Nano Rolling-Circle Amplification for Enhanced SERS Hot Spots in

Oct 9, 2012 - Here we report a new strategy based on nano rolling-circle amplification ... circle amplification (nanoHRCA) to increase “hot spot” ...
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Nano Rolling-Circle Amplification for Enhanced SERS Hot Spots in Protein Microarray Analysis Juan Yan,† Shao Su,† Shijiang He,† Yao He,‡ Bin Zhao,† Dongfang Wang,† Honglu Zhang,† Qing Huang,† Shiping Song,*,† and Chunhai Fan† †

Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China Institute of Functional Nano & Soft Materials (FUNSOM) and Jiangsu Key laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou, Jiangsu 215123, China

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S Supporting Information *

ABSTRACT: Although “hot spots” have been proved to contribute to surface enhanced Raman scattering (SERS), less attention was paid to increase the number of the “hot spot” to directly enhance the Raman signals in bioanalytical systems. Here we report a new strategy based on nano rolling-circle amplification (nanoRCA) and nano hyperbranched rollingcircle amplification (nanoHRCA) to increase “hot spot” groups for protein microarrays. First, protein and ssDNA are coassembled on gold nanoparticles, making the assembled probe have both binding ability and hybridization ability. Second, the ssDNAs act as primers to initiate in situ RCA reaction to produced long ssDNAs. Third, a large number of SERS probes are loaded on the long ssDNA templetes, allowing thousands of SERS probes involved in each biomolecular recognition event. The strategy offered high-efficiency Raman enhancement and could detect less than 10 zeptomolar protein molecules in protein microarray analysis.

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SERS signal harvested from each microspot might be so weak that it can hardly be detected. In such a nanoparticle-based SERS system, the observed large Raman scattering enhancement is mostly attributed to the electromagnetic effect arising from the Ag nanoparticles grown around the fluorophore-labeled AuNPs. Actually, electromagnetic “hot spots” show inordinate SERS intensities, which allow for the ultrasensitive detection of molecules.10−12 Although there are some arguments on “hot spots”, more and more studies have confirmed their existence.13−18 Thus, the increase in the number of “hot spots” affects the SERS intensity in a much more direct way. However, most of previous studies were focused on the fabrication of solid phase substrates containing more “hot spots”. These methods are not able to be used in quantified bioassays such as protein microarrays because such “hot spots” could not quantify biorecognition events.19−21 So it is necessary to directly increase the amounts of “hot spots” in signaling system of protein microarrays. However, such designs have received less attention compared with other effort such as the development of new nanomaterials for Raman labels.22 Here, we have developed a new SERS-based strategy for protein microarray detection that is focused on increasing the number of “hot spots” in signaling system via nano rolling-circle amplification (nanoRCA) and nano hyperbranched rolling-

ver the past twenty years, protein microarrays have been widely used in both basic and applied biological research.1−3 However, protein microarray technology is still suffering “growing pains”.4 Although fluorescent dyes are mostly used in current protein microarrays, they have some insurmountable limitations, particularly low sensitivity. The sensitivity of protein microarrays are often considered insufficient when the detection involves particularly small samples or scarce protein targets.4 Throughout the past decade, functional nanomaterials have been actively explored to greatly enhance the sensitivity of bioassays by exploiting their several unique optical, electronic and catalytic properties.5 For example, we previously developed nanoparticle probes to improve the sensitivity of protein microarrays.6 Coupling with a signal-amplification method based on nanoparticle-promoted reduction of silver(I), the sensitivity of the microarray detection system exceeds that of the analogous fluorophore system by 3 orders of magnitude. However, the gold−silver staining method7 inherently results in a high background and low signal-to-noise ratio. Surface-enhanced Raman spectroscopy (SERS) opens up exciting opportunities for applications of functional nanoprobes in ultrasensitive bioassays.8 Mirkin and co-workers developed a SERS-based DNA and RNA detection system with an analogous gold−silver staining method, using fluorophore-labeled gold nanoparticles (AuNPs) instead of nonlabeled nanoparticles.9 The system had a detection limit of 20 femtomolar and provided high selectivity. However, protein microarrays need much stronger enhancement because the © 2012 American Chemical Society

