Gold-Nanoparticle-Decorated Hybrid Mesoflowers: An Efficient

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Gold Nanoparticles-Decorated Hybrid Mesoflowers: An Efficient Surface-Enhanced Raman Scattering Substrate for Ultra-Trace Detection of Prostate Specific Antigen Sajanlal R Panikkanvalappil, and Mostafa A. El-Sayed J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp505204f • Publication Date (Web): 21 Aug 2014 Downloaded from http://pubs.acs.org on August 23, 2014

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The Journal of Physical Chemistry

Gold Nanoparticles-Decorated Hybrid Mesoflowers: An Efficient Surface-Enhanced Raman Scattering Substrate for Ultra-Trace Detection of Prostate Specific Antigen Sajanlal R. Panikkanvalappil and Mostafa A. El-Sayed* Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, United States

ABSTRACT

We demonstrate a method for making highly sensitive hybrid gold mesoflowers (MFs)-based surface-enhanced Raman scattering (SERS) substrate via programmed assembly of spherical gold nanoparticles (AuNPs) onto a highly anisotropic silica-coated MF. This imparts a new signal enhancing mechanism for the ultrasensitive detection of molecules from highly complex molecular environments. This substrate has been utilized as an ideal platform for the detection of Raman features correspond to the prostate specific antigen (PSA) of concentration as low as 2.5 ng/mL without the aid of any labels. We also demonstrated the possible conformational modifications of PSA on the hybrid MF surface and influence of various parameters on obtaining

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reproducible SERS spectra of proteins. This is critical in SERS based label-free detection of biomarkers from a diagnostic perspective. KEYWORDS: SERS, protein, mesoflower, prostate specific antigen, sensors.

1. INTRODUCTION Detection and identification of structural modifications of biomarker proteins hold great promise for early diagnosis of various diseases including cancer. Though enzyme based immunoassay1 is known for sensitive detection of biomarker proteins, they require comparatively high preparation and analysis time and high production cost. From a diagnostic perspective, blood serum is a more preferable material than tissue specimen as serum is easy to collect and carries biomarkers of several diseases, including cancer. However, the complex nature of serum with large amount of biomolecules such as proteins, lipids, etc. along with very small quantity of over expressed proteins indicative of cancer or any other diseases makes the diagnosis challenging. Hence, more sensitive techniques are required for the precise identification of biomarker proteins. Raman spectroscopy holds great potential in biological research since it is a non-destructive and noninvasive method, which requires minimal time for the sample preparation.2,3 However, the relatively weak signals inherent to normal Raman scattering hampers the clinical applications of this technology. This problem has been solved to a greater extent using surface-enhanced Raman spectroscopy (SERS),4,5 where Raman signal of molecules adsorbed on nanostructured metallic surfaces could be enhanced by tens of orders of magnitude compared to that of free molecules.6-8 Among biological molecules, proteins are the most challenging in terms of obtaining reproducible SERS spectra. Though indirect SERS detection strategy based on certain labels has been used widely, it is more time consuming and expensive.9 Due to this reason, label-free


