; Chen, Y. C. Biosens. Bioelectron. 2014, 61, 88− 94. (18) Kong, H.; Lu, Y. X.; Wang, H.; Wen, F.; Zhang, S. C.; Zhang, X. R. Anal. Chem. 2012, 84, 4258−4261. (19) Link, S.; Beeby, A.; FitzGerald, S.; El-Sayed, M. A.; Schaaff, T. G.; Whetten, R. L. J. Phys. Chem. B 2002, 106, 3410−3415. (20) Liu, G.; Shao, Y.; Wu, F.; Xu, S.; Peng, J.; Liu, L. Nanotechnology 2013, 24, 015503. (21) Kennedy, T. A. C.; MacLean, J. L.; Liu, J. Chem. Commun. 2012, 48, 6845−6847. (22) Chevrier, D. M.; Chatt, A.; Zhang, P. J. Nanophotonics 2012, 6, 064504. (23) Yue, Y.; Liu, T.; Li, H.; Liu, Z.; Wu, Y. Nanoscale 2012, 4, 2251−2254. (24) Chen, T.; Tseng, W. Small 2012, 8, 1912−1919. (25) Guevel, X. L.; Daum, N.; Schneider, M. Nanotechnology 2011, 22, 275103. F
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A Visual Sensor Array for Pattern Recognition Analysis of Proteins Using Novel Blue-Emitting Fluorescent Gold Nanoclusters Shenghao Xu,†,# Xin Lu,‡ Chenxi Yao,† Fu Huang,§ Hua Jiang,† Wenhao Hua,∇ Na Na,† Haiyan Liu,† and Jin Ouyang*,† †
Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, People’s Republic of China # Key Laboratory of Sensor Analysis of Tumor Marker Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People’s Republic of China ‡ National Institutes for Food and Drug Control, Beijing 100050, People’s Republic of China § Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China ∇ Department of Clinical Laboratory, Beijing Ditan Hospital, Capital Medical University, Beijing 100050, People’s Republic of China S Supporting Information *
ABSTRACT: This paper describes a visual sensor array for pattern recognition analysis of proteins based on two different optical signal changes: colorimetric and fluorometric, by using two types of novel blue-emitting collagen protected gold nanoclusters and macerozyme R-10 protected gold nanoclusters with lower synthetic demands. Eight proteins have been welldiscriminated by this visual sensor array, and protein mixtures after one-dimensional polyacrylamide gel electrophoresis also could be well-discriminated. The possible mechanism of this sensor array was illustrated and validated by fluorescence spectra, X-ray photoelectron spectroscopy (XPS), fluorescence lifetime, isothermal titration calorimetry (ITC), and matrixassisted laser desorption/ionization−time-of-flight mass spectrometry (MALDI-TOF MS) experiments. It was attributed to that the adsorption of proteins onto the surface of gold nanoclusters (Au NCs), forming the protein−Au NCs complex. Furthermore, serums from normal and hepatoma patients were also effectively discriminated by this visual sensor array, showing feasible potential for diagnostic applications.