Received: July 2, 2012 Accepted: October 9, 2012 Published: October 9, 2012 9139

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polymerase at 37 °C for 16 h. The enzymes were denatured by heating at 85 °C for 10 min. DNA template was removed by using a centricon filtration device (30 000 Da cutoff). Preparation of RCA and HRCA Products. Ten microliters of biotin-DNA (first primer, 100 μM) was added to the nanoconjugates (Au-SA, 200 μL), and incubated with gentle shaking at room temperature for 30 min. The mixture was repeatedly centrifuged at 4 °C for 20 min (12000 rpm) three times, and the precipitate was redispersed in phosphate buffer (200 μL, 10 mM, and pH 7.4). The nanoconjugate (Au-SAbiotinDNA, 7.5 μL) was mixed with the RCA reaction mixture to produce a final volume of 30 μL; the mixture consisted of circular DNA template (9 μL), dNTPs (3 μL, 10 mM), RCA buffer (3 μL, 10 × ), phi29 polymerase (3 μL, 10 U/μL), MilliQ water (4.5 μL). Then, the reaction was performed at 30 °C for 30 min. After 6 μL Au-SH-complementary DNA was added to the RCA product solution, the mixture was frozen at −20 °C and then incubated overnight at room temperature for hybridization. The solution was centrifuged at 4 °C for 5 min (5000 rpm) once, and the precipitate was redispersed in 20 μL Milli-Q water. At the same time, nanoconjugates (Au-SAbioDNA, 10 μL, and Au-SH-Cy3-DNA, 6 μL) were mixed with the RCA reaction mixture to produce a final volume of 40 μL; the mixture consisted of circular DNA template (12 μL), dNTPs (4 μL, 10 mM), RCA buffer (4 μL, 10 × ), phi29 polymerase (4 μL, 10 U/μL). Then, the reaction was performed at 30 °C for 10 min. The product was centrifuged at 4 °C for 5 min (5000 rpm) and the redispersed in Milli-Q water (20 μL) as well. RCA and HRCA product were examined by atomic force microscopy (AFM). Preparation of Protein Microarray. Secondary antibodies (B8895, 0.2 mg/mL, dissolved in PBS with a concentration of 150 mmol/L, and pH 7.4, 30% glycerol) were deposited on an aldehyde-acivated chip (5 × 5, approximately 150 μm in diameter for each spot; the distance between two spots was 500 μm). After it was incubated overnight at 4 °C, the slide was rinsed with the washing buffer (150 mmol/L PBS, 0.25% Tween20) and blocked with the blocking buffer (150 mmol/L PBS, pH 7.4, 5%BSA) for 30 min. Then, the nanoconjugates (Au-SA-primer, 20 μL) were added to the grids on the surface of the slide and incubated at 37 °C for 30 min. Circle DNA (2 μM, 8 μL), dNTPs (10 mM, 4 μL), amplification reaction buffer (10×, 2 μL), and phi29 polymerase (10 U/μL, 2 μL) BSA (1%, 2 μL) Milli-Q water (2 μL) were added to the mixture (20 μL). RCA was carried out at 30 °C for 30 min. Then, 2 μL Au-SH-Cy3-complementary DNA (dissolved in PBS buffer, 150 mM, and pH 7.4, 20 μL) was added to hybridize with the RCA product after the rinsing step. Solution A and B were mixed in a 1:1 ratio (40 μL) and added to each grid. After 15 min at room temperature, the slide was rinsed with washing buffer (150 mM phosphate, pH 7.4, 0.25% Tween 20) three times and gently blown dry with N2. For HRCA, 2 μL of the second primer was coupled with the other amplification mixture, which contained circle DNA (2 μM, 8 μL), dNTPs (10 mM, 4 μL), amplification reaction buffer (10×, 2 μL), and phi29 polymerase (10 U/μL, 2 μL) BSA (1%, 2 μL). The other steps were the same as those described above for RCA. Characterization of RCA and HRCA Products. RCA and HRCA product were examined by drop-casting approximately 2 μL of the products onto a cleaned and freshly peeled mica surface. After 5−10 min, the sample was rinsed with Milli-Q water droplets and gently blown dry with N2. All samples were imaged under ambient conditions in the tapping mode.

