Ultrasensitive Simultaneous Detection of Multiplex Disease-Related

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Ultrasensitive Simultaneous Detection of Multiplex DiseaseRelated Nucleic Acids Using Double-Enhanced SERS Nanosensors Ruiyan Guo, Fangfei Yin, Yudie Sun, Lan Mi, Lin Shi, Zhijin Tian, and Tao Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06757 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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ACS Applied Materials & Interfaces

Ultrasensitive Simultaneous Detection of Multiplex Disease-Related Nucleic Acids Using DoubleEnhanced SERS Nanosensors †∥

‡∥

†

†

†

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Ruiyan Guo , Fangfei Yin , Yudie Sun , Lan Mi , Lin Shi , Zhijin Tian and Tao Li*, †

†

Department of Chemistry, University of Science and Technology of China, Hefei, Anhui

230026, China. ‡

Division of Physical Biology and Bioimaging Center, Shanghai Institute of Applied Physics,

Chinese Academy of Sciences, Shanghai 201800, China ∥

The contribution of these authors is equal to the research as first authors.

*

Corresponding author. E-mail: [email protected]

KEYWORDS: Nucleic acids, Multiplexed Detection, Double-Enhanced, SERS, Au@Ag nanosnowmen, Gold substrate

ACS Paragon Plus Environment

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ABSTRACT: Developing ultrasensitive probes holds great significance for simultaneous detection of multiplexed cancer-associated nucleic acids. Bimetallic nanoparticles containing silver may be exploited as nanoprobes for disease detection, which can produce stable and strong surface-enhanced Raman scattering (SERS) signals. However, it remains extremely challenging that such SERS nanoprobes are directly synthesized. Herein DNA-mediated grown gold-silver nanosnowmen attached on thiol-containing Raman dyes are successfully synthesized. Stable SERS-enhanced gold substrates are also prepared and used as the enriching containers, where the capture DNAs are tethered to sense the target genes jointly enhanced by the SERS nanoprobes in a sandwich hybridization assay. This means detecting the target gene can obtained the limit detection to 0.839 fM. Such double-enhanced SERS nanosensors are further employed to simultaneously detect the three types of prostate carcinoma-related genes with high sensitivity and specificity, and meanwhile which exhibits robust capacity of resisting disturbance in practical samples. Simultaneous and multiplexed detection of cancer-related genes may provide further biomedical applications with new opportunity.


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1. INTRODUCTION Nucleic acids are critical biomolecules in all living things, which can encode the expression of genetic information and regulate the synthesis of protein1. However, genetic variation resulted from some minor changes of nucleic acid sequence can bring out significant biological influence2. Therefore, sensitive detection and accurate analysis of nucleic acid are fundamentally important and are in urgent requirement in biomedical researches, for example, distinguishing the biomarkers of cancer therapy3-5, revealing the mysteries of genetic information6-7. Many studies demonstrate that more than one nucleic acid sequence is related to a specific disease8-10. In comparison to single biomarker detection, simultaneous differentiation of multiplex targets is found to offer more helpful information to clinical medicine and may promote to understand their function in the field of disease research11-12. Therefore, it’s highly necessary that multiple nucleic acid biomarkers in one sample can be simultaneously detected for early disease diagnosis and therapy13-17. Surface-enhanced Raman spectroscopy (SERS), as an ultrasensitive spectroscopic method has extensive application for detecting nucleic acids18-22, and proven powerful for disease biomarker analysis23-27. SERS provides individual analyte with unique sharp fingerprint signals, enabling simultaneous detection of multiplex nucleic acids at the trace levels28-30. In general, nanoscale silver substrates exhibit the larger plasmonic enhancement for SRES than gold

31-32

,

while great difficulties are always faced in the synthesis of stable uniform nanometallic silver. To overcome this problem, some bimetallic (e.g. Au-Ag) nanoparticles were synthesized for quantitative SERS analysis29, 33-34. For example, Song and colleagues recently reported a DNAmediated approach to synthesize gold-silver bimetallic nanomushrooms for involving in DNA/miRNA detection34. Besides, the gold substrates synthesized by gradual growth of AuNPs