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detection of biomolecules is more desirable for sensing applications.10-12 Owing to the possibilities of conformational modifications of proteins on metal surfaces,13,14 obtaining reproducible SERS spectra, which is critical in medical diagnosis, from them is difficult. Apart from this, temperature also plays an important role in the kinetics of protein adsorption on the nanoparticle surface and their conformations.15,16 Hence, nanoparticle featuring appropriate surface areas for binding protein, coupled with the high electromagnetic field for signal enhancement of Raman vibration is necessary for their SERS detection.17-19 While the anisotropic noble metal nanoparticles are good SERS substrates, their efficiency mainly depends on electromagnetic field enhancement around the nanoparticle. Although it is well known that aggregates of plasmonic nanoparticle creates more electromagnetic hot spots, responsible for high sensitivity in SERS, it is challenging to create such reproducible aggregates thus limiting their practical applications. Consequently, directing the self-assembly of nanoparticles into ordered aggregates is highly desirable to design and develop ideal SERS substrates for biomolecule detection. Anisotropic nanoparticles with sharp protrusions and nanogaps are known for their extremely high SERS activity.18,20,21 Recent report also shows that a thin inert oxide coating over the nanoparticle surface can enhance the intensity of Raman signals to a large extent.22,23 Incorporating all these aspects into a single nanoparticle can bring revolutionary capabilities to the resultant hybrid nanomaterial in view of its SERS activity. More recently, noble metal measo/nano flowers have been introduced as powerful SERS probes for chemical analysis.24,25 Gold mesoflowers (AuMFs) have large number of sharp stems, with high surface area, forming large number of SERS ‘hot spots’ on their surfaces. In this paper, we demonstrate various aspects to obtain reproducible SERS spectra of highly complex protein molecules by using a 3-dimentional (3D) hybrid plasmonic material made up of silica-coated


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AuMFs decorated with spherical gold nanoparticles (AuNPs). The highly anisotropic nature of the amino-functionalized silica-coated AuMFs provides greater surface area per MF, which can accommodate more number of AuNPs. The unique SERS capability of this hybrid material has been utilized for the trace detection of prostate specific antigen (PSA) (a biomarker protein overexpressed in prostate cancer patients) from the serum, which is spiked with multitudes of complex biomolecules such as protein and lipid. Apart from that, adsorption kinetics of PSA protein on the hybrid MF surface and subsequent spectral modifications has been probed using SERS. The critical role of various experimental parameters such as temperature and incubation time (proteins with hybrid MF) on obtaining reproducible SERS spectra of proteins is also studied in detail. 2. METHODS Materials: Hydrogen tetrachloroaurate trihydrate aqueous solution (HAuCl4.3H2O), sodium borohydride (NaBH4), AgNO3, ascorbic acid, cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), trisodium citrate, and prostate specific antigen (PSA) from human semen were purchased from Sigma-Aldrich USA. Insulin was purchased from Lantus insulin glargine (rDNA origin) and 1 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was procured from Avanti Polar Lipids, Inc. All chemicals were used without further purification. Synthesis of silica-coated AuMFs: For making silica coating over AuMFs, we followed a reported procedure.26 In this process, 200 μL of AuMFs was added to 20 mL of 2-propanol. Under constant stirring, 1 mL of ammonia solution and 60 μL of tetraethyl orthosilicate (TEOS) were added. The reaction was allowed to proceed for 1 h at room temperature under continuous stirring. The resultant solution was redispersed in water without adding any surfactant. The core-


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shell particles separated from the reaction medium by centrifugation at 4000 rpm and then redispersed in deionized water. This resulted in the silica shell around the MF surface of thickness around 5-10 nm. AuSiO2MFs with silica shell thickness ~80 nm were synthesized by increasing the amount of TEOS and ammonia added. Synthesis of Au@Citrate nanoparticles: Au@Citrate (AuNPs) with an average diameter of ~20 nm were synthesized by following Turkevich method.27 Here, 5 mL of 5 mM was diluted to 90 mL of DI water and heated until it boil. Afterwards, 5 mL of 0.5% trisodium citrate solution was added to the above solution and continued heating until the solution turned wine red. The solution was cooled and used for further studies. Synthesis of Au@Citrate decorated AuSiO2MFs (AuSiO2MFs@AuNPs): 0.5 mL of 3aminopropyltriethoxysilane (APTES) was added to a suspension of silica-coated AuMFs (2 mg) dispersed in isopropyl alcohol (5 mL), and left for 15 min. The solution was washed repeatedly with isopropyl alcohol to remove excess APTES and centrifuged. Further, Au@Citrate solution (1 mL) was added to the residue and incubated for 30 min. The solution was again centrifuged and the residue was washed with DI water. This process was repeated to ensure the removal of unbound AuNPs from the solution. Synthesis of gold nanorods (AuNRs): Gold nanorods were synthesized by the seed-mediated method reported by Sau and Murphy.28 Briefly, the seed nanoparticles of ~4 nm diameter were prepared by NaBH4 reduction of HAuCl4. Subsequently, the seed solution was added to the growth solution that contains Au3+ ions, AgNO3 and ascorbic acid. The resultant AuNR solution was purified by centrifugation.


