Nanoparticle Surface Specific Adsorption of Zein ... - ACS Publications

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Nanoparticle Surface Specific Adsorption of Zein and Its Selfassembled Behavior of Nanocubes Formation in Relation to On−Off SERS: Understanding Morphology Control of Protein Aggregates Navdeep,§ Tarlok Singh Banipal,*,§ Gurinder Kaur,‡ and Mandeep Singh Bakshi*,† †

Department of Chemistry and Biochemistry, Wilfrid Laurier University, Science Building, 75 University Avenue West, Waterloo Ontario N2L 3C5, Canada ‡ Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, Newfoundland A2V 2K7 Canada § Department of Chemistry, UGC Sponsored Center for Advanced Studies-1, Guru Nanak Dev University, Amritsar-143005, Punjab India S Supporting Information *

ABSTRACT: Zein, an industrially important protein, is characterized in terms of its food and pharmaceutical coating applications by using surface enhanced Raman spectroscopy (SERS) on Au, Ag, and PbS nanoparticles (NPs). Its specific surface adsorption behavior on Ag NPs produced self-assembled zein nanocubes which demonstrated on and off SERS activity. Both SERS characterization as well as nanocube formation of zein helped us to understand the complex protein aggregation behavior in shape controlled morphologies, a process with significant ramifications in protein crystallization to achieve ordered morphologies. Interestingly, nanocube formation was promoted in the presence of Ag rather than Au or PbS NPs under in situ synthesis and discussed in terms of specific adsorption. Zein fingerprinting was much more clear and enhanced on Au surface in comparison to Ag while PbS did not demonstrate SERS due to its semiconducting nature. KEYWORDS: zein, SERS, surface adsorption, self-aggregation

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INTRODUCTION Zein is a corn storage, water insoluble industrial protein with several important applications in food and pharmaceutical industries. It is an odorless and edible protein but poor in nutritional quality because it lacks essential amino acids such as lysine and tryptophan. It contains high proportions of nonpolar amino acid residues, such as leucine, alanine, and proline, which render it water insoluble.1,2 It is, therefore, one of the best bioingredients for moisture resistant biodegradable protein films essential to replace environmentally nonfriendly synthetic alternatives.3−6 Despite its numerous industrial applications, it is still poorly characterized due to its complex nature, which originates from its four different classes, viz. α, β, γ, and δ based on its solubility, amino acid sequences, and surface charges. αZein6 with molecular weight of 21−25 kDa is the most abundant fraction and also contains large amounts of hydrophobic residues. Since zein is frequently used in the surface coating of paper cups, clothing fabrics, adhesives, and binders to make them biodegradable as well as on pharmaceutical drugs, therefore, its surface characterization is an essential aspect to study. Although its surface characterization has been reported previously,7−9 surface enhanced Raman spectroscopy (SERS) on nanometallic surfaces, such as of Au and Ag noble metal nanoparticles (NPs), has not been reported to our knowledge. With increasing applications of zein in the pharmaceutical industry, its surface adsorption on a nanometallic surface is another important aspect to be quantified so as to use zein coated nanomaterials as drug release vehicles, as biolabels, in bioassays, and in clinical diagnosis.10−12 Fingerprinting of zein on nanometallic surfaces © XXXX American Chemical Society

can be used as a spectroscopic tool for its drug binding ability as well as site specific release. Zein coated Au and Ag NPs are the best candidates to study the SERS of zein because of their conduction band electrons interactions with incident light photons (surface plasmon resonance, SPR), which can transfer the absorbed energy to the protein layer to produce the Raman scattering fingerprint spectrum of zein. Similar studies are conducted with zein coated PbS NPs to compare the SERS of zein between noble metal and semiconducting NPs. In order to properly understand the SERS behavior of zein, we have used it in in situ conditions10,11 to synthesize Au, Ag, and PbS NPs, and tried to compare its behavior with non-in situ conditions. Zein, like other model proteins, has the ability to participate in the reduction reaction of Au and Ag to produce NPs whose colloidal stabilization as well as crystal growth is controlled by the surface active behavior of zein.11 This helps us to understand the shape directing effects of zein, and self-aggregation in ordered morphologies and in relation to SERS characterization. The self-assembled behavior of zein in ordered morphologies is a step forward in understanding the protein crystallization and how the participation nanometallic surfaces promote this through the seeding process. These results are expected to help us in exploring the potential use of environmentally friendly, edible, and nontoxic Received: November 18, 2015 Revised: December 24, 2015 Accepted: January 5, 2016