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on treated glasses possess a number of nanoscale gold islands and therefore exhibit an excellent enhancement on SERS signal35. This unique feature have been utilized to construct diversities of SERS sensors for different targets

22, 35-36

. Many nanomaterials have been reported for

biosensing37-43. The sandwich hybridization assays combined with nanomaterials based on SERS have been sensitively detected for cancer-related nucleic acids44-45. In this work, a general strategy based on a sandwich hybridization assay has ultrasensitively simultaneously detected multiplex nucleic acids by combaining dye-attached bimetallic Au@Ag nanosnowmen as SERS nanotags with the plasmonic gold island substrate. The probe DNAmodified SERS nanotags were prepared by growing the Au@Ag nanosnowmen on DNAmodified AuNPs seeds, followed by the attachment of thiol-containing Raman dyes and the probe DNAs onto the nanosnowmen (Scheme 1a). Upon addition of the target DNAs, the SERS nanotags with probe DNA were immobilized onto the capture DNA-modified gold substrate in a sandwich hybridization way (Scheme 1b). In this way, ultrasensitive simultaneous detection of multiplex prostate carcinoma cancer-related genes (miDNA-141, miDNA-21 and miDNA-7d, which are the DNA analogues of miRNA-141, miRNA-21 and let-7d) in the same sample was realized. Note that the SERS signal is here enhanced by both nanotags and gold islands, which is of great significance to ultrasensitive nucleic acid detection. 2. EXPERIMENTAL SECTION Preparation of SERS Nanotags. The SERS active nanotags were made up of Au@Ag nanosnowmen attached to thiol-containing Raman dyes. In brief, 1 mL Au@Ag nanosnowmen (0.1 nM) were mixed with 1 µL as-prepared Raman dyes (20 mM of MPY, 10 mM of NBT, and 20 mM of MBN), respectively. At room temperature, the mixtures were incubated with gentle


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shaking overnight. Then the particles were centrifuged for 15 min at 5000 rpm and washed by using PB buffer to eliminate unmodified Raman dye molecules. Above procedure was repeated three times. Detailed component information of PB buffer, PBS buffer, and PBST buffer can been found in the supplementary experimental section of the Supporting Information. Subsequently, the resulting particles were added to PB buffer and for further use. Specific and detailed synthesis processes of DNA-modified AuNPs and Au@Ag nanosnowmen can see in the supplementary experimental section of the Supporting Information. Preparation of SERS Nanoprobe. SERS nanoprobe was composed of SERS nanotag and thiol-modified DNA, whose preparation procedure was also based on the salt-aging method. Thiol-modified DNA (100 µM, 12 µL) was mixed with corresponding nanotag(1 nM, 40 µL), respectively. Individual mixed solutions contained 3 µM thiol-modified DNA, 0.1 nM nanotag and 10 mM PB, and were incubated with mild shaking for 1 hour. Then 1 M NaCl (4 µL once and 6 µL six times) every 30 min was added to above solutions to obtain a final 0.1 M NaCl. And the resulting solutions were shaked mildly overnight at room temperature. After above reaction was finished, 400 µL nanoprobes were washed three times with PB buffer by centrifuging for 10 min at 5000 rpm to eliminate unmodified thiol-modified probe DNA. After washing, nanoprobes were redissolved to PBS buffer. After the whole process was finished, the SERS probes were placed in a 4 °C of refrigerator for detecting the targets. DNA-Modified of Gold Substrate. At room temperature, the gold substrate was immersed in PBS buffer containing 1 µM thiol-modified capture DNA, incubated for 4 h, and subsequently washed with PBST buffer to remove unmodified DNA. Above procedure was repeated three times. Next, 100 µM thiolated PEG-350 (SH-PEG-350) was incubate for 30 min in the reactor on gold substrate to take up excess position of gold substrate, minimizing the false positives. After


