Surface-Enhanced Raman Spectroscopy Platform with Padlock Probe

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An “OFF” to “ON” SERS platform with padlock probe-based exponential rolling circle amplification for ultrasensitive detection of microRNA 155 Yi He, Xia Yang, Ruo Yuan, and Yaqin Chai Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04082 • Publication Date (Web): 25 Jan 2017 Downloaded from http://pubs.acs.org on January 28, 2017

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Analytical Chemistry

An “OFF” to “ON” SERS platform with padlock probe-based exponential rolling circle amplification for ultrasensitive detection of microRNA 155 Yi He#, Xia Yang#, Ruo Yuan∗, Yaqin Chai∗ Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China

Abstract In this work, an “OFF” to “ON” surface enhancement Raman spectroscopy (SERS) platform was constructed for ultrasensitive detection of microRNA (miRNA) by using magnetic SERS substrate (Co@C/PEI/Ag) and padlock probe-based exponential rolling circle amplification (P-ERCA) strategy. Herein, miRNA 155 could act as primers to initiate rolling circle amplification (RCA) for producing a long repeat sequence, and then the obtained DNA would be cleaved into two kinds of single-strand DNAs in the presence of nickase. One of the DNAs can be new primers to initiate new cycle reactions for obtaining large numbers of the other one (trigger DNA), consequently leading to an exponential amplification. On the other hand, the hairpin DNA (H1), with a Raman label (Cy5) at one end, would form hairpin structure to make the Cy5 closer to the SERS substrates, which could produce a strong SERS signal (“ON” status). Then placeholder DNA (P2) partly hybridized with H1 to open the hairpin structure making Cy5 far away from substrates with a decreased signal (“OFF” status). Nextly, the obtained trigger DNA can complement with P2 to make Raman label reclosed to SERS substrates with a strong SERS signal (“ON” status). From this principle, the strategy could achieve the change from “OFF” to “ON” status. And this SERS strategy exhibited a wide linear range of 100 aM to 100 pM with low detection limit of 70.2 aM, which indicated the proposed SERS platform with

#

These authors contributed equally to this work. Corresponding author. Tel.: +86-23-68252277; Fax: +86-23-68253172. E-mail address: [email protected]; [email protected]. ∗

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potential application value for ultrasensitive bioassay of miRNA.

Introduction MicroRNAs (miRNAs), a single-stranded noncoding RNAs which contain about 19-23 nucleotides, can control the events of cleavage or translational repression for mRNA.1,2 They can also been regarded as a potential biomarker in the fields of diagnosis, prognosis, and treatment of diseases.3,4 However, due to the characteristics of small size, sequence homology for miRNA among family members, low abundance in total RNA samples and susceptibility to degradation, the sensitive detection of miRNAs is still a challenge.5,6 Hence, it is urgent to search a sensitive and specific detection way to rapidly and accurately analyze miRNAs. Surface enhancement Raman spectroscopy (SERS) is a vibrational spectroscopic technology which owns high sensitivity and molecular specificity.7 Based on the advantages of rapid, specific, sensitive and nondestructive nature8, SERS has attracted great interest in the area of biochemistry and life sciences such as detection of antigen, cell, DNA and so on.9-11 SERS can also specifically detect analytes ranging from picomoles to femtomoles, which often served as a signal amplifying technology to sensitively detect miRNA.12,13 There is no doubt that SERS substrates play an important role in SERS technology which would produce and amplify Raman signal for achieving the aim of detecting ultra-trace target. Various nanomaterials combining with noble metal as SERS substrates are successfully synthesized to extremely enhance Raman signal. Such as Wang et al. synthesized double-wall Au nanocage/SiO2 nanorattles as sensitive SERS substrate for drug delivery and photothermal therapy.14 Tang et al. prepared a helically arranged Ag NPs@homochiral MOF for the enantioselective recognition of D/L-cysteine and D/L-asparagine.15 Chen et al. compounded gold nanoparticle coated carbon nanotube ring for theranostic applications.16 It is worth mention that magnetic materials such as Fe3O417,18, CoFe2O419,20, NiFe21 which can combine with Au or Ag were widely applied as SERS active substrates. These magnetic substrates can not only covalently bind dye molecule to produce SERS signal, but also magnetically focus the signal probe to 2


