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Synthesis of Gold Nanoparticles from Au(I) Ions Shuttle to Solidify: Application on the Sensor Array Design Yumin Leng, Kai Jiang, Wentai Zhang, and Yuhui Wang Langmuir, Just Accepted Manuscript • Publication Date (Web): 05 Jun 2017 Downloaded from http://pubs.acs.org on June 6, 2017

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Synthesis of Gold Nanoparticles from Au(I) Ions Shuttle to Solidify: Application on the Sensor Array Design Yumin Leng*†,‡, Kai Jiang‡, Wentai Zhang†, and Yuhui Wang‡

†

College of Physics and Electronic Engineering, Nanyang Normal University, Nanyang 473061,

China ‡

Ningbo Institute of Materials Technology & Engineering (NIMTE), Chinese Academy of

Sciences, Ningbo 315201, China

@ Supporting Information

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ABSTRACT: Metal-mediated interlocking rings won the 2016 Nobel Prize in Chemistry. The metal-directed interlocking rings in macromolecular systems (e.g. proteins) may be similar to the form of Maxwell's electromagnetic waves, the metal ions may shuttle among the rings in the special environment. To verify this hypothesis, we designed a general approach to synthesize the multicolor gold nanoparticles (GNPs) mediated by Au(I)-directed interlocking rings in proteins. The Au(I) ions shuttled among these interlocking rings in the strong alkaline solution. Through the rapid nucleation method, the multicolor GNPs of different morphology and sizes were synthesized in the multiple honeycombed templates. Based on the “three colors” principle of Thomas Young, we extracted the red, green, and blue (RGB) alterations of GNPs to fabricate a visual sensor array for protein discrimination. The fingerprints (∆RGB) were obtained from the target proteins and fed into computer programs. The proposed sensing platform was also applied to detect lysozyme in human tears with satisfactory results. Importantly, we forecasted that lysozyme could be the effective drug for curing the dacryocystitis and nasolacrimal duct obstruction diseases.

KEYWORDS: Au(I)-directed interlocking rings, ions shuttle, gold nanoparticles, proteins, wave, sensor array


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INTRODUCTION Professors Sauvage and Stoddart, the winners of 2016 Nobel Prize in chemistry, synthesized a series of catenates mediated by metal ions.1-4 For example, the dimetallic catenates were prepared through Cu(I)-template assisted synthesis.1 Sauvage et al forecasted that the metal-mediated interlocking rings could in principle be extended to polymers,4 and the macromolecular systems incorporating interlocking rings would lead to interesting systems.5 The metal-directed interlocking rings in macromolecular systems (e.g. proteins) may be similar to the form of Maxwell's electromagnetic waves, as illustrated in Scheme 1. Inspected by the molecular shuttle principle,6 the metal ions may shuttle among the rings under the special environment (e.g. strong alkaline solution). To verify this hypothesis, we designed a general approach to synthesize the multicolor gold nanoparticles (GNPs) mediated by Au(I)-directed interlocking rings in proteins. Actually, the Au(I) ions shuttled among the interlocking rings in the strong alkaline solution. Through microwave boiling the solution, the multicolor GNPs of different morphology, size and distribution were synthesized in the honeycombed templates. The facile strategy from Au(I) ions shuttle to solidify to be GNPs with various colors should enable new approaches to color modulation, nanomaterial engineering and sensing.

Scheme 1. Metal-directed interlocking rings in macromolecular systems (e.g. proteins) may be similar to the form of Maxwell's electromagnetic waves. Owing to the optical characters, GNPs are the natural and promising candidates for visual sensor arrays, and has been exploited for wide detection of heavy metal ions, organic molecules, proteins, and so on.7-12 Recently, GNPs-based multichannel sensing devices have been constructed


