Novel Aptasensor Platform Based on Ratiometric Surface-Enhanced

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Novel Aptasensor Platform Based on Ratiometric Surface-Enhanced Raman Spectroscopy Yan Wu, Fubing Xiao, Zhaoyang Wu, and Ru-Qin Yu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04010 • Publication Date (Web): 10 Feb 2017 Downloaded from http://pubs.acs.org on February 12, 2017

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Analytical Chemistry

Novel Aptasensor Platform Based on Ratiometric Surface-Enhanced Raman Spectroscopy Yan Wu, Fubing Xiao, Zhaoyang Wu* and Ruqin Yu State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan, China. ABSTRACT: A novel aptasensor platform has been developed for quantitative detection of adenosine triphosphate (ATP) based on a ratiometric surface-enhanced Raman scattering (SERS) strategy. The thiolated 3’-Rox labeled complementary DNA (cDNA) is firstly immobilized on the gold nanoparticle (AuNP) surface and then hybridizes with the 3’-Cy5 labeled ATP-binding aptamer probe (Cy5-aptamer) to form a rigid double-stranded DNA (dsDNA), in which the Cy5 and Rox Raman labels are used to produce the ratiometric Raman signals. In the presence of ATP, the Cy5-aptamer is triggered the switching of aptamer to form the aptamerATP complex, leading to the dissociation of dsDNA, and the cDNA is then formed a hairpin structure. As a result, the Rox labels are close to the AuNP surface while the Cy5 labels are away from. Therefore, the intensity of SERS signal from Rox labels increases while that from Cy5 labels decreases. The results show that the ratio between the Raman intensities of Rox labels and Cy5 labels is well linear with the ATP concentrations in the range from 0.1 nM to 100 nM, and the limit of detection reaches 20 pM, which is much lower than that of other methods for ATP detection and is also lower than that of SERS aptasensor for ATP detection. The proposed strategy provides a new reliable platform for the construction of SERS biosensing methods and has great potential to be a general method for other aptamer systems.

including the differences in aggregation degree, the heterogeneity in particle size,16 sharp,17 the adsorption energies, the distribution and orientation of the SERS “reporter” chromophores at various sites on the metal surfaces,18 may lead to the poor reproducibility for SERS signal intensity. To overcome the unfavorable factors, some contributions have been made to improve the stability of “hot spots”. For example, Mir-Simon et al. recently reported a one-spot, inexpensive, and scalable synthetic protocol for the fabrication of SERSencoded nanoparticles with controlled co-absorption of mercaptoundecanoic acid and the SERS code, in which a high colloidal stability was provided during the codification process and a remarkable reproducibility from batch to batch was achieved.19 Even so, how to eliminate the influence of the detrimental factors and develop effective assay platforms for reproducible SERS detections still remains a challenging task. The ratiometric method is an effective way to surmount the unstability of single analytical signal intensity. In the past decades, some ratiometric fluorescent assays20-23 and ratiometric electrochemical strategies24-26 have been developed and they have been demonstrated to permit signal rationing and provide built-in correction for environment effects. Meanwhile, some ratiometric SERS assays were also proposed for quantification of ligand adsorption,27 H2O2 imaging and hypochlorite and glutathione.28,29 However, there is no report about aptasensor based on ratiometric SERS strategy. Herein, we try to propose a novel biosensing platform based on a ratiometric SERS strategy. In the ratiometric sensing strategy, two different Raman-labeled probes are employed, a Ramanlabeled recognition aptamer used as the signal probe and a

Introduction Much attention in surface-enhanced Raman spectroscopy (SERS) has received in recent years due to its unique characteristics of high sensitivity, specific Raman fingerprinting spectra and rapid detection capability without complicated sample preparation. Great progress has also been made in SERS biosensors and they have been employed as effective tools to detect DNA,1,2 small biomolecules,3-5 and proteins.6,7 It is well known that there are two main mechanisms attribute to the SERS effect. One is chemical enhancement (CE),8 which is based on the charge transfer between SERS substrate and analytes, and the other is the electromagnetic (EM) field enhancement.9 The EM field enhancement near metallic nanoparticles is the main contribution to SERS signals. The incident electromagnetic field is significantly enhanced by the light excitation of the localized surface plasmon resonances (LSPR) of metal nanostructures which is called as “hot spots”. Many efforts are mainly focused on the construction of SERS sensing substrates and some promising metal nanomaterials, such as gold microshell,10 nanoporous gold (NPG),11 and silver/gold nanoparticles decorated silicon nanowire array (AgNPs/AuNPs@SiNWAr),5,12 have been used as the efficient substrates of SERS sensors. For most of SERS sensors, the intense EM field enhancements is involved in the mild aggregation of the metallic nanoparticles to form the hot spots.13 These aggregation based SERS methods show good sensitivity, however, it has to face with some difficulties to achieve a highly reproducible SERS signal for quantitative assessment.14,15 This is because many uncontrollable factors,