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improve the sensitivity. Also, large surface area and porous structure can act as advantageous factors to connect more dye and biological molecules, which can produce strong Raman signal to further improve sensitivity of the proposed biosensor. Thus, a new magnetic material Co@C with large surface area and porosity was chosen to combine with Ag nanoparticle forming Co@C/PEI/Ag SERS substrate for ultra-sensitively detecting targets. Rolling circle amplification (RCA), an isothermal enzymatic DNA replication process, has been widely applied in biomedical research owing to its simplicity and high sensitivity.22-24 As a result, RCA attracts lots of attention on the assay of fluorescent25,26, electrochemiluminescent27,28 and electrochemistry29-31 and so on. It is also studied in SERS to promote signal amplification which further improve the sensitivity of the biosensor. Some groups employed linear RCA technique to obtain a long repeat sequence which can be utilized to produce a lot of SERS hot spots for sensitively detecting targets.32-34 Nevertheless, this linear RCA process with limited amplification capability would hinder the further improvement of the sensitivity. In this work, nickase was introduced to solve this problem for it can cleave the long repeat sequence into abundant new primers, which can initiate more new RCA reactions to significantly improve the sensitivity of the biosensor with an exponential amplification mode. Padlock probe-based exponential rolling circle amplification (P-ERCA) is a process including a padlock probe specific ligation with target, linear RCA and nicking reactions. In our work, we have combined this efficient P-ERCA assay with SERS technology to achieve sensitive and effective detection of targets at a low concentration level. Herein, we developed a novel “OFF” to “ON” SERS platform with magnetically active substrate (Co@C/PEI/Ag) and specific padlock probe-based exponential rolling circle amplification (P-ERCA) strategy for sensitive detection of miRNA. Co@C/PEI/Ag as SERS active substrates could obviously enhance the sensitivity of this SERS strategy by magnetic aggregating. Herein, thiol group modified hairpin DNA (H1) with the Raman label molecule (Cy5) was immobilized onto Co@C/PEI/Ag to produce strong Raman signal. Then the placeholder DNA (P2) 3



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could partly hybridize with H1 to open the hairpin structure which would weaken the Raman signal making the sensor at an “OFF” status. On the other hand, a small number of miRNAs can act as primers to initiate P-ERCA reaction, generating new primers to initiate new RCA reaction for the aim of obtaining enough trigger DNA. The generated trigger DNA could grab P2 to form complementary DNA double chain, leading H1 to recover the hairpin structure. By this way, SERS platform recovered “ON” status with a strong Raman signal. The fabrication of this SERS platform was shown in Scheme 1. Based on this magnetically active SERS substrate and P-ERCA amplification strategy, the “OFF” to “ON” SERS platform can detect target miRNA 155 with a wide linear range and low detection limit, providing a promising potential in future for trace miRNA assay.

Scheme 1. (A) MiRNA 155 induced P-ERCA amplification process; (B) Schematic diagram of the construction of the SERS platform

Experimental section Materials and regents. HPLC-purified microRNA was provided by Takara Biotechnology Company Ltd. (Dalian, China). The DNA probes were synthesized by Sangon, Inc. (Shanghai, China) and the nucleotide sequences were listed in Table 1. Phi29 DNA polymerase, T4 RNA ligase 2, Nb.BbvcI and dNTP mixture were purchased from New England Biolabs, Inc. (Beverly, MA, USA). 6-mercapto-1-hexanol (MCH) was obtained from Shanghai Reagent Co., Ltd. (Shanghai, China) 2-Methylimidazole, Polyvinylpyrrolidone (PVP, 4


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K30), silver nitrate (AgNO3), sodium citrate, sodium borohydride (NaBH4), methanol (CH3OH), Polyetherimide (PEI), cobalt chloride (CoCl2·6H2O) and magnesium chloride (MgCl2·6H2O) were purchased from ChengDu Kelong Chemical Reagent Company (Chengdu, China). Table 1. Sequence information for the nucleic acids used in this study Name

Sequences (5′-3′)

miRNA 155 (target)

UUA AUG CUA AUC GUG AUA GGG GU

Padlock probe (P1)