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materials, including dendrimer, DNA, glutathione and proteins, etc.18−21 Among these synthetic methods, because of the threedimensional (3D) complexed structures of proteins which can be easily conjugated with other systems, protein-directed synthesis is especially attractive.22 To date, a variety of different proteins have been developed for the preparation of red fluorescent Au NCs, such as bovine serum albumin (BSA), transferring, insulin, lysozyme, trypsin, horseradish peroxidase, and pepsin.23−28 Nevertheless, to the best of our knowledge, there has been scarce previous studies reporting on proteindirected synthesis of blue-emitting fluorescent Au NCs. Hence, the development of blue-emitting fluorescent Au NCs with lower synthetic demands, which can be used for protein discrimination still remains a challenge. In this work, we report a visual sensor array for pattern recognition analysis of proteins by utilizing two types of novel
rotein analysis plays crucial roles in many areas, such as proteome research, clinical diagnostics, and biomedical research.1−3 Traditional protein detection methods, such as Coomassie Brilliant Blue-R250 (CBB-R250) staining and silver staining, only show the same color for all target proteins and it may be also restricted because of the time-consuming procedures.4,5 Recently, as a rapid, cost-effective, and practical method for protein discrimination, sensor arrays have been successfully applied for the discrimination and recognition of proteins, including using polymers, nanoparticles, graphene oxide, carbon nanotubes, etc.6−9 However, the synthesis or surface modification of these materials are relative complicated or time-consuming. Therefore, it is still very important to develop new materials, which require simple synthetic procedures for protein discrimination. Fluorescent gold nanoclusters (Au NCs) have gained much attention, because of their burgeoning photophysical properties and potential applications in biosensing and bioimaging.10−17 Impelled by their potential applications, fluorescent Au NCs have been synthesized by utilizing many diverse protected © XXXX American Chemical Society
Received: July 17, 2014 Accepted: November 6, 2014
A
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blue-emitting fluorescent gold nanoclusters. Compared with the traditional protein discrimination sensor arrays, three obvious advantages of this sensor array make it particularly attractive: (1) These Au NCs can be rapidly synthesized within 90 s and can be directly applied as a sensor array for protein discrimination without further surface modification; (2) Protein discrimination can be achieved both by fluorescent intensity and color changes; and (3) Only two types of nanomaterials are needed to distinguish eight proteins, and protein mixtures after polyacrylamide electrophoresis could also be well-discriminated, according to the diverse color response pattern. Thus, a visual sensor array with lower synthetic efforts for pattern recognition analysis of proteins was established.
Bruke Autoflex mass spectrometer (Burker Daltonics, Bremen, Germany). All the mass spectra were collected in the reflectron and positive ion mode with an accelerating voltage of 20 kV and proceeded by Flex Analysis 3.4 software (Bruker Daltonics). Synthesis of Col-Au NCs and Mac-Au NCs. All glass bottles were washed with aqua regia and rinsed with ultrapure water. In a typical MW-assisted synthesis experiment, a solution of 40 mg/mL collagen was added to an equal volume of 2.5 mM HAuCl4, and the pH of the resulting solutions was adjusted to 12 by adding NaOH solution. The mixture then was subjected to microwave treatment at 300 W for 90 s. Thus, blue-emitting fluorescent Col-Au NCs were achieved. For the Mac-Au NCs synthesis, a solution of 40 mg/mL Macerozyme R-10 was added to an equal volume of 1.25 mM HAuCl4. The mixture then was subjected to direct microwave treatment at 300 W for 90 s. Protein Discrimination. Twenty microliters (20 μL) of 30 mg/mL target proteins were added to 2 mL of Col-Au NCs and Mac-Au NCs, respectively. They then were incubated at 37 °C for 3 h. Subsequently, the images of the different fluorescent colors were obtained by the bioimaging system at the excitation wavelength of 365 nm. At last, the fluorescent data were processed using linear discriminant analysis (LDA) in SPSS v16.0.