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chloride (HAuCl4), sodium citrate, tris (hydroxymethyl) aminomethane (Tris), MgCl2 and NaCl were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All solutions were prepared with ultrapure water (>18 MΩ. cm) which was obtained from ZOOMWO water purification system. Apparatus. SERS measurements were performed using a Renishaw Invia micro-Raman spectrometer (Renishaw, Wotton under Edge, U.K.) equipped with a Peltier chargecoupled device (CCD) detector, a Leica microscope, and spectra was obtained using a 632.8 nm He-Ne laser at 25 ℃ and a 50×long objective lens. For Raman measurement, the laser output power was 20 mW. The AuNP was characterized by UV-vis absorption spectra using a Shimadzu UV-2450 spectrophotometer (Kyoto, Japan). Synthesis of AuNPs. AuNPs were synthesized according to the seed-growth method.31 First, the Au seed was prepared with Frens method.32 Briefly, a 1.0 mL of 0.024 M HAuCl4 was added to 99.0 mL ultrapure water with a gentle stirring, and then heated to boiling, kept boiling for 30 min. Then 0.6 mL of 0.2 M sodium citrate solution was rapidly added to the boiling solution. The boiling solution was kept stirring for 20 min. When the color of the solution turned into wine red, the solution was stopped heating and cooled to room temperature. The colloidal solution was filtered through a 0.22 µm filter for the subsequent experiments. The Au seed size is 18 nm according to the UV-vis absorption spectra which the surface plasmon resonance (SPR) absorption band is at 517 nm (Figure S3) and TEM images (Figure S4). Second, to obtain the 60 nm AuNP, 0.7 mL of 0.024 M HAuCl4 was mixed with 195 mL ultrapure water which contained 1.3 mL of 18 nm Au seed solution, and then 0.9 mL of 0.01 M MSA solution was added into the solution. The solution was kept stirring for 2 h. The size of the obtained AuNP is about 60 nm from the UVvis absorption spectra which the SPR absorption band is at 536 nm (Figure S3) and TEM images (Figure S4). The morphology of the particles was characterized using TEM (JEM1230, JEOL). Preparation of dsDNA-AuNPs. The cDNA was incubated with 100 mM Tris-HCl buffer (containing 100 mM NaCl and 10 mM TECP, pH 7.4) for 1 h before mixing with AuNPs to avoid the disulfide formation between the thiolated cDNA strands. Then 8.0 µL of 0.05 % FSN was added to 200 µL AuNPs solution and incubated for 1 h. The UV-vis absorption spectrum of the FSN-capped AuNPs was compared with the AuNPs without FSN, and the SPR absorption band is still at about 536 nm (Figure S3). The mixture solution consisting of 208 µL of AuNPs-FSN, 150 µL of 10 µM cDNA and 42 µL of NaCl (the final concentration is 1.0 M) was allowed to incubate for 2 h. The excess cDNA was removed by centrifugation at 12000 rpm for 20 min. The cDNA-AuNPs conjugates were then resuspended in 10 mM PBS (containing 100 mM NaCl, pH 7.4). The procedure was repeated three times for removing unreacted cDNA. The UV-vis absorption spectrum of the cDNA-AuNPs is presented in the Figure S3 (A SPR at 539 nm). A 3 nm of red-shift of the SPR from 536 nm to 539 nm supports the formation of the cDNA-AuNPs. To form the dsDNA-AuNPs conjugates, 150 µL of 20 µM ATPbinding aptamer solution in 10 mM PBS buffer was added to the cDNA-AuNPs conjugates solution and incubated for 16 h.

