Protein Discrimination Using Fluorescent Gold Nanoparticles on

Letter pubs.acs.org/ac

Protein Discrimination Using Fluorescent Gold Nanoparticles on Plasmonic Substrates Hao Kong, Yuexiang Lu, He Wang, Fang Wen, Sichun Zhang, and Xinrong Zhang* Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Department of Chemistry, Tsinghua University, Beijing 100084, P.R. China S Supporting Information *

ABSTRACT: Fluorescent gold nanoparticle (GNP) is an easily synthesized and biocompatible optical platform for sensing and imaging with tunable near-infrared (NIR) emission. However, the relatively low fluorescence (FL) quantum yield limits the further improvement of sensitivity and application. Here, we find that, on plasmonic substrates, the FL intensity of proteindirected synthesized GNPs can be enhanced significantly (∼20-fold). Moreover, protein analytes can interact with GNPs and influence the enhanced fluorescence process so that we can obtain distinct FL image patterns. Then, using the array-based sensing strategy, protein discrimination can be achieved. In our present experiment, five GNPs were used as sensing elements and 10 kinds of proteins at three concentrations (0.2, 0.5, and 1 μM) were successfully identified. This array-based sensing strategy using enhanced-fluorescence from GNPs is highly sensitive and differentiable, expanding the application field of GNPs. ecently, fluorescent gold nanoparticles (GNPs) have attracted great interest due to their advantages in contrast to semiconductor quantum dots (QDs), such as ultrasmall size and biocompatibility and especially being greenly synthesized for protein-directed GNPs.1 Several applications of GNPs have been reported, including detection of ions based on fluorescence quenching,2 detection of H2O2 using active enzyme integrated GNPs,3 detection of trypsin using bovine serum albumin (BSA)−GNPs conjugates,4 investigation of the protein absorbance effect on GNPs,5 visual detection of trinitrotoluene,6 identification of amino acids,7 and cell marking for in vivo imaging,8 etc. For most GNPs, the relatively low fluorescence (FL) quantum yield limits the improvement of sensitivity and application, so the enhancement of GNPs’ FL emission would play a key role in further application of GNPs. Herein, we report an approximately 20-fold enhancement of FL emission from GNPs on plasmonic substrates. The plasmon-enhanced fluorescence (PEF) results from resonant coupling of FL emission with localized surface plasmons on metallic nanostructures.9 To the best of our knowledge, we here report the first case of PEF of protein-directed GNPs. Moreover, we find it more interesting that enhanced fluorescence from different GNPs changes distinctly in the presence of analyte proteins and for each analyte, the FL change profiles are unique just like “fingerprints”. Hence, an array of GNPs can be cross-responsive to proteins as an excellent “chemical nose/tongue” approach10 for convenient, rapid, and low-cost protein sensing. Strategically, as opposed to “lock-key” specific recognition, semiselective and cross-reactive sensor arrays can generate a response pattern that is fed into a computer for analyte discrimination.11 In comparison with previous arraybased sensing strategies employed for biological matrix discrimination,12 fluorescent GNPs are biocompatible and diverse for the improvement of differentiability considering that many

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© 2012 American Chemical Society

biomolecules with semispecific activity such as aptamers could be applied to the construction of fluorescent GNPs.2−4 As a proof-of-concept system, we create a sensor array (Figure 1) of GNPs on plasmonic substrates, which provide differential FL changing patterns that can be used for protein discrimination. Due to the significant enhanced fluorescence and multidimensional signal recording mode, our array is highly sensitive and differentiable, expanding the application field of fluorescent GNPs. First, according to the protein-directed reduction method (see more details in the Supporting Information), five GNPs were synthesized using five proteins as reductants and scaffolds: bovine serum albumin (BSA), human serum albumin (HSA), egg white albumin (EA), lysozyme (lys), and horseradish peroxidase (HRP), and nanostructured silver substrates were made by chemical deposition of Ag coatings on glass slides using silver-mirror reaction, as described in the Supporting Information. As shown in Figure 2, at 405 nm laser excitation, five GNPs directly added on Ag substrate and then blow-dried gave much stronger FL emission than that on bare glass. Owing to the plasmons of the surface of Ag substrates, about a 20-fold increase in FL intensity was observed through a laser scanning confocal microscope (see more details in the Supporting Information). Compared with organic fluorophores and classical QDs, this system needed no spacer between Ag substrates and GNPs. We speculated that protein scaffolds might prevent GNPs from quenching in contact with Ag substrates. We then investigated the FL change profiles in the presence of protein analytes. Ten proteins (Table S1, Supporting Information) Received: March 13, 2012 Accepted: April 30, 2012 Published: April 30, 2012 4258

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Figure 1. Schematic of our sensor array. The presence of protein analytes changes the PEF profiles of five GNPs distinctly, which can be used for protein discrimination.