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EXPERIMENTAL SECTION Chemicals. All reagents were of analytical reagent grade. Collagen (Col), Macerozyme R-10 (Mac), lysozyme (Lys), human serum albumin (HSA), egg white albumin (EA), pepsin (Pep), hemoglobin (Hb), trypsin (Try), catalse (CAT), trandferrin (Tf), and tetramethylethylenediamine (TEMED) were obtained from Sigma (St. Louis, MO, USA). Tris(hydroxymethyl)aminomethane), aminoacetic acid (glycine), Bis(N,N′-methylenebisacrylamide), ammonium persulfate, acrylamide, 3′,3″,5′,5″-tetra bromophenol sulfone phthalein (Bromophenol Blue), and hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O) were purchased from Sino-American Biotechnology Co. (Beijing, China). Sodium hydroxide (NaOH) and glycerine were purchased from Beijing Fine Chemical Factory (Beijing, China). Protein markers (myoglobin, catalase, aldolase, and albumin) were supplied by GE Healthcare (Piscataway, NJ, USA). All solutions were prepared using ultrapure water from a Millipore Simplicity 185 water purification system (Millipore, Bedford, MA, USA). Serum samples of healthy people were obtained from the Affiliated Hospital of the Beijing Normal University. Serum samples of hepatoma patients were obtained from the Beijing Ditan Hospital. Serum samples from thalassemia patients were acquired from Guangzhou Children’s Hospital (Guangzhou, China). These samples were obtained with informed consent from the human subjects. In addition, this sample collection was approved by the Institutional Review Board of Beijing Ditan Hospital. Instruments. An advanced microwave digestion system was used for the microwave pyrolysis (Milestone, USA). Fluorescence spectra was recorded on a fluorescence spectrometer (Perkin−Elmer, Model LS55). High-resolution transmission electron microscopy (HRTEM) images were taken on a JEOL TEM 2100 microscope operating at 200 kV. X-ray photoelectron spectroscopy (XPS) was carried out on a thermoelectron instrument (Thermo-VG Scientific ESCALAB 250). Time-resolved fluorescence measurements were performed using an OB920 single-photon counting fluorometer (Edinburgh Analytical Instruments) with a pulsed nanosecond nitrogen lamp as excitation source. All spectra were referenced to the C 1s peak at 285.0 eV. Dynamic light scattering (DLS) experiment was performed on a Zetasizer Nano-ZS (Malvern Instruments, Malvern, U.K.). The electrophoresis systems consisted of DYCZ-24D and DYCZ-21 vertical electrophoresis tanks (Liuyi Instrument Factory, Beijing, China). The fluorescence images were recorded by a bioimaging system (UVP EC3 Imaging System, UVP, Inc., USA) at the wavelength of 365 nm. MALDI-TOF MS analysis was performed on the
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RESULTS AND DISCUSSION Characterization of Col-Au NCs and Mac-Au NCs. Figures 1A-a and 1B-a present typical transmission electron microscopy (TEM) images of the as-prepared Col-Au NCs and Mac-Au NCs, appearing as spherical particles with good monodispersity. Moreover, the well-resolved lattice planes with a spacing of ∼0.23 nm in the HRTEM image (inset in Figure 1A-a and B-a) demonstrates the excellent crystalline
Figure 1. (A-a) Typical TEM image of Col-Au NCs. The top and bottom insets display size distribution and the HRTEM image of ColAu NCs, respectively. (A-b) Fluorescence excitation (λex = 330 nm, blue line) and emission (λem= 420 nm, red line) spectra of the Col-Au NCs; inset shows a photograph of the Col-Au NCs under sunlight (left) and 365-nm UV light illumination (right). (A-c) XPS spectrum of Au 4f for Mac-Au NCs. (B-a) Typical TEM image of Mac-Au NCs; the top and bottom insets display size distribution and the HRTEM image of Mac-Au NCs, respectively. (B-b) Fluorescence excitation (λex = 366 nm, blue line) and emission (λem = 440 nm, red line) spectra of the Mac-Au NCs, the inset is the photograph of the Mac-Au NCs under sunlight (left) and 365 nm UV light illumination (right). (B-c) XPS spectrum of Au 4f for Mac-Au NCs. B