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for pattern recognition analysis of macromolecules (especially proteins).13-18 For example, Rotello et al developed a series of multichannel sensor arrays for recognition and analysis of macromolecules (including proteins) based on fluorescent polymer or proteins modified GNPs.13,14,18 Liu et al reported a multichannel sensing array for pattern recognition of proteins based on DNA sequences modified gold nanoparticles (DNA−GNPs).17 These efforts have made great progress on the development of GNPs-based sensing array for protein recognition. However, in these GNPs-based colorimetric assays, surface modifications are indispensable, which greatly limit their further applications. In our experiment, the synthesis process of multicolor GNPs is obviously avoiding complicated surface modification. Thereupon we are interested to design a multichannel sensor array for protein discrimination based on the “three colors” principle of Thomas Young, that is “all colors are mixed by red (R), green (G), and blue (B) in different proportions”. The RGB data were extracted from multicolor GNP which is corresponding to a certain protein, and further fed into the Multi-Variate Statistical Package (MVSP) computing software.19 Then the proteins were quantitatively distinguished by using the statistical methods (e.g. hierarchical clustering analysis (HCA) and principal component analysis (PCA)).20,21 As for real application, the proposed sensor array is successfully applied to monitor the level of Lysozyme (Lys) in human tears. RESULTS AND DISCUSSION Preparation of Au(I)-directed Interlocking Rings in Protein. As shown in Figure 1, the preparation of Au(I)-directed interlocking rings involves incubating the protein (e.g. pepsin (Pep) or trypsin (Try)) with Au(III) (e.g. [AuCl4]-) in strong alkaline solution (See methods)22 to produce the colorless solution. As shown in Figure S1 (Supplementary Information (SI)), the maximum absorption wavelength (i.e. 246 nm) of the colorless solution agrees well with that of Au(I) anions (e.g. [AuCl2]-).23,24 The Au(III) complexes are reduced by the protein to be the aqueous Au(I) anions in the alkaline solution.22 According to the principle developed by professors Sauvage and


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Stoddart,1-5 the Au(I) ions should mediate the amino acid residues present in the protein to be interlocked rings, as shown in Figure 1 and Figure S2 (SI). The multiple covalent bonds of Au-O and Au-N are formed between Au(I) ions and the functional groups of proteins (See Figure S2 in SI).

Figure 1. Schematic illustration of preparation of Au(I)-directed interlocking rings in proteins and synthesis of GNPs from Au(I) ions shuttle to solidify. The protein (e.g. Pep and Try) and Au(III) are firstly incubated in strong alkaline solution to generate the Au(I)-protein (e.g. Au(I)-Pep and Au(I)-Try) complex. Addition of the other proteins prompts Au(I) anions shuttling from Pep/Try to other proteins. The interlocked rings of Au(I)-Pep-protein are subjected to microwave reaction to synthesize GNPs, which are produced in the honeycombed templates. Synthesis of GNPs from Au(I) Ions Shuttle to Solidify. Inspected by the molecular shuttle principle,6 the metal ions may shuttle among the rings under the special environment (e.g. strong alkaline solution). To demonstrate this hypothesis, we designed the experiment to synthesize the multicolor GNPs, which might be produced in the honeycombed templates achieved from Au(I) ions shuttle to solidify. To prompt Au(I) ions shuttle, the synthesized Au(I)-Pep/Try complexes are mixed with other proteins, the Au(I) ions shuttled from Pep/Try to other proteins (See Figure 1). The multiple interlocking rings were formed through Au(I) ions directing (See Figure 1). The strong interactions between Au(I) ions and the functional groups of proteins (e.g. -NH and -CO), help the Au(I) ions in rings to be the cores, which could be deposited by the free Au(I) ions. - 5Plus - Environment ACS Paragon

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Through the rapid nucleation method, the Au(I) ions were reduced by the reductive proteins to be GNPs,8 which were synthesized in the honeycombed templates (See TEM images in Figure 2 and Figure S3 (SI)) and show different colors (see Figure 3).