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reaction, gold substrate was washed three times with PBST buffer to remove the unreacted SHPEG-350. Preparation processes of gold substrate and calculation processes of enhancement factor (EF) of gold substrate were provided in the supplementary experimental section of the Supporting Information. SERS Detection of Single DNA. For miDNA-21 detection taken as an example, 20 µL Au@Ag nanoprobe was mixed with 20 µL various concentrations of miDNA-21 in the 600 µL microcentrifuge tube. 100 mM Mg2+ was added to mixed solutions to obtain a final 1.5 mM Mg2+. The resulting mixtures (40 µL) containing 0.1 nM Au@Ag nanoprobe, 1.5 mM Mg2+ and different concentrations of miDNA-21 varied from 10 fM to100 nM were incubated in a 37 ° C of shaker for 2 h with shaking mildly. After reaction, the mixtures were centrifuged three times for 5 min at 4000 rpm to eliminate unreacted miDNA-21. And resulting

mixtures were

redissolved to PBS buffer for further detection. Then mixed solutions were injected to the prepared reactors to form sandwich complexes by incubating for 2 h at 37 °C. After reaction, the gold substrate was washed three times with PBST buffer, followed by drying with nitrogen for collecting SERS signals. As for detecting miDNA-141 and miDNA-7d, experiment procedures were similar to miDNA-21. And concentration detection range was both from 100 fM to 100 nM. SERS Detection of Multiplex DNAs. For the multiplex DNA detection, 30 µL Raman dye (MPY, NBT, and MBN) binded SERS nanoprobes were mixed with miDNA-141, miDNA-21, and miDNA-7d in a 600 µL microcentrifuge tube, followed by adding to Mg2+. The final mixtures (60 µL) containing SERS nanoprobes (0.1 nM of probe-141, 0.1 nM of probe-21, and 0.1 nM of probe-7d), 1.5 mM Mg2+ and individual targets were simultaneously incubated in PBS buffer and shaked gentlely for 2 h at 37 °C. Extra unconnected targets were removed by using centrifugation (4000 rpm, 5 min) for three times, and then various target-connected nanoprobes


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were redispersed in 60 µL of PBS buffer containing 1.5 mM Mg2+, then added to the synthesized reactors coassembled with co-modified capture DNAs (1 µM of capture-141, 1 µM of capture-21, and 1 µM of capture-7d), followed by incubating for 2 h at 37 °C. After reaction, multiplex sandwich structures were formed onto the gold substrates. Washing and drying procedure was the same as above. 3. RESULTS AND DISCUSSION Characterization of SERS Nanotags and Gold Island Substrates. The bimetallic Au@Ag nanosnowmen were prepared by using DNA-modified AuNPs, AgNO3, and other chemicals to obtain Ag structures on the Au shell surface 46. Au@Ag nanosnowmen and AuNP seeds were imaged by transmission electron microscopy (TEM) (Figure 1a). In addition, the snowman-like heterostructure was also characterized by high-resolution transmission electron microscope (HRTEM) and HAADF-STEM elemental mapping (Figure 1b and Figure 1c), indicating that Au heads and Ag bodies had clear boundaries in the nanosnowmen. UV-vis spectroscopy (Figure 1d) indicates that uniform nanosnowmen were formed, which is consistent with previous literature.46 Figure S1 showed more detailed TEM images separately displaying good dispersion and uniform size of the AuNP seeds and Au@Ag nanosnowmen. The size of Au@Ag nanosnowmen was evenly 60 nm in diameter (Figure S2). Au@Ag nanosnowmen were dissolved in PBS buffer, and can store for 1 month in a 4 °C of refrigerator without aggregation (Figure S3). As gold heads were modified with SH-T10, which were used as building blocks for orientedly synthesizing Au@Ag nanosnowmen, and partially covered by silver bodys in the nanosnowmen, thiol-modified DNA easily attached onto the silver bodys can hybridize to their complementary oligonucleotides. In addition, silver exhibits the strong Raman signals, so Au@Ag nanosnowmen are used as favorable SERS-active substrates for detecting nucleic acids.