thiolated Raman-labeled complementary DNA (cDNA) immobilized on the surface of gold nanoparticle (AuNP). The signal probe can hybridize with the immobilized cDNA to form a rigid double-stranded DNA (dsDNA) to construct a ratiometric SERS biosensing platform, in which the Raman labels on the signal probe are close to the AuNP surface and produce a strong SERS signal while the Raman labels on the cDNA are away from and hardly has SERS signal. In the presence of target, the aptamer combines with the target, resulting in the dissociation of the signal probe and the decrease in the SERS signal. At the same time, the change of the cDNA structure from the chain-based structure to the hairpin structure makes its Raman label close to the surface of AuNP and produces another strong SERS signal. The ratio value between the Raman signals of two labels is used to quantify the concentration of target. Thus, a ratiometric SERS sensing strategy is constructed. Adenosine triphosphate (ATP) is used as the model target. To recognize ATP specifically, an aptamer, which possesses specific affinity for ATP is employed.30 To the best of our knowledge, this is the first report for the aptasensor platform based on the ratiometric SERS strategy, which will well improve the reproducibility of SERS signal and is immune to the influence from the variations of the physical/chemical property of SERS substrates and other experiment parameters.

Scheme 1. Schematic illustration of the ratiometric SERS aptasensor for ATP detection. Experimental Section Chemicals and Reagents. The ATP-binding aptamer and its complementary DNA were obtained from TaKaRa Biotechnology Co. Ltd. (Dalian, China). The sequence of the cDNA is 5’-SH-(CH2)6-ACC TTC CTC CGC AAT ACT CCC CCA GGT-(CH2)6-Rox-3’ (Rox: 5-carboxy-X-rhodamine). The four bases at both ends are complementary and the cDNA can form a hairpin structure. The sequence of the ATP-binding aptamer is 5’-ACC TGG GGG AGT ATT GCG GAG GAA GGT-(CH2)6-Cy5-3’ (Cy5: Cyanine 5). The structure of the Rox and Cy5 are listed in Figure S1 in Supporting Information. Na2HPO4, NaH2PO4, 2-mercaptosuccinic acid (MSA), Zonyl FSN-100 (F(CF2CF2)3-8-CH2CH2O(CH2CH2O)x H), adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP) were purchased from Sigma-Aldrich and the molecular structure of ATP, CTP, GTP, and UTP is listed in Figure S2. Gold

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Analytical Chemistry attributes to the ring C-C stretching vibrations of Rox.33 When the Cy5 labeled aptamer is hybridized with the cDNA to form a rigid dsDNA on the AuNP surface, the typical Raman peaks of Cy5 including 1120 cm-1, 1230 cm-1, 1361 cm-1, 1500 cm-1, and 1605 cm-1 corresponding to ν(C-H)ip-bend, ν(C-N)stretch, ν(C=V)ring, ν(C=C)ring-stretch, and ν(C=N)stretch modes of the dye, respectively, can be clearly detected. These peaks observed here are equivalent to those of the Cy5 spectrum ever reported.34 As a result, the ICy5 increases while the IRox is near zero (curve b). The 3’-Rox labels are quite close to the surface of SERS substrate and the strong Raman intensity of up to 25000 is detected when the Rox-labeled cDNA is immobilized on the AuNP surface and forms the hairpin structure (curve c). When the ATP molecules (10 nM) are added into the test solution, the ATP molecules interact with the Cy5-labeled aptamer, making the aptamer dissociate from the dsDNA structure, and then the remaining Rox-labeled cDNA forms a hairpin structure in the presence of Mg2+. The above conformation changes result in the decrease of the SERS signal intensity of Cy5 labels and the increase of the SERS signal intensity of Rox labels (curve d). Furthermore, the SERS spectra for 0.08 nM, 0.1 nM and 1 nM ATP conditions is investigated and the results are shown in Figure S8 in Supporting Information. These results well demonstrate the feasibility of using the IRox /ICy5 values as the quantitative standard for ATP detection.