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structure of the as-prepared Au NCs, which was consistent with the previous report.29 The diameter histograms in Figure 1A-a and 1B-a, calculated by measuring more than 100 particles in the TEM image, reveal that the Col-Au NCs and Mac-Au NCs have average sizes of 1.4 and 2.0 nm, respectively, which was in accordance with the DLS data (see Figure S1 in the Supporting Information). The as-prepared two Au NCs showed a broad absorption band and the absence of localized surface plasmon resonance bands, suggesting the formation of the Au NCs (see Figure S2 in the Supporting Information). In addition, the fluorescence emission were also investigated to confirm the formation of Au NCs. As shown in Figures 1A-b and 1B-b, the emission peak of the as-prepared Col-Au NCs and Mac-Au NCs was located at 420 and 440 nm, respectively. This suggests the presence of Au8 clusters according to the spherical Jellium model,30 which are consistent with those of Au8 nanoclusters reported in the previous studies.31 Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was performed to confirm the production of Au8 clusters (see Figure S3 in the Supporting Information). The quantum yield (QY) was 3.1% and 4.2% (calibrated with quinoline as the reference), and the fluorescence lifetime was 2.65 and 5.82 ns for Col-Au NCs and Mac-Au NCs, respectively (see Figure S4 in the Supporting Information). In order to obtain additional information about the as-prepared Au NCs, X-ray photoelectron spectroscopy (XPS) was conducted and the results are shown in Figures 1A-c and 1Bc. The binding energies (BEs) of the Au 4f5/2 and Au 4f7/2 peaks are 85.18 and 88.58 eV for Col-Au NCs and 84.48 and 87.93 eV for Mac-Au NCs, inferring that both Au0 and Au+ exist in the Au NCs.32 Furthermore, these Au NCs are relatively stable and the fluorescence quenching only occurred slightly after one month of storage in darkness at 4 °C (see Figure S5 in the Supporting Information). In addition, a systematic study was performed to explore the optimal conditions (e.g., reaction time and the concentrations of Col, Mac, HAuCl4, and NaOH) to prepare the fluorescent Col-Au NCs and Mac-Au NCs (see Figures S6 and S7 in the Supporting Information). Meanwhile, we also investigated whether red-shifted Au NCs could be synthesized by changing five different molar ratio of Au/protein (0.02, 0.04, 0.08, 0.16, and 0.33), using Col-Au NCs as an example. However, as shown in Figure S8 in the Supporting Information, red-shifting still could not be observed. We could only observe the change of the fluorescence intensity. Protein Discrimination by Linear Discriminant Analysis (LDA). As a proof-of-concept system, we select eight proteins that have diverse isoelectric points and molecular weights as the sensing targets (see Table S1 in the Supporting Information). As shown in Figure 2A, after adding different target proteins, the as-prepared blue-emitting Col-Au NCs and Mac-Au NCs gave diverse responses to the target proteins and caused a various fluorescent color change, which could be observed by the naked eye. These color differences could provide rough discriminations among the target proteins. For example, HSA, Hb, and Tf caused visible color changes of ColAu NCs from blue to red, green, and pink, respectively. Similarly, these three target proteins caused visible color changes of Mac-Au NCs from blue to pink, yellow, and purple, respectively. In other words, after adding different target proteins, it will also cause different wavelength shifts. These different wavelength shifts, which act as “fingerprints”, encouraged us to exploit a pattern recognition approach for protein discrimination. Therefore, linear discriminate analysis
Figure 2. Array-based sensing of eight proteins: (A) photographs of the fluorescence color change upon addition of protein solutions at 300 μg/mL; (B) canonical score plot for the wavelength shift as obtained from LDA with 95% confidence ellipses; and (C) canonical score plot for the FL response patterns as obtained from LDA with 95% confidence ellipses.
