Aptamer-Based Plasmonic Sensor Array for Discrimination of Proteins

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Aptamer-Based Plasmonic Sensor Array for Discrimination of Proteins and Cells with the Naked Eye Yuexiang Lu,† Yueying Liu,‡ Suge Zhang,‡ Song Wang,† Sichun Zhang,*,† and Xinrong Zhang† †

Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing, 100084, P.R. China ‡ Department of Chemistry, Capital Normal University, Xisanhuan North Rd. 105, Beijing, 100048, P.R. China S Supporting Information *

ABSTRACT: We developed a colorimetric sensor array with reported protein aptamers as nonspecific receptors. We found that different target proteins could make the aptamer-protected gold nanoparticles (AuNPs) exhibit different aggregation behaviors in the presence of a high concentration salt and cause various color change. On the basis of this phenomenon, we applied a series of reported protein aptamers as a receptor array obtaining a distinct response pattern to each target protein. Seven proteins have been well distinguished with the naked eye at the 50 nM level. Cancerous human cells have also been discriminated from noncancerous cells. This method is simple, label-free, and sensitive. It will broaden the application filed of plasmonic nanoparticle-based sensors and give a new direction of developing sensitive array sensing systems.

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unpredictable, a sufficiently large DNA library is needed for identification. Moreover, the fluorescently labeled DNA-based method is complicated, and the detection needs professional instruments. It is highly needed for one to develop appropriate receptors and a corresponding simple and sensitive array sensing system, especially with naked eye detection. Compared to random designed DNA, the selectivity of aptamers is more predictable, as an aptamer could only give affinity to a limited number of target proteins with different degree.14 Therefore, a group of the aptamers binding to a target protein may form a unique pattern, which could be used for the discrimination of the proteins by the sensor array. Higher discrimination ability for proteins could be achieved by limited sensing elements. Moreover, aptamer could bind to the unmodified AuNPs and protect them from salt-induced aggregation.15−17 The competition binding of aptamers by target proteins removes them from the AuNP surface, resulting in the aggregation of AuNP, and the color changes could be observed by the naked eyes.18 Herein, we build a plamonic sensor array for naked eye detection with aptamers as receptors. We found that the aptamer-protected AuNPs exhibited different aggregation behaviors when mixed with target proteins, giving various color change. The variation of the color change as fingerprint could be used to discriminate proteins even at a low level of

n resource-constrained areas, plasmonic nanoparticle-based visual detection can potentially improve the standard of living as it is simple and cost-effective.1−4 Any target that directly or indirectly triggers the wavelength of plasmonic resonance changes can be colorimetrically detected. The most classical colorimetric sensor proposed by Mirkin’s group is based on the target-caused aggregation of functioned gold nanoparticles (AuNPs) and has been used for detection of a broad range of targets.5 Recently, target-guided growth or regrowth of plasmonic particles have been reported and used for unltrasensitive detection of disease biomakers.6,7 However, most of the existing methods are based on a “lock-key” sensing mode, wherein it requires a highly selective receptor for each analyte to be detected. The limited supply of high-specificity receptors forms a major bottleneck for the further application of this method. A chemical sensor array, which is able to respond differentially to a variety of analytes, might be an alternative method to solve this problem. In the “array” strategy, receptors with lower selectivity give a distinct response pattern of each analyte as a fingerprint for classification and identification.8 Although many kinds of receptor systems have been developed for array sensing, such as substituted porphyrins,9 synthetic polymer−nanoparticle systems,10 and nanoparticles,11 their large-scale application is still hampered by the difficulty of the molecular design, synthesis, or chemical modification. Recently, using DNA as receptor has been proposed and applied for array sensing of biotargets by using fluorescently labeled DNA12 or DNA-decorated catalytic AuNPs.13 However, as the affinity between random designed DNA receptors and targets is © 2013 American Chemical Society

Received: May 15, 2013 Accepted: June 25, 2013 Published: June 25, 2013 6571

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Letter

also caused an obvious color change from red to purple, and the k/k0 values were 3.93 and 3.81, respectively. HSA, EA, and Pep gave a lower response signal as the solution color was still red, and the k/k0 values were 0.66, 1.07, and 0.54, respectively. These results demonstrated that an aptamer-protected AuNP could give a different colorimetric response to proteins in the presence of a high concentration salt. On the basis of this phenomenon, a group of reported protein aptamers as receptor array should give a distinct response pattern to each target protein. Two thrombin aptamers (Tro-1 and Tro-2)21,22 and one Human Immunoglobulin E aptamer (HIgE-1)23 with different sequences, structures, and lengths were selected as the receptor array for the discrimination of proteins (Figure 2A). We found

concentration, and cancerous human cells could be discriminated from noncancerous cells.