Figure 2. TEM images at different magnifications of typical GNPs distributed in the honeycombed templates. Insets: the interplanar distances of GNPs determined by using ImageJ software. The protein templates might be similar to the honeycombed substrates.25,26 The circular protein template is proved by the distribution of as-synthesized GNPs (See TEM images in Figure 2). The diverse colors of GNPs are attributed to the different sizes and shapes of GNPs synthesized in the multiple honeycombed templates (See Figure 1, Figure 2 and Figure S3 in SI). As shown in the TEM images of GNPs synthesized from Au(I)-Pep in presence of proteins (e.g. Glu, BHb, Cat and Try), the sizes of GNPs decreases in the order from 4.8 ± 1.1 nm for GNPs-Pep-Glu, 4.6 ± 0.59 nm for GNPs-Pep-BHb, 2.7 ± 0.53 nm for GNPs-Pep-Cat, to 2.5 ± 0.46 nm for GNPs-Pep-Try. The sizes of as-synthesized GNPs are slightly bigger than that of gold nanoclusters.27-29 The small sized GNPs of different colors have some advantages in biomedical applications (e.g. drug deliver). The characterization of UV-vis absorption spectra was performed to reveal the reason for GNPs exhibiting multicolors. The UV-vis absorption spectra of GNPs are found to be characteristic (See Figure S4 in SI). Moreover, the functional groups (e.g. –SH and –NH2) induce proteins to interact


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with GNPs via Au-S and Au-N interactions, their stretching vibrational modes appear in the region of 250 ± 30 cm-1 (See Figure S5 in SI), which are in good agreement with the reported data.30,31 The GNPs are also investigated by using High-resolution TEM (HRTEM), as shown in the insets of Figure 2 and Figure S3 (SI). The HRTEM images illustrate clear lattice fringes separated by 0.20 nm, which can be assigned to the interplanar distance of the (200) plane of GNPs with the face-centered cubic (fcc) structure.32 The strategy to synthesize GNPs is expected to prepare the other noble metal nanoparticles (e.g. platinum, silver and alloy nanoparticles).

Figure 3. The multicolor GNPs synthesized from Au(I) ions shuttle to solidify. The Au(I) ions shuttle from Au(I)-Pep (or Au(I)-Try) to other proteins, and are reduced by proteins to be multicolor GNPs through microwave boiling. The different sizes and shapes of GNPs are solidified in the multiple honeycombed templates. Note: the tested proteins (40 µg/mL) are bovine hemoglobin (BHb), catalase (Cat), glucoamylase (Glu), Try, bovine albumin (BA), Lys, bovine serum albumin (BSA), collagen (Col) and Pep. Construction of Visual Sensor Array for Protein Discrimination. The excellent optical properties of the as-prepared GNPs, show us that they are the perfect and prospective candidates for visual sensing applications. Considering that the content of proteins in human body fluids is the most sensitive indicators for early diagnosis of specific diseases.33-37 We are interested in building a portable and low-cost multichannel sensing device to identify protein balances in human body fluids (e.g. tears). We are interested to design a multichannel sensor array based on the “three


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colors” principle of Thomas Young. We extracted the RGB data of synthesized GNP, which is corresponding to a certain protein, and further fed them into the MVSP computing software, then quantitatively differentiated proteins by using the statistical methods (e.g. HCA and PCA).19-21

Figure 4. Construction of visual sensor array for discrimination of proteins (0.4 µg/mL). Digital photographs of the Au(I)-Pep and Au(I)-Try complexes in the absence (named “before”) and presence (named “after”) of proteins after microwave reaction. For visualization, the color ranges of these difference maps are expanded from 4 to 8 bits per color (RGB range 4−19 expanded to 0−255). For qualitative comparisons of the color responses of the Au(I)-Pep/Try complexes (as indicators) to the target proteins at a much lower concentration (0.4 µg/mL), a scanner is applied to record digital photographs. As shown in Figure 4, from the color images of the as-developed indicators in the absence (named “before”) and presence (named “after”) of proteins after microwave reaction, the obvious color differences are observed to the specific target proteins. The visual sensor arrays seem to be successful at differentiating the target proteins (0.4 µg/mL) as shown in the difference maps, which are obtained through taking the RGB variations from the “before” and “after” images. The unique pattern of difference maps is corresponding to a certain


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protein (See Figure 4). The observations demonstrate that the Au(I)-Pep/Try can potentially determine the target proteins (0.4 µg/mL) via constructing the multichannel visual sensing array.