circle amplification (nanoHRCA). Our previous studies have demonstrated that on-particle rolling-circle amplification (RCA) could provide high sensitivity and a broad detection range of proteins in biological fluids.23 The nanoRCA-SERS strategy integrates the advantages of both the SERS effects of functional nanoprobes and signal-amplification effects of nanoRCA. The detection capability of the system greatly exceeds that of the analogous gold−silver staining system with the same RCA process. The detection limit of the system for protein microarrays is lower than 10 zeptomolar. Moreover, coupling with hyper-branched rolling-circle amplification (HRCA), the system demonstrates a much stronger enhancing effect. Overcoming the limitations of conventional methods, the novel nanoRCA-SERS system offers high-efficiency Raman enhancement as well as high detection sensitivity for protein microarrays.

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EXPERIMENTAL SECTION Materials. DNA oligonucleotides were synthesized and purified by TAKARA Biotechnology (Dalian, China). The sequences of these oligonucleotides are shown in the Supporting Information (Table S1). Gold chloride trihydrate (HAuCl4·3H2O) was purchased from Sigma-Aldrich. Cy3 dye was purchased from Bio Chemical (Dalian, China). Bovine serum albumin (BSA), streptavidin, a silver enhancement kit, and PEG (polyethylene glycol) were purchased from Sigma− Aldrich (St. Louis, MO, USA). T4 DNA ligase, T4 DNA polymerase, and dNTP were purchased from MBI Fermentas, and phi29 polymerase was purchased from NEB. Tween 20 was purchased from Fluka. Other chemicals were of analytical grade, and water was purified using a Millipore filtration system (MilliQ water). Preparation of NanoRCA Conjugates. AuNPs with an average diameter of 15 nm were prepared by using the citrate reduction method.24 After a solution of 15-nm AuNPs (1 mL, pH 7.6) was incubated with streptavidin (20 μg/mL) with gentle shaking for an hour (AuNPs conjugated to SA via noncovalent absorption),25 approximately 110 μL 1% PEG was added to the solution of AuNPs−SA conjugates. The solution was incubated at room temperature overnight. The final solution was repeatedly centrifuged at 4 °C for 20 min (12000 rpm), and the precipitate was redispersed in phosphate buffer (200 μL, 10 mM, and pH 7.4). The solution was stored at 4 °C. Twenty microliters (100 μM) SH-Cy3-DNA was added to the 15-nm AuNP solution (1 mL), which were allowed to sit overnight at room temperature. Then, phosphate buffer (100 mM) was added to the solution to produce a final concentration of 10 mM. The solution was incubated with gentle shaking at room temperature for 30 min. A salting buffer (10 mM PB, 2 M NaCl, pH 7.4) was then added to the mixture to create a final ionic strength of 0.15 M. The solution was incubated at room temperature overnight. The final solution was repeatedly centrifuged at 4 °C for 20 min (12000 rpm), and the precipitate was redispersed in phosphate buffer (200 μL, 10 mM, with 0.15 M NaCl, pH 7.4). The solution was stored at 4 °C. Preparation of Circle DNA Templates. Phosphorylated linear DNA (100 μM) and the ligation template DNA (100 μM) were mixed and incubated at 90 °C for 1 min. When the solution was cooled to room temperature, T4 ligase and T4 ligase buffer were added and the mixture was incubated at 25 °C for 16 h. The enzymes were denatured by heating at 65 °C for 10 min. The ligated circular DNA was then treated with T4 9140

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Scheme 1. Schematic Illustration of NanoRCA-SERS-Based Protein Microarray Strategya

a First, patterns of biotin-secondary antibody were captured onto the surface of a slide; then, after blocking with BSA, Au−SA, and biotin-primer were added step by step for recognition. The amplification mixture (circle DNA, phi29 polymerase, dNTP, and buffer) was then added to initiate the rolling-circle amplification process. Au-Cy3-DNA was then added to hybridize with RCA product (ssDNA). Finally, amplification by silver enhancement was performed.