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SERS measurements: For the SERS studies, 5 µL of AuSiO2MFs@AuNPs solution was mixed with 2 µL of PSA solution of desired concentration and was placed on a Si wafer. The solution was then covered with thin glass cover slip and spectra were collected at different time intervals directly from the suspension, while temperature was maintained at ~37 0C. All the SERS spectra were measured with a 1200 lines/mm grating using a Renishaw InVia Raman spectrometer coupled to a Leica microscope. The 785 nm laser was focused onto the sample by a 50x/ 0.75 N. A. objective, which could provide us a ∼1 µm laser spot size. The back-scattered signals from the samples were collected using a CCD detector. SERS spectra of amino acids and PSA-spiked serum samples were also measured as described above. The spectra were processed by removal of the spectral background. Here, cubic spline interpolation is used for the baseline fit by manually selecting the points representative of the background. Then the spectra were averaged using Origin 8.0. 3. RESULTS AND DISCUSSION 3.1 Characterization of Gold Nanoparticles-Decorated Hybrid Mesoflowers Due to the highly complex structure of proteins, composed of large number of amino acids,29,30 it is important to design a suitable SERS substrate for its label-free detection, which can give all the information regarding its structure and conformation in a reliable and reproducible manner. In view of this, we have developed a new hybrid material, where spherical gold nanoparticles (AuNPs) were closely assembled on the silica-coated AuMFs. The resultant 3D-mesoflower is called as AuSiO2MF@AuNPs, which meets all the major requirements of an ideal SERS substrate. Schematic representation of various stages of preparation of AuSiO2MFs@AuNPs is given in Figure 1A.


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Figure 1. (A) Schematics showing the preparation of AuSiO2MF@AuNPs. (B-D) are the magnified TEM images of a single stem of AuMF, AuSiO2MF, and AuSiO2MF@AuNPs, respectively. Aminopropyltriethoxysilane (APTES)-functionalized AuSiO2MFs were synthesized by following earlier reported procedure,26 which yielded MFs with average size in the range of 1-2 μm (Figure S1A). Afterwards, AuNPs were decorated on its surface by incubating them with APTES-functionalized AuSiO2MFs. The AuNPs were attached firmly on the SiO2-coated MF surface as repeated washing and centrifugation of the MFs did not result in the removal of attached AuNPs from the MF surface. Various stages of formation of AuSiO2MFs@AuNPs were also probed using the transmission electron microscopy (TEM) (Figure 1B-D). Thin silica coating of thickness 5-10 nm is clearly visible in the enlarged TEM image (Figure 1C). The magnified TEM image of closely packed AuNPs on the stem of an AuSiO2MF surface is given in Figure 1D. AuNPs of size ~20 nm were arranged onto the surface of MFs to form large number


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of closely packed AuNPs with very narrow interparticle gap (3 h) with APTEStreated bare AuMFs did not result in their decoration on the MF surface (Figure S2C). 3.2 SERS Efficiency of Hybrid Mesoflowers AuMFs is being used as a template for assembling AuNPs in this study is primarily due to its high SERS activity and stability. The unique capabilities of AuMF on trace SERS detection of analytes were compared with various isotropic and anisotropic noble metal nanoparticles. Here, the SERS spectra of 10-8 M methylene blue (MB) solution were collected using three different SERS substrates such as AuMFs, gold nanorods (AuNRs) and AuNPs (Fig. S4). The extinction spectra of AuNPs and AuNRs are given in figure S5. The concentrations of AuNPs and AuNRs kept constant (2 nM). However, due to the larger size, high scattering and tendency to settle out from the solution, we could not use the same concentration of AuMFs. In order to avoid any effect due to the surface area and particle concentrations, spectra were collected from the aggregated particles. Here, we used NaCl as an aggregating agent to induce aggregation of AuNPs and AuNRs. Well resolved SERS spectra with noticeable enhancement in the intensity of