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DOI: 10.1021/acs.jafc.5b05495 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. (a) FESEM image of Au NPs prepared with in situ reaction of 0.1% zein and HAuCl4 = 1 mM at constant temperature 70 °C for 6 h. (b) TEM micrograph of zein coated Au NPs in a group of three NPs. Block arrows indicate the protein coating in the form of a thin film. (c) EDX elemental analysis of sample (b). (d) XRD patterns showing maximum growth at the {111} crystal plane. (e) UV−visible absorbance of Au NPs due to the SPR. (f) Raman spectra of pure dried zein on a glass substrate. (g) Raman spectra of in situ (0.1%/red, 0.2%/green, and 0.4%/blue) zein coated Au NPs. In situ synthesis of zein coated Au, Ag, and PbS NPs. In a round-bottom flask, 10 mL of (0.1, 0.2, and 0.4%) zein (in 24 mM SDS aqueous solution) was taken along with 1 mM HAuCl4 or AgNO3. The mixture was shaken thoroughly for some time and kept in a thermostatted water bath at constant temperature 70 °C for 6 h in the case of Au NPs, while at 80 °C for 24 h to synthesize Ag NPs, because Ag NPs required more annealing to complete the reduction and crystal growth. Zein initiated the reduction and converted the Au(III) into Au(0), and Ag(I) into Ag(0) with final pink to purple or yellow-brown color. The samples were kept for overnight and purified by centrifugation at 11,000 rpm after washings a couple of times with

zein, in zein coated biomaterials for their possible biomedical applications.

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EXPERIMENTAL SECTION

Chemicals. Zein protein, hydrogen tetrachloroaurate (III) trihydrate (HAuCl4), lead acetate trihydrate, glacial acetic acid, thioacetamide, silver nitrate (AgNO3), and sodium dodecyl sulfate (SDS), all AR grade obtained from Sigma-Aldrich, were used as such without further purification. The double distilled water was used for all sample preparations. B


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Journal of Agricultural and Food Chemistry double distilled water. The purified NPs were stored in a glass container and used for characterization. For the PbS NPs synthesis, 1.0 mL of 0.5 M acetic acid, 0.5 mL of 50 mM lead acetate, and 0.5 mL of 50 mM thioacetamide were added in the above-mentioned 10 mL aqueous zein solution, and the reaction was carried out at 80 °C for 24 h. Samples with black suspensions of PbS NPs were obtained which were purified by centrifugation at 11,000 rpm after washing a couple of times with double distilled water. Seed-growth method for the synthesis of zein coated Au NPs. In this method, we first synthesized Au seeds by taking 1 mM trisodium citrate in 25 mL and 1 mM HAuCl4 followed by an addition of an ice cold 0.5 mL (0.1 M) sodium borohydride solution. The ruby red color confirms the formation of Au seeds. The growth was initiated by taking 0.1 mL of Au seeds in 10 mL of zein solution (0.1 and 0.2%) in the presence of 1 mM HAuCl4 at 70 °C for 6 h. The zein coated Au NPs thus obtained were purified as mentioned above. Polyol process for the synthesis of zein coated Ag NPs. In polyol synthesis of Ag NPs, 10 mL of ethylene glycol was maintained at a constant temperature of 140 °C for 1 h. This is followed by an addition of 2 mL of 3 mM HCl (in ethylene glycol) and simultaneous addition of 6 mL of 94 mM AgNO3 (in ethylene glycol) along with 6 mL of 0.1% zein (in ethylene glycol). The reaction mixture was kept at 140 °C for another 24 h, which produced ocher color zein coated Ag NPs. The purification was done as mentioned above. Synthesis of PbS NPs in the presence of SDS. In this reaction, the synthesis of PbS NPs was carried out in an exactly similar manner as that mentioned in the in situ reaction (explained above), except instead of zein, SDS has been used as a stabilizing and shape directing agent. Zein coating was done on the purified PbS NPs by stirring the NPs suspension in aqueous zein solution overnight at room temperature. The samples were purified as mentioned previously. Instrumentation. UV−visible spectra were taken on a Shimadzu UV1800 spectrophotometer. The baseline correction was done by taking water in a reference cuvette. Transmission electron microscopy (TEM) analysis was done on a JEOL-2100 TEM, Japan. The sample was prepared by placing a drop of suspension on a carbon coated copper grid, dried in air, and analyzed at an operation voltage of 200 keV. Scanning electron microscope (SEM) analysis was done on a Carl Zeiss Supra 55 at operating voltage 10 keV for SEM images and 20 keV for energy dispersive X-rays (EDX). Sample was prepared by placing the suspension on a glass substrate and analyzed after silver coating. Raman Spectra were taken on a Renishaw in-via Raman Microscope, U.K. Sample was prepared by placing a drop of sample suspension on a glass substrate and dried in air. The sample was irradiated with a laser beam of 785 nm, and spectra were collected with the help of wire software. X-ray Diff raction analysis was done on a Shimadzu MAXima-X XRD, Japan. Sample was prepared by placing a drop of sample suspension on a glass substrate and dried in air. XRD patterns were collected at a wavelength of 1.54 Å (Cu Kα), operating voltage of 40 keV, and scan speed of 4 deg per min. Origin 6.1 and Kaleida Graph 3.5 were used to process and plot the spectra.