GAT TAG CAT TAA CCT CAG CGA AAG CGA TCT ATA ATC CCT ACA CCA CCC TCA GCA CCC CTA TCA C

Placeholder (P2)

GAA AGC GAC TCT ATA ATC CCT ACA CCA C

Hairpin probe (H1)

HS-(CH2)6 CTC TAT AAG TGG TGT AGG GAT TAT AGA G-Cy5

Signal-base mismatch (sRNA)

UUA AGG CUA AUC GUG AUA GGG GU

miRNA 21

UAG CUU AUC AGA CUG AUG UUG A

miRNA 141

UAA CAC UGU CUG GUA AAG AUG G

Apparatus. The morphologies of materials were recorded by a scanning electron microscope (SEM, S-4800, Hitachi, Japan). The crystalline structures were characterized with X-ray diffraction (XRD, MAXima_X XRD-7000, Japan). The special surface area was measured by Brunauer-Emmett-Teller measurement (BET, ASAP 2020, USA). SERS spectra were measured by a Raman spectrometer (Renishaw Invia Raman spectrometer, Invia, UK) with the equipment of 633 nm line from a He-Ne laser (17 mW of power on the 50-objective). And the Raman spectrometer was calibrated before the SERS measurement with a clean silicon wafer at 520 cm−1 Raman shift. All the spectra were measured with a single 10 s accumulation. Preparation of Co@C. Firstly, ZIF-67 crystal was synthesized according to the literature35 with a little modification. Simply, 520 mg CoCl2·6H2O and 600 mg PVP were added into 40 mL CH3OH under stirring to form a homogeneous solution. 2.63 g 2-Methylimidazole was dissolved in another 40 mL CH3OH. Then the two solutions were mixed together 5



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and stirred for 5 min to fully mingle with each other. Afterwards, the prepared solution was aged at room temperature for 24 h. The purple precipitates were obtained during the age process. Next, the precipitates were washed by CH3OH several times to remove the un-reacted reagents and dried completely. Following that, the ZIF-67 were carbonized at 600 oC under an argon atmosphere for 6 h with a heating rate of 5 °C·min−1. Then the black powder Co@C was collected for further use. Preparation of Co@C/PEI/Ag. The Co@C/PEI/Ag was synthesized as follows. Firstly, 0.5 mg Co@C was added into 2 mL 1% PEI solution and sonicated for 2 h to form homogeneous complexes. The products were magnetically separated and washed twice with ultrapure water. Subsequently, 1 mL 10 mM AgNO3 was added into Co@C/PEI solution and stirred for 0.5 h. Then 1 mL 2 mM sodium citrate and 1 mL 10 mM NaBH4 were slowly dropped into the mixture. After 20 min stirring, the composites Co@C/PEI/Ag were washed by magnetic separation with ultrapure water. Preparation of P-ERCA products. The ligation reaction of linear padlock probe was performed in 10 µL of reaction mixture containing 1 × ligation buffer (50 mM Tris-HCl, 400 mM ATP, 2 mM MgCl2, 10 mM DTT, pH 7.5), 0.2 U T4 RNA ligase 2, 0.8 U RNase inhibitor, 100 nM linear padlock probe (P1), and different concentrations of miRNAs. The P1 and miRNA mixture were firstly denatured at 65 oC for 3 min and then cooled to room temperature slowly. Next, T4 RNA ligase 2 and the ligation buffer were added into the mixture and incubated for 2 h at 37 oC. Afterwards, 0.4 U Phi29 DNA polymerase, 1U Nb.BbvCI, 400 µM dNTP and 20 mM Tris-HCl buffer (containing 10 mM (NH4)2SO4, 10 mM KCl, 6 mM MgSO4, 0.1% Triton X-100) were added into 10 µL ligation reaction products to form a 20 µL mixture. After the mixture was incubated at 30 oC for 6 h, the products (trigger DNA) can be obtained. Preparation of SERS-Based Platform. Firstly, the thiol group modified H1 was denatured at 95 oC for 5 min and then slowly cooled down to room temperature. Secondly, 0.1 µM denatured DNA was incubated with the prepared Co@C/PEI/Ag in 0.25 mM MgCl2 solution overnight at 6