SERS Measurement. The dsDNA-AuNPs conjugates were incubated with different concentrations of ATP (CTP, GTP or UTP) in 10 mM PBS (pH 7.4) buffer for 30 min (The incubation time of ATP was optimized and the result is shown in Figure S7 in Supporting Information). To induce the formation of stem-loop hairpin structure of cDNA, the cDNAAuNPs conjugates were incubated with a solution with high ionic strength (10 mM PBS containing 150 mM NaCl and 5 mM MgCl2, pH 7.4). Subsequently, the SERS spectra were recorded using a 633 nm laser with 50×objective. Five minute acquisition time was employed, and three times of scanning was accumulated for each measurement with a resolution of 1 cm-1. Each measurement was carried out at least three times, and the control experiments for GTP, UTP and CTP were performed as the same condition as ATP detection. Results and Discussion Principle of the Ratiometric SERS Aptasensor Scheme 1 represents the operational principle of the ratiometric SERS aptasensor for ATP detection. The Rox and Cy5 dyes are used as the Raman labels since they have unique Raman peaks. When the two dyes are combined in a composite sensing system, their characteristic peaks do not overlap and can be easily distinguished visually. More importantly, Cy5 and Rox as the Raman labels have a close excitation wavelength λex = 633 nm but have different absorption maxima at 648 nm and 588 nm, respectively. The cDNA, whose opposite ends are dually labeled with 3’-Rox (served as Raman labels) and 5’-SH, is initially immobilized on the 60 nm AuNP surface via Au-S bonds. The 3’-Cy5 (also served as Raman labels) labeled aptamer is then hybridized with the cDNA to form a rigid dsDNA. After the aggregation of AuNPs under a high concentration of salt, the SERS signal of the close Raman labels can be significantly enhanced. In the absence of ATP, the dsDNA displays a rigid structure, and the 3’-Cy5 labels on the ATP-binding aptamer is close to the AuNP surface while the 3’-Rox labels cDNA is distant from the surface. As a result, the SERS signal of Cy5-labels can be maximally enhanced by the LSPR originating from the aggregation of AuNPs. In the presence of ATP, the ATP molecules intercalate into the aptamers and the structure of the aptamers switches to the aptamer/target complex. The structural switch makes the Cy5 labeled aptamer dissociate from the dsDNA complex into the solution, and thus the 3’Cy5 labels leave the AuNP surface and there is a significant reduction in the SERS signal of Cy5. While the remaining Rox labeled cDNA can form a hairpin structure in the presence of Mg2+, which makes the 3’-Rox labels close to the AuNP surface again and leads to a distinct increase in the SERS signal of Rox. With the increasing of the ATP concentration, the SERS signal of Rox labels continually increases and that of Cy5 labels decreases. As a result, ATP is detected by simultaneously monitoring the Rox Raman signal intensity (IRox) and the Cy5 Raman signal intensity (ICy5). Feasibility of the Ratiometric SERS Aptasensor To demonstrate the feasibility of the ratiometric SERS aptasensor, the SERS response signal for ATP is investigated. As showed in Figure 1, no SERS signal is detected when there is not Raman labeled DNA probe on the surface of the SERS substrate (curve a). The typical Raman peaks of Rox including 1499 cm-1 and 1646 cm-1 can be clearly detected which

Figure 1. Raman spectra obtained from the AuNPs in solution (a). The dsDNA immobilized on the AuNP surface with Cy5labeled in the absence of ATP (b). The Rox-labeled cDNA immobilized on the AuNP surface (c). The ratiometric SERS sensor in the presence of ATP (d). Optimization of the Ratiometric SERS Platform In order to achieve a best performance of the SERS aptasensor platform, the concentration ratio between the aptamer and the cDNA (Captamer/CcDNA) is optimized. The cDNA is designed to hybridize with the ATP aptamer to form a rigid dsDNA. A low Captamer/CcDNA value may result in an inefficient hybridization between the cDNA and the aptamer, which is detrimental to the SERS signal of the Cy5 labels. Therefore, an appropriate Captamer/CcDNA value is crucial for the SERS signal response. The result is listed in the Figure S5 in the Supporting Information, and the IRox/ICy5 value reaches equilibrium when the Captamer/CcDNA value is 2:1. Thus, the Captamer/CcDNA value of 2:1 is chosen for the subsequent investigations.