Figure 5. Fingerprints of 9 selected proteins based on the patterns of the ∆R1G1B1 and ∆R2G2B2 values obtained from the Au(I)-Pep/Try complexes in the presence of the target proteins (0.4 µg/mL) after microwave reaction. All experiments were run in quadruple trials. Quadruplicate data are obtained to evaluate the repeatability of the multichannel visual sensor array to detect the target proteins. As shown in Figure 5, the fingerprints (∆RGB variations) of 9 selected proteins are distinct, suggesting the feasibility of protein identification using the constructed sensor array. The RGB variations of the as-developed indicators in the absence and presence of nine proteins after microwave reaction are listed in Table S1 (SI). The color alterations of the most proteins response to the Au(I)-Pep complex (∆R1G1B1) are positive, whereas to the Au(I)-Try complex (∆R2G2B2) are negative (See Figure 5 and Table S1). To expose the ∆RGB alterations more clearly, the three-channel response patterns are also explored by PCA, which is a statistical treatment used to reduce multichannel data for easier interpretation.20 The canonical colorimetric response patterns (6 channels × 9 proteins × 4 replicates) are clustered into several groups (See Figure 6a). The resulting 2D canonical score plot (Figure 6a) shows clear clustering of the data using the first two principal components (representing 90.8% of the total variance), with excellent discriminatory capacity. The PCA plots for various proteins are not random, but - 9Plus - Environment ACS Paragon

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rather follow certain patterns. As shown in Figure 6a, all the target proteins (0.4 µg/mL) are separated from each other, demonstrating that they are effectively discriminated by PCA based on both the ∆R1G1B1 and ∆R2G2B2 values. The as-developed indicators in the presence of the nine proteins at 0.4 µg/mL are also analyzed using HCA, which is a model-free method based on the grouping of the analyte vectors according to their spatial distances (i.e. Euclidean distances) in their full vector space.19,21 All of the 36 cases (9 samples × 4 replicates) are correctly assigned to their respective groups (Figure 6b). It is noteworthy that the as-developed multichannel sensor array can differentiate the target proteins at 0.4 µg/mL, which is quite lower than the discrimination concentrations at 300 µg/mL and 2 mg/mL acquired by other sensor arrays.9,15

Figure 6. (a) PCA plot and (b) HCA analysis for the discrimination of proteins at 0.4 µg/mL based on the ∆R1G1B1 and ∆R2G2B2 variations. All experiments were run in quadruple trials. Discrimination of Lys in Human Tears. Considering that the level of Lys in human tears is directly related to the diseases of dacryocystitis and nasolacrimal duct obstruction.33-36 We are interested in performance testing the constructed visual sensing device to identify Lys content in human tears. As shown in Figure 7a, comparing with the color responses of Au(I)-Pep/Try to the concentrations of Lys spiked in pure water, the content of Lys in human tear should be in the range of 0.7 – 1.0 mg/mL. The excellent linear relationship between the euclidean distances (EDs = [∆R2 + ∆G2 + ∆B2]1/2) and concentrations of Lys spiked in pure water is given in Figure 7b. Based on the linear relationship and the ED data obtained from human tears, we calculate that the content of


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Lys in human tears is 0.81 ± 0.10 mg/mL. We compare the result with a laboratory grade standard of UV-vis spectrophotometer, which determines the content of Lys in human tear to be 0.83 ± 0.13 mg/mL (See Table 1 and Figure S6 in SI). As listed in Table 1, the obtained data by the asdeveloped sensing platform are in good agreement with the measured data by UV-vis spectrophotometer and the reported value range,38 which strongly prove the high reliability of our designed visual sensor array. Interestingly, it is reported that a linear decline of Lys with age,38 forecasting that Lys could be the effective drug for curing the diseases of dacryocystitis and nasolacrimal duct obstruction.

Figure 7. The constructed visual sensing array for detection of Lys in human tears. (a) Images of the Au(I)-Pep/Try complexes before and after exposure to concentrations of Lys in water and human tears after microwave reaction, and the color difference maps (RGB range 4−19 expanded to 0−255). (b) The total EDs vs concentrations of Lys spiked in water. Note: The calculation method for the content of Lys in human tear: YED

of tear

= 38.77 ± 0.89 = 31.56 +

0.89×X → X = 8.1 ± 1.0 µg/mL → The content of Lys in human tear = (8.1 ± 1.0) µg/mL×100 = 0.81 ± 0.10 mg/mL. Note: the human tear is diluted 100 times by the analytical process. Table 1. Determination of Lys in human tears by using the as-developed visual sensing platform. [Lys] (mg/mL) *

Sample Human tear

Found Naked eye Sensor array 0.7 – 1.0 0.89 ± 0.11

UV-vis

Ref38

0.83 ± 0.13

0.72 – 1.10

*

mean ± standard deviation, n=3.