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For all the nanotags combining Au@Ag nanosnowmen with specific organic Raman dyes, strong and stable Raman signals were obtained. Three kinds of nanotags all generated reproducible SERS signals, whose time-dependent SERS spectra was presented in Figure S4 with 10s of time interval between adjacent Raman spectra. And every nanotag had unique Raman characteristic peak (Figure S5), demonstrating three kinds of nanotags can simultaneously detect multiplex analytes. The synthesized gold substrate producing nanoscale gold islands can highly increase SERS signal, which also can be used us a capture container. The prepared gold substrate looked golden yellow, whose morphology was characterized by SEM (Figure 1e) and obtained different scales SEM images (Figure S6). The SEM images demonstrated that gold substrate uniformly distributed abundant nanogaps largely producing electromagnetic field enhancement effect. EF of gold substrate was calculated on the basis of SERS intensity of NBT separately attaching to gold substrate and glass slide (Figure 1f). The calculated EF was 2.4 × 106, which was consistent with previous literatures22,

35

. The uniformity and reproducibility of gold substrate was

investigated in Figure S7 by randomly selecting 30 spots on gold substrate and glass slide to obtain SERS spectra of NBT. As active SERS substrate has the above outstanding characteristics, capture probes can be immobilized on plasmonic substrate to detect DNA. For the sandwich hybridization assay, the sequences of probe and capture DNAs were optimized. When the end was 8bp at which probe DNA hybridized with capture DNA, the hybridization products had the lower background and the higher yield. So it was adopted for DNA detection in the following experiments. For more details, see Figure S8-S9 in Supporting Information. Detection of Single DNA. The concentrations of capture DNA and probe DNA were separately optimized to achieve best performance at the beginning of SERS detection (Figure S10). The various concentrations of DNA were compared the SERS peaks of NBT at 1335 cm−1 during the


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optimal experimental conditions, and optimal concentrations were 1 µM of capture DNA and 3 µM of probe DNA, respectively. The sensitivity of miDNA biomarker was evaluated by detecting three kinds of prostate carcinoma-related genes (miDNA-141, miDNA-21, and miDNA-7d). Raman spectra of the sandwich complexes was obtained by measuring various miDNA concentrations. Concentration-dependent response in SERS spectra were shown in Figure 2, indicating that the SERS intensity of corresponding peak from Raman dyes (MPY, NBT, and MBN) increased concomitantly with raising concentration of the miDNAs. The characteristic peaks of MPY, NBT and MBN were around 1004 cm-1, 1335 cm-1, 2226 cm-1, respectively. The results of quantitative experiments indicated that Raman intensity was a function of the logarithm of the miDNA concentration, and the calibration curves had a good linearity. ∆I represented that Raman intensity had been deducted against the blank background. Figure 2a showed SERS spectra of miDNA-141 ranged from 100 fM to 100 nM, whose corresponding calibration curve was obtained by plotting the SERS intensity of MPY(∆I1004) versus lg [CmiDNA-141] (Figure 2b). The regression equation was showed as ∆ImiDNA-141 = 818.6 × lg [CmiDNA-141] + 10841.4 with R2= 0.987. The definition of the limit of detection (LOD) was that the signal that target generates is three times larger than the standard deviation of the background signal47-48. The LOD for miDNA-141 was calculated to be 1.019 fM. Figure 2c illustrated SERS spectra of miDNA-21 ranged from 100 fM to 100 nM, and Figure 2d demonstrated that the calibration curve had good quantitative relationship between SERS intensity of NBT and the logarithm of miDNA-21 concentration. The regression equation was calculated as ∆ImiDNA-21 = 1012.5 × lg [CmiDNA-21] + 14431.6 with R2= 0.992 and the LOD of 1.089 fM. Figure 2e showed the SERS spectra of miDNA-7d from 100 fM to 100 nM, whose calibration curve also showed good linearity between ∆ImiDNA-7d and lg [CmiDNA-7d] (Figure 2f). ∆ImiDNA-7d = 491.2 × lg[CmiDNA-7d]


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+ 6533.8 (R2 = 0.982), along with the LOD of 0.839 fM. These results are comparable with previous counterparts (see Table S2 in Supporting Information). In addition, the reproducibility was measured by taking 20 random spots to collect Raman spectra and SERS peaks of NBT at 1335 cm−1. 6.05 % of relative standard deviation (RSD) was obtained (Figure S11), which demonstrated that the test results were very reliable and convincing. Moreover, the stability of the sensor was studied by systematically detecting the prepared sensors after being stored in PBS for one week at room temperature. Figure S12a and Figure S12b separately showed SERS spectra and I1335 by collecting from the fresh sensors and the sensors stored for 1 to 7 days. RSD (4.16%) of I1335 for miDNA-21 was very small. The experimental results above confirmed the good reproducibility and stability along with high sensitivity of our research. Simultaneous Detection of Multiplex DNAs. The application effect of simultaneous multiple target detection was studied. We tested the sensitivity of the multiplex prostate carcinoma-related