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increasing of the ATP concentration. In this assay, the IRox/ICy5 value is employed as quantitative standard, and the increasing trend of the IRox/ICy5 value with ATP concentration is further summarized in Figure 2B. As shown, a distinct increase in the IRox/ICy5 value is observed when the ATP concentration increases from 0.1 nM to 2500 nM. A calibration curve in the concentration range from 0.1 to 100 nM is shown in the inset of Figure 2B. The present LOD is estimated to be 20 pM (the detail is listed in the Supporting Information) and the limit of quantitation (LOQ) is estimated to 67 pM. To the best of our knowledge, the LOD obtained in this ratiometric SERS aptasensor is much lower than that of other methods for ATP detection,37,38 The LOD is also lower than that of SERS aptasensor for ATP detection, which obtained a LOD of 10 nM for ATP. 39 Influence of External Factors on the Ratiometric SERS Aptasensor Performance With the merits of highly sensitivity, nondestructive detection, and unique spectroscopic fingerprint, SERS has been proven to be a useful spectroscopic tool for the detection of chemical and biological species. However, SERS still suffers from variability in enhancement of Raman intensity depending on not only the concentration of analyte but also the physical/chemical properties of SERS substrates, making SERS a qualitative or semiquantitative detection technique at present. To well understand the sensing performance of the ratiometric SERS aptasensor platform, therefore, the influence of external factors on the ratiometric SERS aptasensor platform is investigated in detail. Influence of Salt Concentration For most of SERS biosensors, the SERS signal of the Raman labels can be significantly enhanced by the interparticle plasonic coupling with the aggregation of the metallic nanoparticles and then the aggregation degree of the metallic nanoparticles is affected by the salt concentration in solution. Therefore, the salt concentration plays a crucial role in the formation of the SERS signals. To investigate the influence of salt concentration on the sensor performance, three different salt concentrations are used and the results are shown in Figure 3. Figure 3A is the Raman spectra of the different salt concentrations (the salt concentrations from a to c are 200 mM, 100 mM, and 50 mM, respectively). It can be easily observed that the SERS intensities continually decrease from a to c with the decrease of salt concentration. Figure 3C is dynamic light scattering (DLS) analysis and UV-vis absorbance spectra of the AuNPs aggregates under the corresponding salt concentrations. Through the DLS signal readout, we can clearly observe the gradual decrease in the average diameter of the AuNPs, showing continually decreasing aggregation degrees from a to c. After the addition of salt, the spectrum exhibited a red shift with decreased absorbance. It seems to be able to conclude that higher salt concentration can lead to higher degree of aggregation and further facilitates higher Raman intensity. That is to say, the use of a relatively high salt concentration can improve the intensity of Raman signal and benefit the SERS determination. On the other hand, it also suggests that it is difficult to achieve accuracy quantitative determination with SERS methods since the aggregation degree of AuNPs is not easy to control. However, the IRox/ICy5 values are 0. 570, 0.533, and 0.546,

The single-stranded DNA self-assembly on the AuNP surface via Au-S bond is a feasible and common method. However, the traditional method for preparing the cDNAAuNPs conjugates needs a time-consuming salt-aging procedure.35 Herein, a facile method to immobilize the cDNA on AuNP surface is adopted,36 which the nonionic fluoro surfactant, i.e., Zonyl FSN-100 is employed to stabilize AuNPs. It has ever been reported that the non-specific adsorption of cDNA can be effectively inhibited by the FSN capping layer and makes the one-step loading of the cDNA on the AuNP surface possible. Therefore, Zonyl FSN-100 is used to immobilize single-stranded DNA on the AuNP surface instead of the traditional self-assembly method in this experiment. Ratiometric SERS Aptasensor for ATP Detection

Figure 2. (A) Raman spectra of Cy5 and Rox with different ATP concentrations. The concentrations are (from a to l) 0.1 nM, 1 nM, 10 nM, 30 nM, 50 nM, 80 nM, 100 nM, 500 nM, 1000 nM, 2500 nM, 5000 nM, and 10000 nM. (B) Dependence of the IRox/ICy5 value on different ATP concentrations. The inset shows a dynamic range and linearly fitted line. Error bars show the standard deviation of three experiments. Figure 2A shows the response of the SERS aptasensor to the variation of the ATP concentration. Meanwhile, the typical peak at 1361 cm-1 of Cy5 and the typical peak at 1646 cm-1 of Rox are used as the characteristic peak to quantitatively evaluate the Raman response of ATP. It is clearly observed that the ICy5 decreases while the IRox increases with the