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room temperature. Nextly, 0.1 mM MCH were added in the mixture for 0.5 h to passivate the metal surface. The functionalized platforms were washed with Tris-HCl buffer (10 mM, pH=8.0) containing Tween 20 (0.01%) three times and then suspend in Tris-HCl buffer (10 mM, pH=8.0). Finally, 0.1 µM P2 was incubated with platforms at 37 oC in the PBS buffer overnight. The excess P2 was washed by PBS buffer through magnetic separation. Ultimately, the as-prepared SERS platform was stored at 4 oC for further use. SERS Strategy Assay Procedure. The SERS platform was incubated with the trigger DNA prepared by section 2.5 for 50 min at 37 oC. And then the platform was washed three times with PBS 7.0. Nextly, the platform was measured by Renishaw InVia Raman microscope equipped with a 633 nm HeNe laser.

Results and Discussion Principle of the SERS Strategy. From Scheme 1, the platform was composed of two parts. For the part of P-ERCA, a specific designed P1 can be ligated by miRNA 155 in the presence of T4 RNA ligase 2. Then Phi29 DNA polymerase would catalyze complex to synthesize a long concatenated repeat sequence by linear rolling circle amplification. In the presence of Nb.BbvCI, the repeat sequence can be cleaved into two kinds of signal-stranded DNAs (ssDNA). One can be new primers whose sequence were copied with target miRNA to initiate new RCA reaction, the other one can act as triggers to induce strand-displacement on SERS substrates, which can be a key factor of an “OFF” to “ON” switch. By this way, one miRNA can convert to a large number of trigger DNA, which obviously achieved the aim of enhancing the sensitivity of this proposed platform. For another part, Co@C as a magnetic carrier owns the advantages of regular morphology, large surface area and good magnetism which can combine with Ag nanoparticles to act as SERS substrates. This SERS substrate not only process significant Raman enhancement ability but also simply experiment operation for its 7



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magnetism. The corresponding Raman spectra of Co@C and Co@C/PEI/Ag were shown in curve a and b of Figure 1A. When H1 connected on the SERS substrates (Co@C/PEI/Ag) through covalent bond between Ag and thiol group, signal molecule Cy5 were close to Co@C/PEI/Ag substrate to produce a strong Raman signal (Figure 1A, curve c). This phenomenon can further demonstrate the feasibility of the proposed strategy. Nextly, P2 can open H1 to make the Cy5 far away from SERS substrate, resulting in the platform with “OFF” status (Figure 1B, black curve). However, trigger DNA obtained from P-ERCA can grab the P2 to form complementary DNA double chain which will leave the platform. Then H1 could recover hairpin structure, leading to the platform at “ON” state (Figure 1B, red curve). By this way, the proposed SERS platform with P-ERCA amplifying strategy and magnetic substrates can be constructed for quantitative and sensitive detection of miRNA 155.

Figure 1. (A) SERS spectra of Co@C (a), Co@C/PEI/Ag (b), Co@C/PEI/Ag-H1 (c); (B) SERS platform on the state of “OFF” (black curve), “ON” (red curve).

Characterization of Materials. Figure 2A was the SEM image of the ZIF-67, in which we can easily see the well-defined rhombic dodecahedral shapes. The ZIF-67 was uniform and the size was about 5 µM. Figure 1B was the SEM image of Co@C nanoparticles which were generated after carbonization of ZIF-67. From Figure 2B, the morphology and size of particles were mainly kept the same as ZIF-67 but with a concave surface. After in-situ producing AgNPs on the Co@C particles, we can see some small and white points were grown on the Co@C as shown in Figure 2C. 8


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Figure 2. SEM of (A) ZIF-67; (B) Co@C; (C) Co@C/PEI/Ag.

The X-ray diffraction (XRD), Brunner-Emmet-Teller (BET) and hysteresis loop were also characterized to prove the properties of Co@C particles. As shown in Figure 3A, the XRD pattern of Co@C was similarly to the XRD graph from the literature25, which indicates the successful synthesis of Co@C. To prove the large surface area of the Co@C compounds, the N2 physisorption measurements were tested. As shown in Figure 3B, the Co@C possessed a large BET surface area of 192.201 m2/g. In addition, to confirm the magnetic property of Co@C, the hysteresis loop was characterized. From Figure 3C, we can see Co@C owns good magnetism with saturation magnetization value of 87 emu/g.