remarkably enhanced in comparison to that of 0.2 and 0.4% (Figure 1g) because of the relatively much thinner coating that effectively responds to the SPR energy transfer on the Au NPs surface. The band near 296 cm−1 (which is absent in control, Figure 1f) represents the S−Au covalent interaction between Au NPs and dissociated disulfide groups of the zein protein.13 The bands at 498 and at 515 cm−1 in the range of 495−545 cm−1 are due to the S−S bonds,13−17 and their presence suggests that the adsorbed zein is not in the fully unfolded state and still retains the mixed secondary or tertiary structure even on the surface of NP. Zein is a highly hydrophobic and robust protein, and it is not expected to fully unfold by a simple surface adsorption process.18 C−S stretch bands at 657 and 734 cm−1 in the region of 630−670 and 700−745 cm−1 (absent in the control) demonstrate the presence of free cysteine thiol groups due to partial unfolding.17 Because of this, tyrosine appears as a single band near 835 cm−1 rather than a well-defined doublet in the range of 800−866 cm−1 representing the equilibrium between hydrogen bonding of the hydroxyl group of tyrosine to a negative acceptor such as CO2− and hydrogen bonding of the oxygen of the hydroxyl group to an external proton.13−16,19−21 Aliphatic residues with a C−C and C−N stretch in the region of 901−960 cm−1 of adsorbed zein on NP as well as on glass do not show any significant change,13,14,16 while the C−C stretch at 978 cm−1 of Leu and Ala as well as of the H-twist of Tyr near the region of 964−975 cm−1 (which is absent in the control) demonstrates significant SERS due to their close vicinity to the NP surface and, hence, is influenced by SPR.19 Bands at 999 cm−1 in the region 1000−1010 cm−1 and at 1457 cm−1 in the region 1424−1465 cm−1 are the characteristic bands of phenylalanine and CH2 deformation (CH2 and CH3) which are also present in the control (at 1005 cm −1 and 1450 cm−1)13−15,17−20 (Figure 1f). The region from 1583 to 1587 cm−1 corresponds to Tyr, Trp, and Phe, and is observed in all samples but absent in the control.13,14,16 These aromatic residues are usually buried deep in the hydrophobic region, and hence, their SERS suggests the unfolding of the zein on the NP surface and the enhancement of their signals due to the SPR. Important information can be extracted from the amide I (α-helix) band at 1650−1661 cm−1 which is absent for adsorbed zein on NP while it is present in the control (at 1658 cm−1), indicating the fact that most of the α-helical structure of zein has changed into a mixed structure of α-helix, β-pleated sheets, and random coils.13,14,17,20,22 Amide III in the region of 1210−1235 cm−1 of the peptide backbone representing the β-pleated sheet15,17,20,22 is also observed in the sample prepared with 0.1% zein at 1218 cm−1, while the amide III region 1240−1255 cm−1 due to the random coil is depicted for 0.2% zein at 1243 cm−1.15,17,20,22,23 All characteristic bands have been summarized in Table 1. Increasing the amount of HAuCl4 from 1 to 1.5 mM produced almost similar morphologies but with larger size: 97 ± 13 nm (Figure 2a,b). They also show almost similar behavior, as explained above, with maximum enhancement in the presence of 0.1% followed by 0.2% and 0.4% zein (Figure 2d). Some of the important points are listed as follows. The band near 961 cm−1 (964−975 cm−1 representing Leu and Ala C−C stretch, CH3 rock, and Try ring twisting19) shifts toward higher wavenumber (977 cm−1) for 0.1% zein and becomes prominent for 0.2% and 0.4% zein. The shift is mainly due to a change in the environment when Lys and Ala interact with Au NPs, as mentioned previously from the molecular dynamics studies.10 The amide III β-pleated sheet at 1225 cm−1 in the region (1210−1235 cm−1)15,17,20,22 is only observed for 0.2% zein, while the amide III random coil is observed at 1243 cm−1 for 0.1% zein and at 1240 cm−1 for 0.4%

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RESULTS Surface characterization of zein on in situ synthesized Au NPs. Zein coated Au NPs synthesized through in situ reaction (see Experimental Section) are shown in Figure 1a. They are mostly cubehedron in shape and 67 ± 13 nm in size. The zein coating indicated by block arrows in Figure 1b is clearly visible on each NP, and EDX analysis indicates the presence of C, N, O, and S, apart from Au (Figure 1c). XRD patterns demonstrate the fcc geometry with maximum growth on {111} crystal planes (Figure 1d). The UV−visible spectrum shows a characteristic peak of Au NPs close to 520 nm due to SPR (Figure 1e). The surface adsorption of zein on Au NPs can be very well characterized from the SERS. Figure 1f and g show the SERS of zein on the glass slide as control and on Au NPs prepared under in situ conditions by taking 1 mM of HAuCl4 in the presence of 0.1, 0.2, and 0.4% of zein. SERS of 0.1% zein is C