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respectively, and keep good precision with a RSD of 3.4 %. It thus clearly indicates that the proposed ratiometric SERS strategy is effectively immune to the effect of salt concentration on the Raman signal intensity and it paves a new way to develop the signal-stability SERS sensors.

under different temperatures (Figure 4). Figure 4A shows the Raman spectrum under different temperatures and Figure 4 B exhibits the IRox/ICy5 value with different temperature. The IRox/ICy5 values from a to e are 0.549, 0.558, 0.545, 0.529 and 0.563, respectively, and the RSD is 2.4 %. As is well known, high temperature is disadvantageous to DNA hybridization, therefore, the IRox/ICy5 value is highest when the temperature is 50℃. Although there is small fluctuate for the Raman spectrum, the IRox/ICy5 values are hardly changed under different temperatures. The results indicate that the developed ratiometric SERS aptasensor can effectively eliminate the influence of the temperature on the sensor performance.

Figure 4. (A) Raman spectra of Cy5 and Rox with different temperatures. (B) The IRox/ICy5 value with different temperatures. Influence of pH Value on the SERS Performance The AuNPs aggregation may also be affected by pH value of the system which further affects the sensor performance. To evaluate the influence of the pH value on the ratiometric SERS aptasensor performance, ATP detection is performed under different pH values of the system (Figure 5). Figure 5A exhibits the Raman spectrum under different pH values and Figure 5B shows the IRox/ICy5 value with different pH values. The IRox/ICy5 values from a to d are 0.542, 0.525, 0.550, and 0.555, respectively, and the RSD is 2.5 %. The reason for the strong Raman intensity depends on the fact that the acidic condition can lead to a higher aggregation degree of AuNPs. Therefore, the pH value less than 7.0 can contribute to

Figure 3. (A) Raman spectra of Cy5 and Rox with different salt concentrations. (B) The IRox/ICy5 values with different salt concentrations. (C) DLS analysis and UV-vis absorbance spectra with different salt concentrations. Influence of Temperature on the SERS Performance The AuNPs aggregation may also be affected by temperature which further affects the sensor performance. To evaluate the influence of the temperature on the ratiometric SERS aptasensor performance, ATP detection is performed



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be obtained from the ratiometric SERS aptasensor method comparing with those results assessed by the IRox value or the ICy5 value alone.

the higher degree of aggregation and further results in the strong Raman intensity. As shown in Figure 5A, the intensity for a is strongest while d is weakest. Although there is small fluctuate for the Raman spectrum in the condition of different pH values, the IRox/ICy5 values hardly change, indicating the proposed ratiometric SERS aptasensor can effectively eliminate the influence of the pH value of system on the sensor performance.

Figure 6. SERS spectra of ATP at ten different samples. Selectivity and Stability of the Ratiometric SERS Aptasensor

Figure 7. Selectivity of the SERS ATP aptasensor (concentration of UTP, CTP, and GTP are 1 µM and ATP is 100 nM). In order to evaluate the selectivity of the SERS aptasensor toward ATP, control experiments are performed by incubating the SERS aptasensors in PBS buffer containing different concentrations of UTP or CTP, GTP, and ATP. The concentrations of UTP, CTP and GTP are all 1 µM, while the ATP concentration is 100 nM. As shown in Figure 7, it can be acquired that the ATP can give a distinct change of the SERS signal (the IRox/ICy5 is 1.33), while the signal change caused by other three analogues are small (the IRox/ICy5 is 0.15 for GTP and the values are even smaller for other analogues), showing a good selectivity. Furthermore, the SERS aptasensor remains available for ATP detection after the sensor is kept at 4℃ for two weeks. The IRox/ICy5 values are 1.33 and 1.30 for zero day and two weeks, respectively, having no obvious changes, while the respective Raman intensities of labels have an evident change and the Raman intensity decreases about 35 % (see Figure S6). The result suggests that the developed SERS aptasensor has predominant storage stability over these with single Raman label.