Instrumentation. Silylated slides (aldehyde) were obtained from CEL Associates (Pearland, U.S.A.). SmartGrid 12-sample Grid was from CapitalBio (Beijing, China). The protein microarray slide was examined by optical microscopy. The Raman spectra was obtained with a NT-MDT confocal Raman microscopic system (laser wavelength 473 nm and laser spot size is 0.4 μm). SERS analysis was performed using a HR800 Raman microscope instrument (HORIBA, Jobin Yvon, France) using the standard 633-nm HeNe 20 mW laser with a laser spot of 1 μm . All of these SERS spectra were obtained using the same parameters (e.g., objective: 50× NA 0.7, acquisition time ∼3s, hole∼400, slit ∼100, grating ∼600 g/mm). LabSpec 5 software was used for Raman data acquisition and data analysis. Protein microarrays were fabricated with MicroarrayersSpotBot2 (Arrayit Corporation, USA). Atomic force microscopy (AFM) (Veeco Dimension-Ico System, Veeco, U.S.A.).

proteins, followed by the binding of biotin-Cy3-oligonucleotides (recognizing remaining subunits of streptavidin on AuNPs) to form Au-biotin-Cy3-DNA conjugates. The oligonucleotides act as primers to initiate the RCA reaction in the presence of circle DNA templates, nucleotides and specific phi29 polymerases. The RCA process produced long single-stranded DNA (ssDNA) in situ. AuNPs functionalized with Cy3-labeled oligonucleotide strands (Au-Cy3-DNA) hybridized with each long ssDNA to form a necklace-like structure. After silver staining, all Au-Cy3-DNA on the ‘necklaces’ were coated with layers of Ag nanoparticles, producing gray microspots on the chip that are visible to the naked eye. The gray microarrays were then measured by a Raman spectrometer. Intensity of Raman signal reflects the amount of proteins in this detection system. Before Ag enhancement, the nanoparticle probes were invisible to the naked eye. It was agreed that no distinct Raman scattering signal could be detected without Ag enhancement due to a lack of electromagnetic-field enhancement for the undeveloped nanoparticles.9 Figure 1a showed four separate protein microarrays after Ag enhancement. The thickness of the Ag nanoparticle layers around the AuNPs depends on the time allowed for silver enhancement. For the same reaction time, optical imaging showed that continuous and uniform microspots were obtained (Figure 1a). Although the images of the microarray subjected to the RCA process showed no differences from the control microarrays that were

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RESULTS AND DISCUSSION The design of the nanoRCA-SERS system for protein microarrays is illustrated in Scheme 1. Proteins (antibodies with biotin groups) were spotted and immobilized on a chip substrate in a humidity chamber at room temperature. Free proteins on the chip were removed by washing. The chip was blocked with blocking reagents to prevent nonspecific adsorption and thus reduce the background signal. Then, streptavidin-conjugated AuNPs (SA-AuNPs) first bound to 9141