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1650−1661 cm−1 is absent in all but present in control. 13,14,17,20,22 Similarly, the S−S stretch (495−545 cm−1)13−17 is not visible for 0.2% and 0.4% zein, while the C− S stretch at 651 and 735 cm−1 in the region (630−670 cm−1, 700−745 cm−1)17 is present, which is again due to the presence of the free cysteine thiol group. CH2 deformation near 1345 cm−1 in the region (1320−1340 cm−1)14,20 is observed for all cases but absent in control, while CH2 deformation at 1457 cm−1 for 0.2% and at 1463 cm−1 for 0.4% zein in the region (1424−1465 cm−1)14,15,17,20 demonstrates a shift toward higher wavenumber for 0.2% and 0.4% zein due to a greater number of NPs, which consequently increased the amount of surface area with a thinner protein film. Comparison of SERS with seed−growth Au NPs. The SERS of zein on Au NPs synthesized under in situ conditions has been compared with those synthesized by the seed−growth method.24−27 In the former case, zein is simultaneously adsorbed on the Au NPs during the single step reduction reaction due to its stabilizing and surface active behavior, while in the latter case, it was allowed to adsorb on growing citrate stabilized Au seeds of less than 10 nm (Figure 3a) prepared previously in the absence of zein (see Experimental Section). The overall morphology of the NPs is somewhat similar to that of in situ synthesized with average size of 107 ± 16 nm (Figure 3b). Surface adsorbed zein (indicated by block arrows) is clearly visible on each NP in Figure 3c. The Raman enhancement of zein on seed−growth Au NPs has been compared with that of in situ in Figure 3d. Some of the contrasting differences have been mentioned here. The region (901−960 cm−1)13,14,16 corresponding to Cα−C−N, C−C, C− Cα‑helix (904, 931, 946, and 957 cm−1, respectively) is more resolved on seed-growth NPs rather than on in situ synthesized Au NPs (913 cm−1). This is due to the fact that, in the seedgrowth method, citrate stabilized seeds possess negative charge while zein bears global positive charge (isoelectric point of zein is ∼6.8)18,28,29 due to a low pH ∼ 3 of the solution in the presence of HAuCl4. This allows a favorable adsorption of zein on growing NPs in the seed−growth method, as evident from Figure 3c. This is not the case when zein is involved in the in situ reaction, where it is first involved in the reduction of Au(III) into Au(0)30,31 and, then, in the stabilization of the nucleating centers by adsorbing on the NPs surface simply by close Van der waals interactions.31 Thus, a reduction and stabilization process may cause a drastic decrease in the α-helix structure of zein, and hence, it is not fully resolved, in contrast to the zein adsorbed on growing Au seeds. Similarly, the band at 1255 cm−1 in the region 1240−1255 cm−1 (amide III random coil) is observed on seed-growth Au NPs but is not visible in the in situ sample because we do not expect these conformations to remain prominent in in situ surface adsorption. Likewise, the band at 1409 cm−1 near the region 1403−1405 cm−1 corresponding to the symmetric ν CO2− stretch) is observed on seed-growth Au NPs but absent for in situ while the region 1424−1465 cm−1 corresponding to the CH2 deformation of CH2 and CH3 is observed in both cases but with higher intensity for in situ NPs. Similar comparative Raman enhancement has been studied for the samples with 0.2% zein (Figure 3e). The band at 665 cm−1 in the region 630−670 cm−1 and at 737 cm−1 in the region 700− 745 cm−1 corresponding to the C−S stretch demonstrates higher intensity for in situ Au NPs rather than the seed−growth sample. The bands at 1057 cm−1 corresponding to the C−C, C−N stretch, at 1177 cm−1 corresponding to tryptophan and phenylalanine, and at 1221 cm−1 corresponding to the amide III β-pleated sheet (Table 1) are observed for seed−growth Au

Table 1. Typical Wavenumbers of Raman Bands and General Assignments of Zein Protein Structurea S. No.

Wavenumber/ cm−1

Assignment

1 2 3 4 5 6 7 8

296 495−545 621 642 630−670 700−745 750−760 800−866

S−Au S−S Phe Tyr C−S strech C−S strech Trp Tyr

9 10 11

877−880 901−960 964−975

12

1000−1010

Trp C−C, Cα−C−N, C−C (α-helix) “Leu and Ala” C−C strech, CH3 rock and Trp ring H-twisting Phe

13 14 15 16

1010−1035 1055−1059 1177−1207 1210−1235

RB of Trp, Phe in-plane bending C−C, C−N strech Tyr + Phe Amide III β-pleated sheet

17

1240−1255

Amide III random coil

18

1265−1300

Amide III α-helix

19 20 21 22

1320−1340 1340−1360 1403−1405 1424−1465

CH2 deformation Trp Symmetric ν CO2− CH2 deformation of CH2 and CH3

23 24 25 26 27 28 29

1484 1535−1550 1550 1583−1587 1605 1615 1650−1661

Phe, Tyr, Trp Trp amide II Tyr, Trp, Phe Phe Tyr amide I (α-helix)

30 31 32

1667−1673 1665 ± 3 1685

β-sheet disordered structure (solvated) disordered structure (H-bonded)

ref 13 13−17 13, 17 13 17 17 13, 14, 19 13−16, 19−21 13, 17 13, 14, 16 19 13−16, 19−21 13, 15, 16 13−16, 20 14 15, 17, 20, 22 15, 17, 20, 22, 23 15, 17, 20, 22, 23 14, 20 13, 17 20 14, 15, 17, 20 16 13, 17 14 13, 14, 16 13, 14 14, 20 13, 14, 17, 20, 22 17 17 17

a

RB = Ring Breathe, bolded rows for characteristic peaks, Tyr = Tyrosine, Trp, Tryptophan, Phe = Phenylalanine.