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CONCLUSIONS In summary, we report a conceptually new strategy to synthesize the multicolor GNPs mediated by Au(I)-directed interlocking rings in proteins. The Au(I) ions shuttled among the interlocking rings in the strong alkaline solution. The Au(I)-directed interlocking rings in proteins may be similar to the form of Maxwell's electromagnetic waves. The GNPs were prepared from Au(I) ions shuttle to solidify through microwave boiling. The strategy is expected to synthesize the other noble metal nanoparticles (e.g. platinum, silver and alloy nanoparticles). Moreover, the facile strategy to prepare multicolor GNPs should enable new approaches to nanomaterial engineering, color modulation and sensing. The as-proposed multichannel visual sensing method based on the principle “three colors” of Thomas Young, shows the rapid, sensitive and efficient differentiation analysis of proteins. Compared with the reported sensing arrays, the as-designed visual sensing array presents the unprecedented advantages. Firstly, the detection does not require complicated modification and expensive instrumentation, which increases the operability and reduces the cost potentially improving the standard of living in resource-constrained areas. Secondly, the developed sensor array provides a new way to detect the proteins contained in the complex matrix (e.g. blood, urine and milk), showing great promise for biomedical diagnostics and milk safety in the future. Significantly, we forecast lysozyme would be the effective drug for curing the dacryocystitis and nasolacrimal duct obstruction diseases. The calculation to prove the Au(I)-directed interlocking rings (See Figure 1) in proteins will be performed in future research. EXPERIMENTAL SECTION Materials and Instruments. Pep, Try, BHb, BA, Lys and chloroauric acid tetrahydrate (HAuCl4•4H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). BSA was obtained from Gibco (Grand Island, USA). Cat, Col and Glu were bought from Aladdin Reagent Co. Ltd (Shanghai, China). 96-well plates (Corning 3632) were obtained from Genetimes Technology. - 12Plus - Environment ACS Paragon

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For the fabrication of visual sensing array, color images were taken with a scanner in 96-well polystyrene plates. The absorption spectra of GNPs were measured using a UV-vis spectrophotometer from Perkin Elmer (Lambda 950). TEM samples were prepared on coppersupported carbon grids by depositing a drop of solution and allowing it to dry. TEM images were recorded with a Tencai F20 instrument operating at an acceleration voltage of 200 kV. FT-IR spectroscopy was recorded with a Nicolet 6700 spectrometer. Preparation of Au(I)-directed Interlocking Rings in Proteins. The protein (e.g. Pep and Try) and HAuCl4 were incubated at alkaline pH to produce the colorless solution of Au(I) ions. The Au(I) should mediate Pep (or Try) to be interlocking rings.1-5 The final concentrations of Au(I) and NaOH in the mixture is 0.5 mM and 0.5 M, respectively. The optimal concentrations of Pep and Try at 0.4 mg/mL were used throughout the experiment. The colorless solution of Au(I)-Pep/Try was collected and stored at 4 °C for further use. Synthesis of Multicolor GNPs from Au(I) Ions Shuttle to Solidify. To prompt Au(I) ions shuttle, BHb, Cat, Glu, Try, BA, Lys, BSA, Col and Pep (20 µL, 4 mg/mL) was added to the colorless solution of Au(I)-Pep/Try (2 ml), respectively. To solidify and reduce Au(I) ions, the mixture was subjected to microwave treatment at 300 W for 90 s. Thus, the multicolor GNPs were synthesized in the multiple honeycombed templates. Fabrication of Visual Sensor Array for Protein Discrimination. 20 µL of 40 µg/mL target protein (BHb, Cat, Glu, Try, BA, Lys, BSA, Col or Pep) was added to the colorless solution of Au(I)-Pep/Try (2 ml). The mixture was subjected to microwave treatment at 300 W for 90 s. Au(I) ions were reduced by proteins to be GNPs,8 and the multicolor GNPs were synthesized. To construct the visual sensing array, the Au(I)-Pep/Try complexes in the absence (named “before”) and presence (named “after”) of proteins after microwave reaction were loaded into a 96-well polystyrene plate. Later the “before” and “after” images of GNPs were recorded using a scanner. Difference maps were obtained by extracting the color alterations (∆RGB) from the “before” and “after” images using the Photoshop software. Four replicates were tested for each - 13Plus - Environment ACS Paragon