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their high content of hot spots,9,26 the Au-Cy3-DNA coated with Ag nanoparticle layers could be regarded as “a group of hot spots”. Obviously, in the microarrays not subjected to the RCA process, each protein molecule bound to Au-biotin-Cy3DNA had only one hot-spot group. Thus, there was almost no significant enhancement in the SERS signal. However, after rolling-circle amplification was carried out, each primer of the Au-biotin-Cy3-DNA conjugate might have created a long ssDNA that hybridizes with a number of Au-Cy3-DNA. Thus, each protein molecule recognized by Au-biotin-Cy3-DNA contains a large number of hot-spot groups after silver staining. Additionally, we have detected microarrays containing different amounts of protein molecules in the nanoRCA-SERS system, while the SERS signal of the microarrays not subjected to the RCA process act as control systems (Figure 1c). The nanoRCA-SERS system exhibited typical dose−response changes and good reproducibility in Raman intensity. We did not find overlap of 1331 cm−1 peak from Cy3 with major scattering band from DNA often occurred in DNA detection (Figure S1, Suporting Information).27 The results showed that the SERS signal of 10 zeptomolar protein molecules in a microspot was significantly different from that of blank microspots, demonstrating that the zeptomolar-level detection limit for protein microarrays can still be achieved (See more details for the detection limit calculation in the Supporting Information). The results also showed that nanoRCA was much more sensitive than conventional RCA methods with a single primer28 and did not need any solid-phase gold substrates.29 Although the wide concentration range of nanoRCA strategy might not be suitable for profiling sample distribution in proteomics, it is very important for medical diagnostics, especially primary screening of patient samples. Considering its limitation of narrow dynamic range, the nanoRCA system could be cooperated with other quantitive methods to perform the detailed analysis of analyte concentration. Thus, the accuracy level of the system could also be improved. This type of detection method not only suitable for biotin-streptavidin system, but also for nonbiotinconjugated proteins such as antibody−antigen system simple by assembling antibodies on AuNPs with DNA primers together instead of avidin. The grayscale of the silver-stained microarrays was measured to compare the signal-amplification performance of the gold− silver staining system with that of the nanoRCA-SERS system. For microarrays containing the same amounts of protein molecules (0.44 fmol), the results showed that there was no significant difference between the grayscale signals of the microspots subjected to nanoRCA and those not subjected to nanoRCA (Figure 2a), demonstrating the high background signal of the silver-staining amplification system. Ag nanoparticle deposition on microspots might not depend on the number of AuNPs because the self-deposition of Ag nanoparticles often occurs during silver staining.7 Figure 2b showed that the SERS signal of microspots subjected to nanoRCA was much stronger than that of microspots not subjected to nanoRCA. This significant difference demonstrates that the nanoRCA-SERS system has an excellent signal-to-noise ratio and thus is much more sensitive than the gold−silver staining system. Comparison between SESR and fluorescence method was also carred out and the results showed the higher sensitivity of nanoRCA-SERS system (Figure S2, Supporting Information).

Figure 1. Flatbed-scanner images of protein microarrays with nanoparticles after Ag enhancement (a) and corresponding Raman spectrum (b). The peaks marked by the star (*) correspond to the following bands: ∼ 906.6 and ∼1331.3 cm−1 of Cy3. Top: partially enlarged drawing of a. Insert: Scan area for Raman intensity. (c) Comparison of the Raman intensity of nanoRCA-SERS and no amplification-SERS systems with the biotin-sencondary antibody concentration range from 3.4 × 105ng/mL to 0, respectively.

not subjected to the RCA process, the Raman spectra of the two arrays were quite different from each other. We chose a few of the microspots at random from each microarray for SERS detection and obtained their average values from an equivalent area showing features typical of Cy3 (Figure 1b). Cy3 dye molecules were chosen as indicators because they have large Raman cross section. The intensities of two peaks (∼906.6 and ∼1331.3 cm−1 of Cy3, marked with asterisks) were selected for comparison. A strong SERS signal was observed in the microarray subjected to the RCA process, while no significant enhancement in the SERS signal was observed in the control microarrays. The differences in the arrays’ SERS spectra are the result of the different amounts of hot spots among the microarrays. Because silver nanoparticle aggregates have been found to be responsible for the SERS enhancement, because of 9142

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Figure 2. Grayscale results (a) and Raman intensities (b) for blank, no-amplification and nanoRCA silver-stained microarrays. Insert: Silver-stained microarrays. From left to right: blank, no-amplification system, and with nanoRCA-SERS system.

Figure 3. (a) Schematic illustration of nanoHRCA-SERS-based protein microarray strategy. First, biotin-secondary antibodies were captured onto the surface of a slide, and Au-SA and biotin-DNA (first primer) were then added. The second primer (Au-Cy3-DNA) and amplification mixture (circle DNA, phi29 polymerase, dNTP and buffer) were then added for hyperbranched rolling-circle amplification. Enlarged drawing of HRCA strategy: Au-SA-bioDNA acts as the first primer. The first primer hybridizes with circle DNA and extends isothermally to create a long single strand of DNA, which hybridizes with and serves as the template for the second primer (Au-Cy3-DNA). With phi29 polymerase, the second primer extends further and downstream DNA displacement. The displaced Au-Cy3-DNA will in turn bind to the first primer. (b) AFM images of nanoRCA products and(c) nanoHRCA products adsorbed on solid substrate. (d): Comparison of Raman intensities; the peaks marked by the star (*) correspond to the following bands: ∼906 and ∼1331.3 cm−1 of Cy3. (e) The relative Raman intensities of Cy3 at different Raman peaks (1049.4, 1126.6, 1234.7, 1331.3, 1451.0 cm−1); the signals without amplification are set to “1”.