genes

(miDNA-141,

miDNA-21,

and

miDNA-7d)

using

Au@Ag

nanosnowmen separately modified with MPY, NBT and MBN (Scheme 1b). The formed multiplex sandwich complexes were separated by the common gold substrate functionalizing with three capture DNAs, followed by simultaneously measuring SERS signals in the same sample. As shown in Figure 3, miDNA-141 (10 pM-100 nM), miDNA-21(1 pM to 10 nM), and miDNA-7d (10 pM-100 nM) were simultaneously sensed, and corresponding characteristic characteristic fingerprint signal of MPY (1004 cm-1), NBT (1335 cm-1), MBN (2226 cm-1) enhanced with the increasing concentration of the specific miDNA (Figure 3a). Figure 3b−d illustrated that calibration curves of three targets all had the good linearity, and Raman intensity was the function of the logarithm of the miDNA concentration. Moreover, the observed SERS signal was down to 10 pM. According to the calculation formula (3N/S), the LODs for miDNA-


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141, miDNA-21 and miDNA-7d were separately calculated to be 0.707, 0.758, and 1.103 pM. The dynamic range of the multiplex assay for miDNA-141, miDNA-21, and miDNA-7d was up to 5 orders of magnitude. Specificity. The specificity of the SERS testing platform was evaluated. Specific analysis for single and multiplex miDNA was determined in Figure 4. For miDNA-21 specificity detection taken as an example, Figure 4a and Figure 4b separately showed Raman spectra and signal quantification in existing target miDNA-21, 1-base mismatched miDNA-21, 2-base mismatched miDNA-21, a non-complementary sequence (miRNA-141) and blank sample under the same concentration of 100 nM. And 30 random spots were separately selected to obtain SERS spectra during each measurements (Figure S13). The results indicated that one mismatch in miDNA-21 sharply decreased SERS signal compared with target miDNA-21, and the second mismatch further decreased SERS signal. The non-complementary sequence (miRNA-141) produced extremely low signal, just like the blank sample. Figure 4c and Figure 4d indicated SERS spectra and Raman intensity in the presence of the mixture-3 (miDNA-141, miDNA-21, miDNA-7d, NC1, NC2 and NC3), the mixture-2 (miDNA-141, miDNA-2 and miDNA-7d), the mixture-1 (NC1, NC2 and NC3) and the blank sample by selecting ∆I1004, ∆I1335, and ∆I2226 for detecting miDNA-141, miDNA-21, and miDNA-7d. The results demonstrated that the mixture-3 of six miDNAs generating SERS signal exhibited very slight change in SERS intensities in comparison with the target miDNA mixture (mixture-2). The nonspecific miDNAs (mixture-1) produced the negligible signals compared with the blank solution. These observations demonstrated that the designed SERS testing platform achieved good selectivity, when many kinds of disturbed miDNAs simultaneously existed. Thus, The SERS sensor has a great application prospect for specificity detecting bioanalytes in complex biological samples.


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Practicality. The practicality of proposed SERS sensors was explored by performing the experiment in 20% human serum sample (Figure 5). The concentration of target miDNA-21 was from 10 fM to 100 nM. Figure 5a showed the dose-response SERS spectra of miDNA-21, whose spectra morphology rarely changed. We found that SERS signal had no significantly difference between the samples in PBS buffer and in 20% human serum (Figure 5b). Thus, the above results indicated that our designed testing platform had strong capacity of resisting disturbance. Au@Ag nanosnowmen probes had good reliability of practical application in 20% human serum. 4. CONCLUSIONS In summary, a SERS sensing platform was designed for ultrasensitively simultaneously detecting multiplex prostate carcinoma cancer-related genes, which made full use of Raman dyemodified Au@Ag nanosnowmen and the gold substrate as SERS-active substrates and capture container. Uniform gold-silver nanosnowmen prepared attached onto Raman dyes and thiolmodified DNAs formed SERS nanoprobes. We prepared three types of nanotags that generate highly intense and reproducible Raman signal with characteristic spectroscopic fingerprint, demonstrating that the SERS nanotags can sensitively detect multiplex analytes. In addition, the synthesized gold substrate were not only used as the capture reactor, but also significantly enhanced SERS signal because of abundant nanoscale gold islands on the surface. Therefore, the proposed SERS sensors which went through dual Raman signal amplification exhibited extremely high sensitivity. The results of quantitative experiments of the single miDNA indicated that the calibration curves had good linearity. The SERS sensors were also simultaneously used to detect the three prostate carcinoma-related genes with the LODs of the picomolar range. In addition, our SERS assay exhibited excellent specificity both in single and simultaneous detection, with the strong anti-interference ability in 20% human serum. Thus, the