Figure 3. (A) XRD of Co@C; (B) N2 adsorption-desorption isotherms of Co@C; (C) Hysteresis loop of Co@C.

Optimization of assay conditions. To increase the sensitivity and selectivity of the SERS-based platform, the detection conditions such as concentration of dNTP substrates, phi29 DNA polymerase and the incubation time were investigated in this study. The concentrations of dNTP and phi29 DNA polymerase are the key factors affecting the trigger DNA amplification by P-ERCA. From Figure 4 (A and B), we can easily see when concentration of dNTP and phi29 DNA polymerase were respectively 600 µM 9



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and 0.4 U, the Raman intensity can reach a steady value. Therefore, 600 µM dNTP and 0.4 U phi29 DNA polymerase were used for the P-ERCA. Also, the incubation time of trigger DNA was chosen with 50 min to make the platform at “ON” state. (Figure 4C)

Figure 4. Optimization of (A) Concentration of dNTP; (B) Concentration of Phi29 DNA polymerase; (C) Incubation time of trigger DNA.

Analytical performance of the SERS-based platform for detection of miRNA 155. The analytical performance of the SERS-based strategy was characterized on the basis of the optimal conditions by detecting the target miRNA 155 with various concentrations for three times. As depicted in Figure 5A, Raman signal intensity gradually increased with increasing concentration of miRNA 155 in the range of 100 aM to 100 fM. We chose the peak at 556 cm-1 as criteria to judge the linear relation between Raman intensity and concentration of miRNA 155. Figure 5B presented an excellent linear relationship between the Raman intensity at 556 cm-1 and the logarithm of miRNA 155 concentration. And the corresponding linear regression equation was y = 1294lgc – 1099 (R2 = 0.9959), where y was the peak intensity at 556 cm-1 and c was the concentration of miRNA 155. The detection limit for miRNA 155 calculated according to IUPAC recommendation was 70.2 aM, which was estimated by LOD = 3Sb/m, where Sb is the standard deviation of the blank signals (nB = 11), m is the analytical sensitivity estimated as the slope of the calibration plot at lower concentration ranges.36,37 Compared with the previously reported biosensors for the detection of miRNA 155, the developed SERS strategy showed better performance as shown in Table 238-44, which illustrated that it would provide a cogent evidence for highly sensitive detection of miRNA. This SERS strategy also compared with other 10


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SERS-based sensors focus on the miRNAs detection45-49 which revealed a lower detection limit as shown in Table 3.

Figure 5. (A) SERS-based platform with the increasing concentration of miRNA 155 (aM) at the peak of 556 cm-1 from a to h: (a) 0, (b) 102, (c) 103, (d) 104, (e) 105, (f) 106, (g) 107, (h) 108; (B) The calibration plot of Raman intensity vs lgc at 556 cm−1 (error bars = SD, n = 3).

Table 2. The comparison of the proposed SERS-based platform with other biosensors for miRNA 155 detection. Analytical method

Detection limit

Linear range

refs

DPV

0.6 fM

2 fM-8 pM

38

SWV

12 fM

50 fM-30 pM

39

Amperometric

0.14 fM

1 fM-100 pM

40

Fluorescent

10 pM

0.01 nM-200 nM

41

Fluorescent

100 pM

0.1 nM-200 nM

42

nanopore

0.1 pM

0.1 pM-100 pM

43

ECL

0.83 fM

2.5 fM-50 pM

44

SERS

0.07 fM

100 aM-100 pM

This work

Table 3 The comparison of the proposed SERS-based platform with other SERS sensors for miRNA detection. Detection