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vibrations was observed when AuMF was used as SERS substrate (Figure S4), which confirmed the effectiveness of AuMFs as a better SERS substrate than AuNRs and AuNPs. In the case of hybrid mesoflower, AuSiO2MFs@AuNPs with an ultrathin silica shell exhibited the highest SERS enhancement. The magnified TEM image of the AuSiO2MFs@AuNPs with shell thickness ~80 nm and SERS spectra collected from the hybrid mesoflowers of different shell thicknesses are given in Figure S6. As expected, the hybrid superstructures prepared by using shell-isolated MFs with a ~10 nm silica shell showed a higher SERS activity than those prepared with a ~80 nm silica shell because of the efficient plamonic coupling between the large MF core and the satellite AuNPs. Furthermore, to demonstrate the capability of AuSiO2MFs@AuNPs in detecting molecules in complex environments, Raman spectra of a mixture of four amino acids (10-8 M) were collected. For this study, we randomly chose arginine, lysine, tyrosine and cysteine. Due to the high sensitivity of the AuSiO2MFs@AuNPs, we could successfully distinguish the Raman bands corresponding to each amino acid from their mixture even at very low concentrations (10-8 M). The SERS spectra of each amino acids and their mixture are shown in Figure S7. This indicates the high sensitivity of the AuSiO2MFs@AuNPs in yielding reliable and reproducible SERS signals from a complex environment. The enhanced SERS activity of AuSiO2MFs@AuNPs could be attributed to the following reasons. The sharp tips and edges of each stem of the MF are capable of enhancing the intensity of Raman vibrations of the analyte molecules.31 Also, thin silica coating on the MF can enhance the Raman signals via the phenomenon called shell-isolated nanoparticle enhanced Raman scattering (SHINERS).22 Here, the anisotropic nature of MF provides larger surface area for


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assembling more number of AuNPs on them. Similar dense assemblies of smaller nanoparticles on SiO2 shell are known for their high SERS activity.32 Weak van der Waals forces between proteins and silica may help to hold the proteins on the MF surface.33 The strong Au–NH2 interaction tends to closely pack the AuNPs on the SiO2 surface, leading to coupling of plasmonic field34 between the neighboring nanoparticles (AuNP-AuNP coupling). Apart from this, the plasmonic field of the closely assembled AuNPs on the MF surface can possibly couple with the inherent huge electric field around the sharp edges and tips35 of the MFs (AuSiO2MFAuNPs coupling). This can also provide a major contribution towards the greater SERS activity. PSA has been recognized as an important serum biomarker protein for screening prostate carcinoma.36 However, screening of PSA may also lead to false positive results and overdiagnosis of prostate cancer. Hence, the detection of PSA at ultralow concentration is important for the early stage detection of prostate cancer. We found that AuSiO2MFs@AuNPs can detect PSA solution even at very low concentration (2.5 ng/mL) with high spectral purity (Figure 2 and Figure S8). Though bare AuSiO2MFs themselves are known for their high SERS activity,26 the Raman intensities of PSA were not resolvable indicating the important role of the closely assembled AuNPs on the MF surface in enhancing the Raman signals of the protein (Figure S9). For collecting the SERS spectra, PSA solution (2.5 ng/mL) was incubated with hybrid MF at 37±2 0C for 30 min. The solution was gently vortexed prior to the use. Afterwards, 10 microliter of this solution was transferred onto a silicon wafer and spectra were collected from different areas of the same sample by focusing the laser onto the clustered hybrid MF (Fig. S8A). Highly reproducible spectra were collected from various regions of the same sample, which demonstrate the high sensitivity of AuSiO2MFs@AuNPs in measuring reproducible SERS spectra (Fig. S8A). A plot showing the ratio of intensities of different vibrations such as I644/I679,


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I810/I843 and I974/I1000 bands is given in Fig. S8C. Here, spectra were collected from the six different regions of the same sample.