Figure 5. (A) Raman spectra of Cy5 and Rox with different pH values. (B) The IRox/ICy5 value with different pH values. In conclusion, the developed assay in this paper employing the IRox/ICy5 value as the signal quantitative standard is hardly affected by the external environment. Therefore, the developed strategy provides a platform for highly stability SERS detection. Reproducibility of the Ratiometric SERS Aptasensor The reproducibility of SERS signals is further investigated since it is a highly important property for a SERSactive sensing platform. Figure 6 shows the SERS spectra for ten different parallel samples with the same ATP concentration. To get a statistically meaningful result, the RSD of the Raman intensity of the major peaks for Rox and Cy5 are estimated. The RSDs in SERS intensity of two prominent peaks at 1646 cm-1 and 1361 cm-1 are 19.4 % and 18.1 %, respectively. While the RSD is 3.4 % when the IRox/ICy5 values are used to estimate the reproducibility of the Raman signals. It indicates that a distinctly enhanced reproducibility result can


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Analytical Chemistry (8) Albrecht, M. G.; Creighton, J. A. J. Am. Chem. Soc. 1977, 99, 5215-5217. (9) Jeanmaire, D. L.; Van Duyne, R. P. J. Electroanal. Chem. 1977, 84, 1-20. (10) Han, D.; Lim, S. Y.; Kim, B. J.; Piao, L.; Chung, T. D. Chem. Commun. 2010, 46, 5587-5589. (11) Zhang, L.; Chang, H.; Hirata, A.; Wu, H.; Xue, Q.-K.; Chen, M. ACS Nano. 2013, 7, 4595-4600. (12) Huang, J.-A.; Zhao, Y.-Q.; Zhang, X.-J.; He, L.-F.; Wong, T.-L.; Chui, Y.-S.; Zhang, W.-J.; Lee, S.-T. Nano Lett. 2013, 13, 5039-5045. (13) Halas, N. J.; Lal, S.; Chang, W.-S.; Link, S.; Nordlander, P. Chem. Rev. 2011, 111, 3913-3961. (14) Jones, J. C.; McLaughlin, C.; Littlejohn, D.; Sadler, D. A.; Graham, D.; Smith, W. E. Anal. Chem. 1999, 71, 596-601. (15) Schneider, S.; Grau, H.; Halbig, P.; Nickel, U. Analyst 1993, 118, 689-694. (16) Emory, S. R.; Haskins, W. E.; Nie, S. J. Am. Chem. Soc. 1998, 120, 8009-8010. (17) Mock, J. J.; Barbic, M.; Smith, D. R.; Schultz, D. A.; Schultz, S. J. Chem. Phys. 2002, 116, 6755-6759. (18) Laserna, J. J.; Campiglia, A. D.; Winefordner, J. D. Anal. Chem. 1989, 61, 1697-1701. (19) Mir-Simon, B.; Reche-Perez, I.; Guerrini, L.; Pazos-Perez, N.; Alvarez-Puebla, R. A. Chem. Mater. 2015, 27, 950-958. (20) Deng, J.; Yu, P.; Wang, Y.; Mao, L. Anal. Chem. 2015, 87, 30803086. (21) Zhang, X.; Xiao, Y.; Qian, X. Angew. Chem. Int. Ed. 2008, 47, 8025-8029. (22) Sun, J.; Mei, H.; Wang, S.; Gao, F. Anal. Chem. 2016, 88, 73727377. (23) Xu, Z.; Singh, N. J.; Lim, J.; Pan, J.; Kim, H. N.; Park, S.; Kim, K. S.; Yoon, J. J. Am. Chem. Soc. 2009, 131, 15528-15533. (24) Chai, X.; Zhou, X.; Zhu, A.; Zhang, L.; Qin, Y.; Shi, G.; Tian, Y. Angew. Chem. Int. Ed. 2013, 52, 8129-8133. (25) Du, Y.; Lim, B. J.; Li, B.; Jiang, Y. S.; Sessler, J. L.; Ellington, A. D. Anal. Chem. 2014, 86, 8010-8016. (26) Zhang, L.; Han, Y.; Zhao, F.; Shi, G.; Tian, Y. Anal. Chem. 2015, 87, 2931-2936. (27) Zhang, D.; Ansar, S. M. Anal. Chem. 2010, 82, 5910-5914. (28) Peng, R.; Si, Y.; Deng, T.; Zheng, J.; Li, J.; Yang, R.; Tan, W. Chem. Commun. 2016, 52, 8553-8556. (29) Wang, W.; Zhang, L.; Li, L.; Tian, Y. Anal. Chem. 2016, 88, 9518-9523. (30) Huizenga, D. E.; Szostak, J. W. Biochemistry 1995, 34, 656-665. (31) Niu, J.; Zhu, T.; Liu, Z. Nanotechnology 2007, 18, 325607325613. (32) Frens, G. Nature 1973, 241, 20-22. (33) Ye, S.; Yang, Y.; Xiao, J.; Zhang, S. Chem. Commun. 2012, 48, 8535-8537. (34) Novara, C.; Petracca, F.; Virga, A.; Rivolo, P.; Ferrero, S.; Chiolerio, A.; Geobaldo, F.; Porro, S.; Giorgis, F. Nanoscale. Res. Lett. 2014, 9, 1-7. (35) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (36) Zu, Y.; Gao, Z. Anal. Chem. 2009, 81, 8523-8528. (37) Wang, Y.; Wang, Y.; Liu, B. Nanotechnology 2008, 19, 415605. (38) Zuo, X.; Song, S.; Zhang, J.; Pan, D.; Wang, L.; Fan, C. J. Am. Chem. Soc. 2007, 129, 1042-1043. (39) Chen, J.-W.; Liu, X.-P.; Feng, K.-J.; Liang, Y.; Jiang, J.-H.; Shen, G.-L.; Yu, R.-Q. Biosens. Bioelectron. 2008, 24, 66-71.