Detection

Detection

method

Target

limit

Linear range

11


Refs


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SERS

miRNA 205

0.5 fM

1 fM -100 n M

46

SERS

Let-7b

0.3 fM

1 pM - 10 nM

47

SERS

miRNA21

5 pM

15 pM - 60nM

48

SERS

miRNA 141

0.17 fM

1 fM-100 nM

49

SERS

miRNA 155

0.07 fM

0.1 fM-100 pM

Our work

Selectivity of the P-ERCA-based SERS strategy. To investigate the selectivity of the P-ERCA-based SERS strategy, three possible interferences including one-base mismatched DNA, miRNA 21, and miRNA 141 were assessed under the same experimental conditions for three times. And the results were exhibited in Figure 6. When the proposed strategy were incubated in 100 pM one-base mismatched DNA, miRNA 21 and miRNA 141, respectively, there is no obvious change compared with the blank sample (no target miRNA). However, when target miRNA 155 (100 fM) were coexisted with the interferences, the Raman intensity was nearly the same as that with only miRNA 155. The results indicated this strategy possess high specificity for miRNA 155 assay.

Figure 6. Selectivity of the SERS platform for different targets: blank; one-base mismatched stand (100 pM); miRNA 21 (100 pM); miRNA 141 (100 pM); miRNA 155 (100 fM); a mixture containing one-base mismatched stand (100 pM), miRNA 21 (100 pM), miRNA 141 (100 pM) and miRNA 155 (100fM).

Reproducibility of the SERS-Based platform. To evaluate the reproducibility of the proposed platform, the coefficient of variation of the proposed SERS strategy was investigated. Firstly, SERS spectra of the 12


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platform at 20 different spots were collected and the peak at 556 cm-1 was used as a standard. As shown in Figure 7A, the 20 different curves were basically consistent. And the relative standard deviation (RSD) was 2.75% which was shown in Figure 7B. When the SERS platform was investigated by 5 different SERS substrates to test the same concention of miRNA 155(100 fM), the RSD was 2.02% shown in Figure 7 (C and D). These results all demonstrated that the proposed SERS-based strategy possessed good reproducibility.

Figure 7. (A) SERS spectrum measured from 20 different spots with a same SERS substrate; (B) The intensity at 556 cm−1 (cmiRNA 155 = 100 fM); (C) SERS spectrum measured with 5 different SERS substrates with the same condition; (D) The intensity at -1

556 cm (cmiRNA 155 = 100 fM)

Preliminary analysis of real samples. To investigate the analytical reliability and application potential of the proposed SERS platform, the recovery experiments were performed by adding various concentration of miRNA 155 into the 100-fold diluted healthy human real serum sample (acquired from Xinqiao Hospital of Third Military Medical University, China). The results shown in Table 4 exhibited good recoveries from 93.56% to 104.8% and the RSD values were from 2.1 to 3.5, which indicated that the prepared platform have potentiality for detection of miRNA 155 in clinical analysis. 13



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Table 4 Determination of miRNA 155 added in human blood serum (n = 3) with the proposed platform. Serum Sample

Concentration of miRNA 155 added/ fM

Concentration obtained with platform / fM

Recovery /%

RSD /%

1

1

1.033

103.4

3.4

2

10

10.49

104.8

2.1

3

100

98.63

98.63

3.5

4

1000

935.6

93.56

4.1

5

10000

9735

97.36

2.9

Conclusion In summary, a novel “OFF” to “ON” SERS platform combining magnetic Co@C/PEI/Ag SERS substrate with the P-ERCA nucleic acid signal amplification strategy has been successfully constructed for detecting miRNA 155. This proposed strategy can achieve a detection limit of 70.2 aM and a detection range from 100 aM to 100 pM. The good performance of the strategy may be attributed to the following reasons. Firstly, Co@C/PEI/Ag as SERS substrates not only effectively enhanced the Raman signal of the Cy5 but also simplified the experiment operation process. Secondly, P-ERCA as a signal amplification strategy can obviously enhance the sensitivity of this SERS strategy by amplifying the amounts of primers DNA. Moreover, DNA strand-displacement hybridization process can further improve the sensitivity of the platform with the “OFF” to “ON” switch. Based on the above advantages of the proposed SERS platform, we hope that the high sensitive and specific platform can be possibly applied in clinical applications.

Acknowledgements This project has been financially supported by the NNSF of China (51473136， 21575116, 21675129), the Fundamental Research Funds for the Central Universities, China (XDJK2015C099, SWU114079), China Postdoctoral Science Foundation (2015M572427) and Chongqing Postdoctoral Research Project (xm2015019). 14


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