These differential responses like “fingerprints” prompted us to develop a pattern recognition strategy for protein discrimination, so the raw data of FL intensities (Table S2, Supporting Information) were then subjected to linear discriminant analysis (LDA, performed using SPSS v16.0).15 The FL response patterns were transformed into canonical scores which were visualized as a well-clustered three-dimensional plot (Figure 3C). All of the 50 cases (10 proteins × 5 replicates) were correctly assigned to their respective groups. The discriminant ability was further tested by sensing protein analytes at different concentrations (0.2, 0.5, and 1 μM). As shown in Figure S5 and S6 (Supporting Information), the sensing of protein analytes at 0.2 and 1 μM was also achieved accurately. To study the similarity of protein analytes with the concentration change, hierarchical cluster analysis (HCA), a routine model-free statistical classification method based on Euclidean distance, was performed by SPSS v16.0.16 From the dendrogram generated by HCA (Figure 4), we found that the influence of trypsin to GNPs changed most obviously with the increase of concentration as discussed above. For the other protein analytes, the similarity between each other did not change a lot, but protein analytes could not give linear responses dependent on the concentration, making the simultaneously qualitative and quantitative analysis difficult. Lastly, 60 protein samples at three concentrations referred above were prepared for the blind tests. According to their shortest Mahalanobis distances,12f these unknown cases (see Table S3 in the Supporting Information for original data and the result of assignment) were identified with 100% accuracy based on the training matrix (10 known protein samples × 3 concentrations × 5 replicates, listed in Table S2, Supporting Information). It was demonstrated that the sensitivity and differentiability of our technique was high enough to detect and identify proteins at 0.2 μM, more sensitive than many previously proposed methods based on “chemical tongue” strategy (>1 μM).12a,b,g,17 In summary, we observed that the FL intensity of GNPs is enhanced about 20-fold on Ag substrate as compared to that on glass. Furthermore, analyte proteins can interact with GNPs and influence the enhanced fluorescence process so that we can obtain distinct FL image patterns, using which protein discrimination can be achieved. Given the good biocompatibility and diversity of GNPs, we foresee this “proof-of-principle” study will broaden the application field of GNPs in biosensing and imaging. First, the detection sensitivity of the previous reports based on GNPs2−4 may be improved since the FL emission can be enhanced about 20-fold. Second, the FL enhancement on plasmonic substrates seems to be universal for GNPs synthesized using different proteins, promising us to design enhanced-fluorescent GNPs integrated with diverse functionalized proteins for biomarker detection. In our ongoing studies, we are exploiting both new semispecific biomolecule scaffolds for GNP construction and plasmonic substrates with higher performance to further improve the differentiability and sensitivity and apply this methodology to complex biomatrixes.

Figure 2. FL images of five GNPs on glass (left) and Ag substrates (right). (A) BSA−GNPs, (B) HSA−GNPs, (C) EA−GNPs, (D) Lys− GNPs, and (E) HRP−GNPs. For the screening of the extremely low FL signal from GNPs on bare glass, the contrast is adjusted so that the FL intensity scale is different. The intensity scale bar is 0-1023 for GNPs on glass and 0-4095 for on Ag substrates.

with diverse characteristics were chosen as the sensing targets. As illustrated in Figure 3A, the presence of analyte proteins (0.5 μM) resulted in a variety of FL responses. This result confirmed that the interaction between a given analyte protein and varying fluorescent GNPs would influence the PEF process and lead to distinct change profiles. As shown in Figure 3B, when the protein analyte was the same as GNP’s protein scaffolds, the FL emission barely changed, but other protein analytes obviously increased or decreased the signal. The primary cause was speculated that protein analytes might displace the protein scaffolds of GNPs and construct GNPs with higher FL emission or during this process GNPs would aggregate, resulting in FL quenching.3 For example, the intrinsic FL intensity of HRP− GNPs was the lowest among the five fluorescent GNPs while HSA and Pap could significantly amplify the emission, and lysozyme enhanced the FL emissions of BSA−GNPs and HSA−GNPs but quenched ones of EA−GNPs and HRP−GNPs. We also studied the influence of proteins to GNPs in solutions. As shown in Figure S7 in the Supporting Information, without two exceptions (BSA against BSA−GNPs and Try against Lys− GNPs) taken into consideration, the FL change trends in solutions were consistent with the ones on plasmonic substrates, but the FL change extents were not exactly the same. We speculated that the protein spacer between plasmonic substrates and the luminescent Au center might also affect the FL intensity.13 Additionally, to our astonishment, instead of quenching, trypsin increased the FL emission tremendously except HRP−GNPs. In order to study the cause, we used trypsin as scaffolds to directly synthesize GNPs. As shown in Figure S8 (Supporting Information), we used trypsin to directly synthesize fluorescent trypsin−GNPs. Accordingly, we speculated that, after addition of trypsin, brighter GNPs might be constructed, but excess trypsin might destruct protein−GNP conjugates and decrease FL emission since 1 μM trypsin decreased the FL emission of most GNPs (Figure S6, Supporting Information). Notably, the three metal-containing proteins (Hem, Myo, and TRF) decreased the FL emission of all the GNPs except the case of TRF against Lys−GNP. We thus speculated that iron contained in proteins might have the ability of FL quenching.14 4259

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Figure 3. (A) FL change profiles of GNPs (from left to right: BSA−GNPs, HSA−GNPs, EA−GNPs, Lys−GNPs, and HRP−GNPs) in the presence of protein analytes (0.5 μM). (B) FL intensity change patterns as an average of 5 parallel measurements. I0 is the average FL intensity of GNPs in the absence of the protein analytes, and ΔI is the difference value of the average FL intensity in the presence and absence of the protein analytes. (C) Canonical score plots for the first three factors of FL response patterns analyzed by LDA.

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ASSOCIATED CONTENT

21125525) and the Tsinghua University Initiative Scientific Research Program.

S Supporting Information *

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Experimental details and additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax:(+86)106278-7678. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support of the National Natural Foundation of China (Grant No. 21027013 and No. 4260

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