zein in the region (1240−1255 cm−1).15,17,20,22,23 The symmetric stretch of COO− at 1399 cm−1 for 0.2% zein and at 1414 cm−1 for 0.4% zein near the region (1403−1405 cm−1)21 also demonstrates the Raman shift for 0.2% and 0.4% zein, which is again due to the change in the environment as the size of the nanoparticles increases. The region from 1600 to 1750 cm−1 for adsorbed zein is observed as a broad band spread over the whole range, suggesting the mixed structure of zein on the nanoparticle surface after adsorption. Interestingly, further increase in the amount of HAuCl4 to 2 mM increases the surface enhancement, especially in 0.2 and 0.4% of zein in terms of more clear peaks in comparison to that observed in Figure 2d because the corresponding amount of Au NPs is increased against the fixed amount of zein (Figure 2e). Now a thinner layer of adsorbed zein is present on the NP surface which generates more hot spots for energy transfer from the SPR, and hence, the enhancement is also evident at higher amounts of zein, i.e. 0.2% and 0.4%. Among the important features, amide I at 1658 cm−1 (α-helix) in the range of D


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Figure 2. (a) FESEM image of Au NPs prepared with in situ reaction of 0.1% zein and HAuCl4 = 1.5 mM at constant temperature 70 °C for 6 h. (b and c) TEM micrographs of a Au NPs sample (a) and those prepared with HAuCl4 = 2 mM. (d) Raman spectra of in situ (0.1%/red, 0.2%/green, and 0.4%/ blue) zein coated Au NPs prepared with HAuCl4 = 1.5 mM. (e) Raman spectra of in vitro (0.1%/red, 0.2%/green, and 0.4%/blue) zein coated Au NPs prepared with HAuCl4 = 2 mM.

under similar conditions (see Experimental Section) shows mainly cubic morphologies of zein conjugated Ag NPs where small Ag NPs are embedded in the zein nanocubes of 200 nm in size, as evident from the SEM images (Figure 4a,b). Such kind of morphology was not obtained in the presence of Au NPs (Figure 1 and 2) due to the reasons discussed later in the Discussion section. TEM images confirm the presence of almost transparent nonmetallic zein nanocubes loaded with metallic dark polyhedral Ag NPs of less than 50 nm (Figure 4 c,d). Most of the Ag NPs are embedded in the surface of nanocubes, and this is confirmed by the HRTEM image (Figure 4e). The selected area diffraction image (inset) shows the characteristic diffraction patterns of face centered cubic geometry (fcc) of Ag. EDX analysis of Ag NPs

NPs and not for in situ Au NPs. Overall, from the above results, the spectra are better resolved for seed−growth NPs rather than the in situ sample, though enhancement is much greater on the in situ synthesized NPs, which is obviously expected in terms of better coating when zein is involved in the nucleation and stabilization process simultaneously during the in situ synthesis. Thus, better zein coating is more prone for energy transfer from the SPR, and hence, better enhancement is observed.32,33 Surface enhancement of zein on Ag NPs and its selfaggregation in nanocubes. Ag NPs, such as the Au NPs, also demonstrate the SERS due to their SPR.13,34,35 Here, we present somewhat different results for zein coated Ag NPs. In situ synthesis of Ag NPs in a ternary zein + AgNO3 + water system E


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Figure 3. (a) TEM images of Au seeds and (b) Au NPs prepared by seed the growth method with 0.1% zein and HAuCl4 = 1 mM at constant temperature 70 °C for 6 h. (c) HRTEM image of zein coated Au NPs. Block arrows indicate the zein coating in the form of a thin film. (d) Comparative Raman spectra of in situ (green) and seed growth methods (magenta) for 0.1% zein coated Au NPs. (e) Comparative Raman spectra of in situ (green) and seed growth methods (magenta) for 0.2% zein coated Au NPs.

band close to 1380 cm−1 which originates from a combination of various bands, viz. 1316, 1411, 1450, and 1533 cm−1, corresponding to CH2 deformation, symmetric νCO2− stretch, CH2 deformation of CH2 and CH3, and tryptophan stretching, respectively (Table 1). Each spectrum is not fully resolved, and no structural information can be extracted because zein has selfassembled in the form of nanocubes and much smaller Ag NPs are entrapped with practically whole SPR screened under several layers of zein. Hence, surface enhancement of zein is not observed.36,37 However, if zein is not allowed to self-assemble in nanocubes entrapping Ag NPs but rather is surface adsorbed on the Ag NPs just like that of Au NPs discussed above, surface enhancement is observed. For this purpose, we synthesized Ag NPs by using the

embedded zein nanocubes (Figure 4f) shows the presence of C, N, and O in addition to Ag. Also, if we dissolve the Ag NPs embedded zein nanocubes in pure ethanol, zein nanocubes dissolve, leaving behind metallic Ag NPs with characteristic absorbance around 410 nm, as shown in the next figure (Figure 5d). Thus, different analyses confirm the presence of zein nanocubes carrying Ag NPs. The mechanism of their formation will be discussed later in the Discussion section. The nanocubes become well-defined when we increase the amount of zein from 0.2% (Figure 4a) to 0.4% (Figure 4b) against a constant amount of AgNO3 = 1.5 mM. Since the Ag NPs are embedded in the nanocubes, therefore, no surface enhancement is expected. Figure 4g compares the Raman spectra of nanocubes with different amounts of zein. All spectra show a prominent broad F