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proposed multiplex SERS sensors hold a great application prospect for simultaneous detection of multiple bioanalytes in early disease screening and therapy.

ASSOCIATED CONTENT Supporting Information. Supporting Information Available: Chemicals and Reagents, Instrumentation, Characterization of sandwich structure, Detailed preparation processes, morphology and performance characterization of Au@Ag nanosnowmen and Gold substrate, Optimization of the experimental conditions. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China [No. 21575133], the National Key Research and Development Program of China [No. 2016YFA0201300] and the Recruitment Program of Global Experts.


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Scheme 1. (a) Schematic Illustration of the preparation of SERS nanotag with probe DNA; (b) Schematic Illustration of simultaneous detection of multiplex miDNAs. Figure 1. (a) TEM images of Au@Ag nanosnowmen and 30 nm AuNP seeds. (b) HRTEM image. (c) HAADF-STEM image and elemental mapping images. (d) UV-vis spectra of Au@Ag nanosnowmen and 30 nm AuNP seeds. (e) SEM images of the gold substrate. (f) SERS spectrums of NBT on the gold substrate and the glass substrate. Figure 2.

SERS assays for detecting singlet miDNA. Representative SERS spectra was

collected from the (a) miDNA-141, (c) miDNA-21, and (e) miDNA-7d sensors with different concentrations. The calibration curves of the SERS intensity (∆I1004, ∆I1335, and ∆I2226) as a function the logarithm of miDNA concentrations for (b) miDNA-141, (d) miDNA-21, and (f) miDNA-7d, respectively. Error bar: standard deviation of three measurements. Figure 3. SERS assays for detecting multiplex miDNAs. (a) Representative SERS spectra was simultaneously collected from multiplex miDNAs with different concentrations in the same sample. The calibration curves of the SERS intensity (∆I1004, ∆I1335, and ∆I2226) as a separate function the logarithm of miDNA concentrations of (b) miDNA-141, (c) miDNA-21, and (d) miDNA-7d. Error bar: standard deviation of three measurements. Figure 4. Specificity assays. (a) SERS spectra of specific analysis for miDNA-21 in the presence of target miDNA-21, 1-base mismatched miDNA-21, 2-base mismatched miDNA-21, a noncomplementary sequence (miRNA-141) and blank sample. The concentration of each nucleic acids was 100 nM. (b) Signal quantification of the SERS intensity for the cases in (a). (c) SERS spectra of specific analysis for multiplex miDNAs in the presence of the mixture-3 (miDNA-141 + miDNA-21 + miDNA-7d + NC1 + NC2 + NC3), the mixture-2 (miDNA-141 + miDNA-21 +


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miDNA-7d), the mixture-1 (NC1 + NC2 + NC3) and the blank sample. The concentration of each nucleic acid sequence was 100 pM. (d) Signal quantification of the SERS intensity for the cases in (c). The NC represented the noncognate DNA. Error bar: standard deviation of three measurements. Figure 5. Practicality assays. (a) SERS spectra of miDNA-21 ranged from 10 fM to 100 nM in diluted (20%) human serum. (b) Raman intensities at 1335 cm−1 peak of detecting the target miDNA-21 in PBS buffer and in 20% human serum.


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Scheme 1


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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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For Table of Contents Only


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Ultrasensitive Simultaneous Detection of Multiplex Disease-Related

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