A#

25#min#

B#

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Intensity((a.(u.)(

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Raman(shi1((cm31)( Figure 2. A and B are different regions of the SERS spectra of PSA collected as a function of time using AuSiO2MFs@AuNPs substrate.


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3.3 Investigation of Possible Conformational Changes in PSA Using SERS The interaction of PSA with hybrid MF and subsequent conformational modifications were also studied in details. SERS spectra collected after 5 min of incubation yielded inconsistent spectra with substantial variations in the intensities of vibrations (Figure S10), which clearly indicated the vital role of incubation time of nanoparticles with proteins during SERS measurements in getting reproducible spectra. We also noted that SERS measurements at lower temperature (~23 0

C) require comparatively higher incubation time (>3 h) to get reproducible SERS spectra of

PSA. The SERS results also show a conformational rearrangement of PSA upon interaction with the hybrid MF. In order to probe this, the spectra were collected at 37±2 0C in a time dependent manner (Figure 2) after incubating AuSiO2MFs@AuNPs with PSA solution. Substantial variation in the extent of adsorption of PSA on the hybrid MF surface was reflected as modifications in the intensities of Raman vibrations in the SERS spectra (Figure 2 and Figure 3A). The proteins are more likely to interact with AuNPs-decorated on the MF surface through preferential adsorption sites, presumably due to greater surface curvature of the spherical particles.37 Due to the high binding affinity of sulfur atom towards AuNPs surface, amino acid residues containing sulfur atom present in PSA is likely to interact with Au atoms. Highly anisotropic nature of AuMF can provides high surface area for accommodating more AuNPs in a single MF, thereby to accommodate more number of proteins in the vicinity of high electromagnetic field. As shown in Figure 2, the two bands observed at ~644 and ~679 cm-1 indeed correspond to the C-S stretching vibrations of two rotamers of cystine and methionine.3840

Due to the S–S moiety in cystine different conformations of the molecule are possible,

depending on the dihedral angle of the C–S–S–C bonds,41 which was also confirmed by spectral


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changes in the region of the C-S modes (600-700 cm-1). The subtle structural changes in the conformation of PSA upon adsorption onto the AuNPs surface were identified by the C-S band analysis. The disulfide bonds are considered to be the entity that lock proteins into particular folds or configurations.39 At the beginning of adsorption (up to 10 min), the intensity of the 679 cm-1 band was relatively high compared to the Raman band at 644 cm-1 (Figure 3). These bands are mainly attributed to the vibrations corresponding to two possible conformation of C-S bond, trans and gauche internal rotation about the –CH2–S– bond.40

Ratio of intensities

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I644/679' B I1220/1282' D

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Figure 3. (A) Plot showing variation in the ratio of intensities of various Raman vibrations. (B) Crystal structure of PSA complexed with an activating antibody (PDB 2ZCH)30. The enhanced signals of the N–H in plane deformation of amide III bands (also contribution from overlapping bands from side chain vibrations) usually appear in between 1210-1300 cm-1 can also be taken as evidence for additional interactions of the protein with the metal through the amide group of the amino acids.42 The amide III band of protein composed of three vibrations corresponds to the α-helix, random coil and β-sheet conformations, which appears generally


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around 1260-1300, 1245-1255, and 1210-1235 cm-1, respectively.42,43 Among the three amide III vibrations, the Raman band of the β conformer appeared as the prominent band (I1220/I1282> 1) in the beginning (

Gold-Nanoparticle-Decorated Hybrid Mesoflowers: An Efficient

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