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Because long ssDNA “strings” the hot-spot groups together in the nanoRCA-SERS system, the number of hot-spot groups depends on the in situ increase in the number of long ssDNA. Hyperbranched rolling-circle amplification (HRCA) is a more efficient isothermal amplification technology than RCA.30−32 It was reported that the powerful homogeneous HRCA reaction is capable of 1012-fold signal amplification33,34 higher than that achieved by RCA technology, because of its capability to create longer ssDNA. To achieve better SERS performance, we improved our system by employing a nanoHRCA strategy (Figure 3a). Biotin-Cy3-DNA bound to SA-AuNPs act as the first primer for HRCA. The primer hybridized with a special region of circle DNA and extended isothermally to create a multimeric ssDNA, which served as the template for the second primer, oligonucleotides on Au-Cy3-DNA conjugates, and were further amplified by phi29 polymerase through the second primer extension and downstream DNA displacement. The displaced single strands would in turn contain multiple binding sites for the first primer. Thus, the strand-displacement process generated a continuously expanding pattern of Au-SA-bio-DNA and Au-Cy3-DNA branches, which could be connected by AuNPs and ssDNA to form networks. An increasing number of Au-Cy3-DNA probes were “woven” into the networks via the HRCA process. Atomic force microscopy (AFM) imaging provided direct evidence of in situ nanoRCA and nanoHRCA. In the image of the nanoRCA system (Figure 3b), a long ssDNA molecule was stretched from AuNPs and linked to several other AuNPs, creating a necklace-like structure. It is important to note that in the image of the nanoHRCA system (Figure 3c), long ssDNA molecules are linked to each other by AuNPs, forming a network. Obviously, more Au-Cy3-DNA probes are involved in the nanoHRCA system than in the nanoRCA system. After silver enhancement, all of these probes become hot-spot groups. To investigate the SERS performance of nanoHRCA for protein microarrays, the system was tested in the same way as that for the nanoRCA-SERS system. It is important to note that the Raman spectrum of nanoHRCA showed the highest Raman intensity, much higher than that of the nanoRCA system from the same microarray (Figure 3d). Therefore, the nanoHRCA strategy generates a much stronger SERS signal for protein microarrays than nanoRCA. To calculate the magnitude of the Raman enhancement, we chose the SERS intensity of Cy3 without amplification as the normalization reference (set to 1). The relative intensity of 5 feature peaks (1049.4, 1126.6, 1234.7, 1331.3, 1451 cm−1.) of Cy3 for nanoHRCA and nanoRCA in protein microarrays are as follows: 42, 53, 72, 205, 120 (nanoHRCA) and 12, 11, 22, 59, 48 (nanoRCA), which were higher than those previously reported.35 According to the results, we can conclude that the RCA-based SERS system (nanoRCA or nanoHRCA) has obvious advantages in improving the Raman intensity of protein microarrays. Actually, the enhancement factors (EF) for nanoHRCA-SERS and nanoRCA-SERS are approximately 1.3 × 108 and 3.7 × 107, respectively. (See more details for the EF calculation in the Supporting Information.) In particularly, the nanoHRCA system, which uses two primers in the amplification process to create hot-spot networks, shows excellent SERS performance.

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CONCLUSION In summary, we have developed a novel SERS strategy based the nanoRCA or nanoHRCA technology. The strategy can greatly enhance the Raman intensity of microarrays by creating hot-spot groups. This nanoRCA-SERS system has a detection limit lower than 10 zeptomolar for protein microarrays, which makes the prospective application in the area of single-molecule detection and proteomics possible.

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ASSOCIATED CONTENT

* Supporting Information S

Additional material as described in the 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]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of China (2012CB932600 and 2011AA02A120) and the National Science Foundation of China (91127037 and 91123037).

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REFERENCES

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