(LDA), which is a statistic approach,33 was used to differentiate the wavelength shift of the Col-Au NCs and Mac-Au NCs with target proteins. For each protein, we tested its wavelength shift against the two Au NCs three times, generating a matrix of 2 Au NCs × 8 proteins × 3 replicates. LDA analysis transforms the matrix into canonical factors and the two factors can be visualized in a two-dimensional (2D) plot shown in Figure 2B under the 95% confidence ellipses. As shown in Figure 2B, all target proteins were separated from each other, demonstrating that they were effectively discriminated by LDA, based on wavelength shift. Moreover, adding different target proteins to the Col-Au NCs and Mac-Au NCs could also result in diversity of fluorescent intensity (FL) responses (see Figure S9 in the Supporting Information). (Here, the change of the fluorescence intensity refers to the fluorescent intensity change before and after adding the target proteins to the Au NCs. Before adding target proteins to the Au NCs, the fluorescence intensity was the intensity of the Au NCs. After adding target proteins to the Au NCs, the target proteins adsorbed onto the surface of Au NCs, forming the target protein−Au NCs complexes. The fluorescence intensity was the intensity of the target protein− Au NCs complex. For example, the emission peak of Au NCs used in Figure2B was 630 nm.) Similarly, these different FL responses could also be a “fingerprint” map for the protein discrimination. As shown in Figure 2C, all target proteins were separated from each other demonstrating that they were effectively discriminated according to the diverse fluorescent intensity (FL) responses. It is noteworthy that the quantum yield of Mac-Au NCs (4.2%) is higher than that of Col-Au NCs (3.1%). However, in Figure 2A, the emission intensity of MacAu NCs is weaker than that of Col-Au NCs. We presented the relevant explanation in the Supporting Information (Figure S10). In addition, target proteins could still be discriminated with a low protein concentration (10 μg/mL) (see Figure S11 in the Supporting Information). Moreover, protein mixtures after polyacrylamide electrophoresis could also be welldiscriminated, according to the diverse color response pattern (see Figure S12 in the Supporting Information). Discussion of the Possible Mechanism. Subsequently, we studied the possible mechanism, taking Col-Au NCs as an C
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example. First, in order to understand the interaction between Au NCs with target proteins, an isothermal titration calorimetry (ITC) experiment using Col-Au NCs and HSA was conducted as an example. As we know, the thermodynamic parameters ΔH and ΔS could be used to describe the binding affinity between Au NCs and target proteins, which can be classified into different models.34 In detail, the interaction is mainly attributed to hydrophobic bonds when ΔH > 0 and ΔS > 0, while the interaction is mainly attributed to van der Waals forces and hydrogen bonding when ΔH < 0 and ΔS < 0. Furthermore, the interaction is mainly attributed to electrostatic interactions when ΔH ≈ 0 and ΔS > 0.34 In this experiment, as shown in Figure 3, ΔH1 < 0, ΔS1 < 0, K1 = 7.24
Figure 4. (A) Fluorescence emission spectra of the Col-Au NCs after adding different concentrations of HSA; inset shows the corresponding photos under UV light. (B) Fluorescence emission spectra of ColAu NCs (spectrum a), Col-Au NCs + CAT (spectrum b), Col-Au NCs + Hb (spectrum c), and Col-Au NCs + HSA (spectrum d). (C) XPS spectrum of Au 4f for Col-Au NCs (spectrum a), Col-Au NCs + CAT (spectrum b), Col-Au NCs + Hb (spectrum c), and Col-Au NCs + HSA (spectrum d). (D) Fluorescence lifetime of the Col-Au NCs (spectrum a), Col-Au NCs + CAT (spectrum b), Col-Au NCs + Hb (spectrum c), and Col-Au NCs + HSA (spectrum d), respectively.