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Figure 4. (a) FESEM image of zein nanocubes with embedded Ag NPs prepared under in situ conditions with 0.2% zein and 1.5 mM AgNO3 at 80 ± 2 °C for 24 h. (b) FESEM image of fine nanocubes prepared with 0.4% zein. (c, d) TEM images of zein nanocubes embedded with small Ag NPs. (e) HRTEM image of a zein nanocube showing most Ag NPs are embedded on the surface of a zein nanocube. (f) EDX elemental analysis of nanocubes with emission from C, O, N, and Ag. (g) Comparison of Raman spectra of Ag NPs embedded zein nanocubes with different amounts of (0.1%/red, 0.2%/ blue, and 0.4%/green) zein. (h) Comparative Raman spectra of zein coated Ag NPs (olive) and in situ synthesized Au NPs (red) with 0.1% zein.

polyol process at 140 °C in the presence of zein (see Experimental Section). No nanocubes of zein entrapping Ag NPs are formed when the reaction is conducted in ethylene glycol medium rather than in water. In this case, polyhedral Ag

NPs of less than 200 nm coated with a thin layer of zein are obtained (Supporting Information, Figure S1a,b) which show significant surface enhancement. XRD patterns (Figure S1c) demonstrate a fcc crystal structure. A characteristic UV−visible G


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Figure 5. (a, b) FESEM images of in situ zein coated PbS NPs prepared 0.1% zein at 80 ± 2 °C for 24 h. (c) TEM image of the sample from part (a). (d) Magnified image showing zein coating on PbS NPs. (e) EDX analysis demonstrating the presence of C, N, O, Pb, and S. (f) XRD pattern of sample showing the fcc geometry of the unit cell. (g) Raman spectra of in situ zein coated PbS NPs with varying amounts (0.1%/red, 0.2%/blue, and 0.4%/ green) of zein.

phase drives an effective surface adsorption on Au NPs in comparison to that on Ag NPs in ethylene glycol. Some prominent differences in the Raman enhancement are discussed as follows. The bands at 1031 cm−1 in the region 1010−1035 cm−1 corresponding to the ring breathe of Trp and Phe in-plane bending, at 1184 cm −1 in the region 1177−1207 cm −1 corresponding to Tyr and Phe, and at 1218 cm−1 in the region 1210−1235 cm−1 corresponding to the amide III β-pleated sheet (Table 1) are observed only in zein coated Au NPs, but they are absent in zein coated Ag NPs. This is mainly due to the greater solubility of zein in ethylene glycol rather than in water, which reduces the surface activity of zein on the Ag NP surface.38 Hence, the energy transfer due to the SPR to zein film is not

absorbance is observed at 410 nm (Figure S1d) due to SPR of Ag NPs. Figure 4h compares the Raman spectra of zein on Ag NPs with that of Au NPs. Overall, the surface enhancement is higher on the Au surface rather than on the Ag surface, which can be mainly attributed to the polyol medium of Ag NPs in comparison to the aqueous medium of Au NPs as well as a much higher reaction temperature (140 °C) for the synthesis of Ag NPs. The surface activity of zein is expected to be more prominent in the aqueous phase rather than in the ethylene glycol38 because its relatively low solubility promotes amphiphilicity in a polar aqueous phase with much higher relative permittivity in comparison to that in less polar ethylene glycol with greater solubility. Thus, stronger amphiphilicity of zein in the aqueous H


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Figure 6. Schematic representation of the growth cycles, protein adsorption, and Raman scattering. (a) Demonstrating the selective adsorption of zein on the {111} crystal planes of gold nucleating centers that results in the icosahedral morphologies bound by {111} facets. SPR is shown as a pink band that provides energy transfer to the zein adsorbed layer in the event of hv incident light producing SERS. Reaction (b) represents the involvement of zein coated Ag nucleating centers in the seeding process which promotes the self-aggregation of growing zein coated Ag NPs in the form of zein nanocube formation embedded with small Ag NPs. (c) Selective adsorption of zein on the {100} lattice planes of PbS nucleating centers, which results in the zein coated PbS cubic morphologies.