Conclusions

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In summary, a novel ratiometric SERS aptasensor has been developed for ATP detection. It has a good linear relationship between 0.1 nM and 100 nM and the LOD is estimated to 20 pM. Most importantly, the developed method is immune to the influence from external factors, such as temperature, salt concentration and pH value, showing an improved SERS performance. In addition to the excellent sensitivity and adaptable reproducibility, this kind of ratiometric SERS aptasensor has no strict requirement for the SERS substrate comparing with other SERS sensors. In terms of these advantages, it can be expected that this ratiometric SERS biosensing strategy may offer a new direction in the development of high performance SERS aptasensors for quantitatively monitoring ATP and other small molecules.

ASSOCIATED CONTENT Supporting Information Supporting Information Available: [The molecular structure of Cy5, Rox, ATP, CTP, GTP and UTP, UV-vis spectra, TEM images, the Raman Intensity of Cy5 with the ratio between the concentrations of aptamer and cDNA (Captamer/CcDNA), the stability of the ratiometric SERS aptasensor, the incubation time of ATP with the value of the IRox/ICy5, Raman spectra obtained from different ATP concentrations and Calculation of limit of detection (LOD).] This material is available free of charge via the Internet at http:// pbus. acs. org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This study was financially supported by the International Scientific and Technological Cooperation Projects of China (2012DFR40480) and the National Natural Science Foundation of China (21175037, 21277042).

REFERENCES (1) Barhoumi, A.; Halas, N. J. Am. Chem. Soc. 2010, 132, 1279212793. (2) Kang, T.; Yoo, S. M.; Yoon, I.; Lee, S. Y.; Kim, B. Nano Lett. 2010, 10, 1189-1193. (3) Kim, N. H.; Lee, S. J.; Moskovits, M. Nano Lett. 2010, 10, 41814185. (4) Shafer-Peltier, K. E.; Haynes, C. L.; Glucksberg, M. R.; Van Duyne, R. P. J. Am. Chem. Soc. 2003, 125, 588-593. (5) Sun, B.; Jiang, X.; Wang, H.; Song, B.; Zhu, Y.; Wang, H.; Su, Y.; He, Y. Anal. Chem. 2015, 87, 1250-1256. (6) Feng, J.; Wu, X.; Ma, W.; Kuang, H.; Xu, L.; Xu, C. Chem. Commun. 2015, 51, 14761-14763. (7) Bonham, A. J.; Braun, G.; Pavel, I.; Moskovits, M.; Reich, N. O. J. Am. Chem. Soc. 2007, 129, 14572-14573.



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For TOC only


Novel Aptasensor Platform Based on Ratiometric Surface-Enhanced

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