solution (1.25 mM, 600 μL), the emission peak of Col-Au NCs at 420 nm was gradually decreased while a new emission peak at 630 nm appeared and gradually increased with increasing HSA concentration. We noticed that the emission peak of ColAu NCs at 420 nm was almost disappeared when the concentration of HSA was higher than 2.2 mg/mL (0.032 mM). Meanwhile, the intensity of the new emission peak at 630 nm was almost no longer increased. It was concluded that the adsorption process had a tendency to be saturated when the concentration of HSA was higher than 2.2 mg/mL (0.032 mM). That is to say, 0.16 × 10−9 mol HSA absorbs on the surface of 750 × 10−9 mol Au NCs, forming protein−Au NC complexes. In other words, 0.0002 mol HSA absorbs on the surface of 1 mol Au NCs forming protein−Au NC complexes. In addition, the HSA-induced fluorescence color change (from blue to red) was so obvious that it could be easily observed by the naked eye under an ultraviolet (UV) lamp (see the inset in Figure 4A). Since HSA is nonfluorescent in this spectral region, we speculated that the generation of the new fluorescent peak resulted from the adsorption of HSA onto the surface of ColAu NCs, forming the HSA-Au NCs complex. Similarly, new emission peaks at 450 and 510 nm also appeared after adding CAT and Hb, respectively, to the Col-Au NCs (see Figure 4B). To support this result, first, XPS experiment was carried out to verify this speculation. As shown in Figure 4C, after adding the same concentrations of CAT, Hb, and HSA, respectively, the binding energy of the Au 4f5/2 and Au 4f7/2 decreased by varying degrees. According to the previous reports, a red shift would happen when the energy decreases.38,39 This might be the reason for the resulting fluorescence red shift from 420 nm to 455 nm (blue color), 520 nm (green color), and 630 nm (red color), leading to the fluorescence color changes. The
Figure 3. Isothermal titration calorimetry (ITC) profile for Col-Au NCs titration into HSA at 310 K. The concentration of Col-Au NCs is 0.5 mM and the concentration of HSA is 0.03 mM.
× 105 M−1, and ΔH2 > 0, ΔS2 > 0, K2 = 3.62 × 103 M−1, The spontaneity for ΔH1 < 0 and ΔS1 < 0 might be that when Au NCs insert into the binding sites of hydrophobic part of target proteins, the hydrophobic interactions could lead to exothermic phenomenon. In addition, the rigid structure of target proteins may also be destroyed leading to the exothermic phenomenon.34 At the same time, The spontaneity for ΔH1 > 0 and ΔS1 > 0 might be that when Au NCs close to the binding sites of target protein, the hydration layer of the target protein around the binding sites are partly damaged, resulting in heat absorption phenomenon.35 This indicates that Col-Au NCs and HSA interacted with a hydrophobic force mainly via hydrogen bonding and van der Waals interactions. Therefore, we speculated that the target proteins are adsorbed onto the surface of Au NCs, forming the target protein−Au NCs complexes. In addition, previous works have been reported that the photophysical properties of the Au NCs could be altered when proteins adsorbed to its surface, forming the target protein− NCs complexes.36,37 As shown in Figure 4A, after adding different concentrations of HSA (5 μL) to Col-Au NCs D
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binding energy shift strongly support our claim that the photophysical properties of the Au NCs are directly altered by proteins binding to the Au NCs surface,40 forming the protein− Au NCs complexes. Furthermore, fluorescence lifetime measurement was also performed to further illustrate the underlying mechanism. As shown in Figure 4D, the average lifetime of the Col-Au NCs increased from 2.65 ns to 3.85, 4.17, and 1.22 μs, respectively. This change might originate from the longest-lifetime component, associated with the Au(I)−thiol complex on the particle surface.41 Moreover, the interactions between the target protein and the Au NCs surface could affect the luminescence process of the Au(I)−thiol complex.41,42 Furthermore, upon target protein adsorption onto the Au NCs, the binding energy shift of Au 4f 5/2 and Au 4f7/2 may be another reason for the resulting variation in the lifetimes of Au NCs.40 This result also indicated that the photophysical properties of the Au NCs was altered by the interactions between target proteins and the Au NCs surface,43 forming the target protein− Au NCs complexes. Furthermore, according to the spherical Jellium model, the wavelength of fluorescent AuNCs is also dependent on the size of the Au NC cores.30 In detail, smaller Au NCs emit at shorter wavelengths, while larger Au NCs emit at longer wavelengths (e.g., blue for Au8, green for Au13, and red for Au25 emission). As shown in Figure S13 in the Supporting Information, the average particle size of the protein−Au NCs complex gradually increased. According to the previous report, compared with other target