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RESULTS AND DISCUSSION In our preliminary experiment, we selected seven proteins that have different molecular weight (MW), isoelectric point (pI), and metal/nonmetal containing properties as the sensing targets (Table 1) and studied the response behavior of target Table 1. Physical Properties of Proteins proteins

MW (kDa)

pI

lysozyme (Lys) hemoglobin (Hem) human serum albumin (HSA) egg white albumin (EA) pepsin (Pep) papain (Pap) myoglobin (Myo)

14.4 64.5 69.4 44.3 35.0 23.0 17.0

9.6−11 6.8 5.2 4.6 1.0−2.5 9.6 7.2

proteins on a lysozyme aptamer (Lys-1), which is the aptamer of the target protein (Lys).19 Water-soluble 13 nm AuNPs were prepared through the classical citrate-reduction process with no further chemical modification.20 The probe solution of 10 μL of aptamer mixed with 100 μL of AuNPs was added into the seven protein solutions, respectively. Then, 70 μL of NaCl (0.2 M) was added and shaken mildly for 15 min. We found that the aptamer (Lys-1) gave a different response to target proteins and caused a various color change (Figure 1). The color change of

Figure 2. (A) Sequences of three aptamers. (B) Fingerprints of seven selected proteins based on the patterns of the corresponding values of k/k0 obtained from Tro-1, Tro-2, and HIgE-1.

that, for the same DNA receptor, the colorimetric response varied with the addition of different proteins. On the other side, for the same protein, the colorimetric response was dependent on different DNA receptors (Figure 2B). The response patterns categorize the seven proteins into two groups. The first group contains Lys, Hem, Pap, and Myo. The k/k0 value of these proteins are greater than 3.00. While, for the second group (HSA, EA, and Pep), all the k/k0 values are less than 2.00. The biggest difference between these two groups is isoelectric point (pI). The pI value of proteins in the first group is higher than 6.8, while the value is lower than 5.2 for the second group. Generally, the unmodified AuNPs are loosely capped by negatively charged citrates. As a result, they show high affinity to flexible, positively charged molecules but much less affinity to rigid, negatively charged ones.2,15 The pH value of the probe solution was about 7.0. That means the proteins with higher pI could be positively charged and bind to these AuNPs more effectively, resulting in removing the protection from DNA receptors. Moreover, as these aptamers have different length and secondary structures, they could respond differently to proteins by various interactions such as hydrogen bonding, electrostatic interaction, and π−π stacking. Furthermore, it has been reported that some proteins could protect the AuNPs from aggregation too.18 On the basis of these reports and our results, we attribute the final response signal to the equilibrium among AuNPs, DNA, and proteins.

Figure 1. (A) Schematic illustration of the transduction principle of the AuNPs-DNA colorimetric sensing array. (B) Photograph of the color change upon addition of different protein. The DNA receptor is Lys-1 (5′-ATCAGGGCTAAAGAGTGCAGAGTTACTTAG-3′), and the protein concentration is 50 nM.

the solutions was caused by different aggregation behaviors and could be observed by naked eyes (Figures S1 and S2, Supporting Information). The colorimetric signals could also be obtained by recording the absorbance of the mixture at 520 and 620 nm (Figure S3, Supporting Information). Lys, which is the corresponding protein of Lys-1, gave a very strong response, and the solution color became blue. While the response signal of Hem (k/k0 = 4.81, k = OD620 nm/OD520 nm) was even higher than that of Lys (k/k0 = 4.44). Pap and Myo 6572

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Linear discriminate analysis (LDA), a statistic method for recognizing the linear combination of features that differentiate two or more classes of object, was used to differentiate quantitatively the response patterns of target proteins.24 Three canonical factors were generated (97.8%, 1.9%, and 0.3%) that represent linear combinations of the response matrices obtained from the response patterns (three DNA × seven proteins × five replicates). The 35 training cases were separated into seven respective groups, with 100% accuracy according to the classification matrix derived from the analysis of subsets of the data sets, and the two factors that were most significant are plotted in 2D (Figure 3). The performance of our sensor array Figure 4. Canonical score plot for the response patterns as obtained from LDA for four cells.

sensing array will be a platform for detecting a broad range of targets involved in diagnosis and environmental monitoring in poorly equipped areas. The present work will broaden the application filed of plasmonic nanoparticle-based sensors and give new direction of developing sensitive array sensing systems.

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ASSOCIATED CONTENT

S Supporting Information *

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

Figure 3. Canonical score plot for the response patterns as obtained from LDA for seven proteins at 50 nM.

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was tested by the discrimination of unknown samples. The discrimination accuracy of 23 unknown samples at 50 nM was found to be 100% (Tables S1 and S2, Supporting Information). It meant that our technique could detect and identify proteins at a concentration as low as 50 nM, which is much lower than the recently proposed methods based on “chemical tongue” strategy (0.2−5 μM).12,25−27 To test the ability of our sensor array to analyze complex samples, the array was used for the detection of cells which is important for diagnosis of cancers.28 It is expected that the DNA array should provide different interactions with cell surface functionalities, e.g., lipids, proteins, and polysaccharides, resulting in different response patterns. We chose three types of human cancer cells (Junkat, Reh, and Raji) and one type of normal human cell (WIL2-S) as sensing targets. The 20 training cases were separated into four respective groups with 100% accuracy (Figure S4, Supporting Information). The three canonical factors are 70.0%, 27.4%, and 2.6%, and the plot of the first two factors is presented in Figure 4. We then tested the system against unknown samples, and the discrimination accuracy of 17 unknown samples was 100% (Tables S3 and S4, Supporting Information).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: +86 010 62787678. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the National Natural Foundation of China (Grant No. 21027013, No. 21125525, and No. 21105066) and the Tsinghua University Initiative Scientific Research Program.

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CONCLUSION In conclusion, we found that an aptamer-protected AuNP could give a different response to target proteins. On the basis of this phenomenon, we applied three reported protein aptamers as receptor array obtaining a distinct response pattern to each target protein. This method is simple, label-free, and sensitive, and only limited sensing elements are needed for achieving a high discrimination ability of proteins. As DNA receptors can bind not only with biological macromolecules but also with small molecules, metal ions, and other targets, this colorimetric 6573

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