surface contributes little toward the SPR and, hence, practically little Raman scattering occurs. The broad bands near 685 cm−1, 887 cm−1, and 1167 cm−1 correspond to the C−S stretch, ring breathe modes of aromatic residues, and tryptophan and phenylalanine, respectively (Table 1), which are not properly resolved. We tried to further evaluate the SERS of zein coated PbS NPs by taking the presynthesized NPs prepared in the presence of SDS rather than zein (see Experimental Section).43 These NPs after purification were coated with zein by stirring the solution overnight in the presence of zein. TEM images of these NPs are shown in Figure S2a−c. Figure S2a shows a low resolution image of PbS NPs where predominantly cubic shapes close to 100 nm in size have appeared though they are not perfectly cubes, as shown in Figure 5a−d, synthesized in the presence of zein. Thus, zein, a predominantly hydrophobic protein is a better shape directing agent in comparison to SDS to produce PbS cubic morphologies.31 Figure S2b,c show the close up images of zein coated NPs where a clear coating of zein (indicated by block arrows) is shown. A colloidal suspension of cubic PbS NPs shows a broad absorbance close to 750 nm (Figure S2d), as observed previously for similar morphologies.39 The size dependent UV− vis absorbance for PbS NPs arises from the large Bohr radius of PbS, which is close to 18 nm and demonstrates a contrasting difference from other semiconductor materials with a much smaller Bohr radius. That is why the latter materials do not show any significant shape and size dependent optical properties. Raman spectra of these samples are shown in Figure S2e. The band near 650 cm−1 corresponds to the C−S stretch while the other bands are too broad. The broad band around 1380 cm−1 is a combination of various bands (1316, 1411, 1450, and 1533 cm−1), and resolution is very poor due to almost insignificant

effectively contributing toward the surface enhancement on Ag NPs. Surface enhancement of zein on PbS NPs. In situ zein coated PbS NPs were synthesized to compare the surface enhancement of zein on Au and Ag NPs with that of PbS NPs. This reaction has to be conducted in acidic medium to promote the synthesis of PbS NPs (see Experimental Section), and thus, no zein nanocube formation is observed. Rather, PbS NPs exist in the cubic morphologies, as observed previously in the presence of highly hydrophobic surfactants under identical reaction conditions.39 Thus, because of the predominantly hydrophobic nature of zein, it proves to be an excellent shape directing agent just like that of surfactants.40 Figure 5a,b and 5c,d show the SEM and TEM images of PbS fine nanocubes, respectively, synthesized in the presence of the zein with clear zein coating. One can compare the crystalline dark PbS nanocubes of Figure 5d with semitransparent amorphous zein nanocubes of Figure 4d from TEM images of comparable magnifications. EDX analysis shows the presence of protein with elemental emission from C, N, O, along with Pb and S (Figure 5e). Sharp XRD patterns indicate the presence of a crystalline fcc cubic geometry (Figure 5f). XRD measurement is done by dissolving the zein coated PbS NPs in pure ethanol to remove zein and then drying on a glass slide for XRD analysis (see Experimental Section). Although it has been observed that the energy transfer from the SPR of noble NPs to surface adsorbed zein contributes toward the SERS;36,37 similar enhancement is not expected in the case of zein coated PbS NPs due to their semiconducting nature.41−43 Therefore, this sample produces an entirely different Raman spectrum (Figure 5g) from that of Au and Ag NPs discussed previously. The entire range shows few broad bands due to the overlapping of many Raman peaks. This happens because the semiconducting I


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lattice structure. The {100} crystal facets are made up of both Pb and S atoms and are without any charge polarization. Contrary to this, the {111} crystal planes are predominantly occupied by either Pb or S atoms and, hence, result in an overall charge separation that establishes a dipole. Thus, a zero dipole moment provides nonpolar properties to the {100} crystal planes, turning them into suitable platforms for predominantly hydrophobic zein adsorption,51,52 which eventually leads to the nanocubic morphologies bound with the {100} crystal planes. Thus, the lack of SERS on the PbS NPs surface apart from its semiconducting nature is also related to the preservation of nonpolar character.53,54 In contrast, the highly unfolded form of zein with a highly surface active nature due to its predominantly highly amphiphilic character is more susceptible to the energy transfer from the SPR of Au and Ag55 and, thus, shows significant SERS (Figure 6). Thus, the SERS of proteins is highly dependent on the characteristic features of lattice planes as well as the surface activity of the protein. A proper coating is achieved only for highly surface active protein31,56 because protein does not adsorb like conventional surface active monomeric molecules, rather it adsorbs as a whole polymeric species whose hydrophilic as well as hydrophobic domains must achieve proper compatibility as an amphiphilic species with the surface. Thus, a proper surface active behavior is the key for energy transfer in the event of the SPR effect for the SERS activity. The above results conclude that Raman fingerprinting of industrially important zein is determined on the Au, Ag, and PbS nanometallic surfaces in terms of their use as biomaterials in biological applications such as biolabeling, bioassay, and clinical diagnosis. Due to its predominantly hydrophobic nature, zein is an excellent shape directing protein to obtain ordered morphologies. It is well characterized on the Au NPs, and the fingerprinting is quite clear and enhanced on in situ synthesized NPs as well as on the seed-growth synthesized Au NPs though it is more resolved in the latter case. This is primarily related to the preferential adsorption of zein on low energy {111} crystal planes of growing nucleating centers under the in situ reaction conditions which provide greater SERS activity. In the case of Ag NPs, in situ reaction conditions induce a relatively lesser amount of unfolding, amphiphilicity, and surface activity of zein due to a higher surface energy of the {111} crystal planes of Ag than that of Au, and that induces the seeding among zein coated Ag nucleating centers to produce zein nanocubes embedded with small Ag NPs. The SPR of Ag NPs in such morphologies are significantly screened, and hence, no SERS is observed. However, if zein nanocube formation is hindered by changing reaction conditions, the resulting zein coated Ag NPs demonstrate SERS just like that of Au NPs. Although fine zein coated PbS cubic morphologies are produced by the shape directing effects of zein, they do not show the clear zein Raman spectrum, mainly due to the semiconducting nature of PbS. Thus, several different factors participate in an appropriate surface adsorption of zein on nanometallic surfaces and contribute toward the SERS activity, resulting in the synthesis of biomaterials for their best possible uses in biological applications. The formation of the zein nanocubes in the presence of Ag NPs is a step forward in shape controlled morphologies of protein self-assembled structures in amorphous as well as crystalline forms. A self-assembled protein in an ordered geometry helps us to understand the mechanism involving the protein−protein interactions with ramifications in critical illnesses such as Alzheimer’s and Parkinson’s.