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protein. The ∆RGB data (fingerprints, See Table S1 in SI) were used to generate the 2×9×4 (two indicators × nine proteins × four replicates) training matrix. The training matrix was processed using the MVSP, HCA and PCA.19-21 Lys Detection in Human Tears. The content of Lys in human tears was taken to tentatively test the feasibility of the as-developed sensing array for biomedical diagnostics. To test unknown content of Lys in human tears, Lys at various concentrations spiked in pure water were prepared at first. 20 µL of human tears and concentrations of Lys were added to 2 ml of the Au(I)-Pep/Try solution. The mixture was subjected to microwave treatment at 300 W for 90 s, and the multicolor GNPs were synthesized. The color corresponding to human tear was compared with those of the standard Lys samples. The color changes of the sensor array were quantitatively illustrated by the total euclidean distances (EDs = [∆R2 + ∆G2 + ∆B2]1/2). According to the linear relationship between the EDs and concentrations of Lys, we obtained the content of Lys in human tears. All the experiments were performed in accordance with Zhenhai Hospital Ethics Committee’s guidelines and regulations. Ethics Statement. All experiments and procedures were performed in accordance with the appropriate guidelines. All procedures were approved by Nanyang Normal University and NIMTE. Informed consent was obtained from all volunteers before being enrolled in the study. ASSOCIATED CONTENT @ Supporting Information The Supplementary information (Figures S1-S6 and Tables S1) is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] or [email protected] (Y. Leng)


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Author Contributions Y.L. conceived the idea, performed the experiments, analysed the data and wrote the manuscript. K.J. characterized GNPs. W.Z. drew Scheme 1 and Figure 1. Y.W. analyzed the data and discussed the paper. Notes The author declares no competing financial interests. ACKNOWLEDGMENTS This work is supported by the Natural Science Foundation of China (21607083), Natural Science Foundation of Henan (162300410206), Technicians Troop Construction Projects of Henan Province (No. C20150029), Scientific and Technological Project of Henan Province (162102310484), Natural Science Foundation of Ningbo (2016A610262) and China Postdoctoral Science Foundation (2015M581970). Y. Leng thanks for Prof. Jane-Marie Lehn's good suggestions on this paper. Y. Leng also thanks Professors Hengwei Lin, Yujie Xiong, Xing Li and Dr. Ling Zhang for discussions, and Dr. Muhammad Zubair Iqbal for checking the manuscript. REFERENCES (1) Sauvage, J.; Weiss, J. Synthesis of Dicopper(I) [3]Catenates: Multiring Interlocked Coordinating Systems. J. Am. Chem. Soc. 1985, 107, 6110−6111. (2) Nierengarten, J.; Dietrich-Buchecker, C. O.; Sauvage, J. Synthesis of a Doubly Interlocked [2]Catenane. J. Am. Chem. Soc. 1994, 116, 375−376. (3) Amabilino, D. B.; Dietrich-Buchecker, C. O.; Livoreil, A.; Pérez-García, L.; Sauvage, J.; Stoddart, J. F. A Switchable Hybrid [2]-Catenane Based on Transition Metal Complexation and π-Electron Donor-Acceptor Interactions. J. Am. Chem. Soc. 1996, 118, 3905−3913.


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Graphical Abstract

Schematic illustration of preparation of Au(I)-directed interlocking rings in proteins and synthesis of gold nanoparticles (GNPs) from Au(I) ions shuttle to solidify. The protein (e.g. Pep and Try) and Au(III) were firstly incubated in strong alkaline solution to obtain the Au(I)directed interlocked rings. Addition of the other proteins prompted Au(I) anions shuttling from Pep/Try to other proteins. The Au(I)-Pep-protein complexes of interlocked rings were subjected to microwave reaction to synthesize GNPs, which were produced in the multiple honeycombed templates.


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Synthesis of Gold Nanoparticles from Au(I) Ions ... - ACS Publications

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