proteins, HSA possesses a relatively large internal space at alkaline pH values.24,44−46 Therefore, it could provide larger internal space to assemble Au8 clusters, eventually forming Au25 clusters.24,26 This may be the reason for the relatively large size distribution of Col-Au NCs + HSA. Meanwhile, this phenomenon was also consistent with the previous research that the binding energy of Au NCs decreases with increasing cluster size.47 We also have used MALDI-TOF MS to characterize the atomic composition of the Au NC cores before and after adding Hb to the Au NCs solution. As shown in Figure S14 in the Supporting Information, after adding Hb to the Col-Au NCs solutions, blue-emitting Au8 clusters have changed to green-emitting Au13 clusters. The results demonstrated that the fluorescence color change was really dependent on the size of the Au NC cores, that is to say, aggregation of Au NCs might also be another reason leading to the change of fluorescence color of Au NCs (from blue to green), which was in accordance with the previous report.26 Discrimination between Serums from Hepatoma Patients and Healthy People. In order to further explore the application of this sensor array to real samples, it was used to distinguish between serums from hepatoma patients and healthy people. Here, serums from hepatoma patients have already been diagnosed by the standard method of chemiluminescent microparticle immunoassay. When serums from hepatoma patients and healthy people were added to this sensor array, different FL responses will be generated. As shown in Figure 5A, the as-prepared blue-emitting Col-Au NCs and Mac-Au NCs generated different responses to the serums from normal and hepatoma patients, respectively, which could be observed by the naked eye under a UV lamp. Clinically, the content of some proteins in the serum of hepatoma patients will be altered, to a certain degree (such as Hb,48 haptoglobin (Hp),49 alpha fetoprotein,50 etc.), compared to those of the healthy people.51,52 Therefore, these different FL responses
Figure 5. Array-based sensing of human serums from five normal people, five hepatoma patients, and five thalassemia patients: (A) Photographs of the fluorescence color change upon the addition of 10 μL of human serums from five normal people (panels a, b, c, d, and e), five hepatoma patients (panels f, g, h, i, and j), and five thalassemia patients (panels k, l, m, n, and o) to the 600 μL pure Au NCs. (B) Canonical score plot for the FL response patterns, as obtained from LDA, with 95% confidence ellipses.
may result from the alteration of certain protein contents in hepatoma patients’ serums.53,54 LDA analysis revealed that serums from normal and hepatoma patients were effectively discriminated (see Figure 5B). This result, which was provided by our sensor, was consistent with those obtained by the standard method. Consequently, this sensor array could provide an auxiliary method for the clinical standard method for the distinction between hepatoma patients and healthy people, which should be beneficial for the clinical diagnosis of liver cancer. Furthermore, in order to find other applications for real clinical diagnosis with this sensor array, we use this sensor to distinguish between serums from thalassemia patients and healthy people. As shown in Figure 5B, serums from thalassemia patients and healthy people could also be discriminated. Different protein contents will change for different diseases, so this may cause the different FL responses of the sensor array. Therefore, this sensor array has the potential to discriminate serums from different diseases.
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CONCLUSIONS In summary, we have first synthesized novel blue-emitting ColAu NCs and Mac-Au NCs and successfully used them as a visual sensor array to discriminate eight proteins, based on the diverse fluorescence color and intensity responses patterns. Five experiments were performed to illustrate and validate the possible mechanism of this sensor array. Furthermore, it was also successfully applied to distinguish between serums from hepatoma patients and healthy people, showing feasible potential in the clinical diagnosis. The findings demonstrated in this study may be of interest to researchers in fields such as nanochemistry, cluster chemistry, biosensors, and biochemistry. We expect that the present study may contribute to future experiments that are focused on the synthesis of fluorescent nanomaterials with diverse colors and research on the interaction between proteins and nanomaterials. E
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Fax: +86-10-62799838. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the support from the National Nature Science Foundation of China (Nos. 21175014, 21475011) and National Grant of Basic Research Program of China (No. 2011CB915504).
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