SPR. The NPs coated with 0.1% zein, though, show a maximum intensity followed by those coated with 0.2% zein, while there is practically no Raman response for the NPs coated with 0.4% zein, and hence, little structural information can be obtained from these plots. The difference in the Raman patterns between Figures 5g and S2e is quite evident, presumably due to the different modes of zein coating. In Figure 5g, the zein is coated during the in situ synthesis due to its colloidal stabilizing as well as shape directing effects31 whereas in Figure S2e zein is simply coated on purified PbS NPs previously synthesized in the presence of SDS.

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DISCUSSION Zein is a predominantly hydrophobic protein, and each cylindrical hydrophobic domain of zein is created by an antiparallel arrangement of nonpolar amino acid residues (mainly alanine, leucine, phenylalanine, valine, and proline).6,44 Different hydrophobic domains are further associated with each other in a three-dimensional arrangement through hydrogen bonds which operate between the three pairs of polar amino acids (glutamine, asparagine, and serine) located along the helical surface of each cylinder.44 Protein surface adsorption is mainly driven by the low lattice energy of the NP surface through hydrophobic (folded form) or amphiphilic (unfolded) interactions,45−47 since zein is mainly a hydrophobic protein but its unfolded form is highly surface active and amphiphilic in nature. This allows zein to favorably adsorb on the low energy crystal planes to induce surface passivation.11 The surface passivation is different on Au nucleating centers than that of PbS, although both possess fcc crystal geometry (Figure 6). In the former case (Figure 6a), the final morphology is bound with {111} crystal planes (icosahedral) while in the latter case it is bound with {100} (cube) (Figure 6c), which suggests the preferential adsorption of zein on respective planes. Adsorption on the low energy {111} crystal planes45−47 seems to cause greater zein unfolding and provides better surface activity with strong amphiphilicity rather than on the high energy {100} planes. The surface energy for a crystallographic plane depends on its packing density. The higher packing density has a greater number of nearest-neighbor atoms with more atomic bonds which provides lower surface energy. The planar densities of {100} and {111} crystal planes are given by 1/4r2 and 1/2r2√3, respectively, where r is the atomic radius. Since the planar density of the {111} crystal planes is higher, therefore, it has the lower surface energy. Probably due to this reason Ag nucleating centers induce instant aggregation among the zein coated nucleating centers through a seeding process to produce Ag NPs entrapped zein nanocubes (Figure 6b) because the {111} crystal planes of Ag possess higher surface energy than that of Au due to a lower atomic density of Ag than Au. The higher surface energy imparts less favorable surface adsorption with relatively less unfolding45−47 and, consequently, less amphiphilicity and surface activity. Thus, partially unfolded hydrophobic domains on Ag nucleating centers are more susceptible for seeding,31 which subsequently leads to the formation of zein cubic morphologies entrapping small Ag NPs (Figure 6b). Parallel control reactions by taking NaNO3 and KNO3 instead of AgNO3 do not induce zein nanocube formation under identical reaction conditions, thus eliminating the effect of NO3− ions on zein unfolding and self-aggregation. The preference for the {100} crystal planes of PbS nucleating centers originates from the zero dipole moment (Figure 6c).48−50 Zero dipole moment is primarily associated with the J


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05495.

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Additional TEM images (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Funding

Navdeep gratefully acknowledges the financial support under the University Grants Commission’s Scheme of University With Potential for Excellence. Use of various instruments under this scheme is highly acknowledged. These studies are partially supported by financial assistance under Article 28.8 of the CAS agreement of WLU, Waterloo. Gurinder Kaur thankfully acknowledges the financial support provided by the Research and Development Council (RDC) of Newfoundland and Labrador, NSERC, and the Office of Applied Research at CNA. Notes

The authors declare no competing financial interest.

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