Multicolor GoldâSilver Nano-Mushrooms as Ready-to-Use SERS

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Multicolor Gold-Silver Nano-Mushrooms as Ready-to-Use SERS Probes for Ultrasensitive and Multiplex DNA/miRNA Detection Jing Su, Dongfang Wang, Lena Nörbel, Jianlei Shen, Zhihan Zhao, Yanzhi Dou, Tianhuan Peng, Jiye Shi, Sanjay Mathur, Chunhai Fan, and Shiping Song Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04729 • Publication Date (Web): 25 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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Analytical Chemistry

Multicolor Gold-Silver Nano-Mushrooms as Ready-to-Use SERS Probes for Ultrasensitive and Multiplex DNA/miRNA Detection ‖

‖

Jing Su, † Dongfang Wang, † Lena Nörbel,‡ Jianlei Shen,† Zhihan Zhao,† Yanzhi § Dou,† Tianhuan Peng,† Jiye Shi, Sanjay Mathur, *‡ Chunhai Fan† and Shiping Song*† †Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China ‡Institute of Inorganic Chemistry, University of Cologne, D-50939 Cologne, Germany §Kellogg College, University of Oxford, Oxford OX2 6PN, UK Corresponding Author *E-mail: [email protected]. Tel: (86) 21 39194065. Fax: (86) 21 39194702. *E-mail: [email protected]. Tel: (49) 221 4704107. Fax: (49) 221 4704899. ABSTRACT: Uniform silver-containing metal nanostructures with strong and stable surface-enhanced Raman scattering (SERS) signals hold great promise for developing ultrasensitive probes for biodetection. Nevertheless, the direct synthesis of ready-to-use such nanoprobes remain extremely challenging. Herein we report a DNA-mediated gold-silver nano-mushroom with interior nanogaps directly synthesized and used for multiplex and simultaneous SERS detection of various DNA and RNA targets. The DNA involved in the nanostructures can act as not only gap DNA (mediated DNA) but also probe DNA (hybridized DNA), and allow it have the inherent ability to recognize DNA and RNA targets. Importantly, we were the first establishing a new method for the generation of multicolor SERS probes using two different strategies. First Raman-labeled alkanethiol probe DNA was assembled on gold nanoparticles and second thiol-containing Raman reporters were co-assembled with the probe DNA. The ready-to-use probes also give great potential to develop ultrasensitive detection methods for various biological molecules.

INTRODUCTION

high-efficiency SERS probes for ultrasensitive

Surface enhanced Raman spectroscopy (SERS) has

detection of these biomolecular targets. One

been considered as a very powerful vibrational

challenge remains in the design of suitable and

spectroscopy technique which could give great

reproducible

potential for bio-detection, especially in the field of

because ‘hot spots’ existing in exterior nanogaps

SERS

active

nanostructures,13,14

For example it allows the detection

between metal nanostructures are usually randomly

of trace levels of analysts such as important

distributed leading to a wide distribution of

biomolecules like nucleic acids, proteins or small

enhancement factor values which goes ahead with

1-5

biomedicine.

Nevertheless,

a large variability and uncertainty in the SERS

there are still two main challenges to develop

signal15-18. Recently, more and more studies

6-12

molecules related to life sciences.

1

ACS Paragon Plus Environment


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focused on the generation of interior nanogaps.19-23

not like the gold-gold core-shell nanostructures

To address this problem, several approaches were

with fully closed interior nanogaps. The not fully

developed in our laboratory and others for the

closed gaps of nano-mushrooms cannot constantly

synthesis of gold core-shell nanostructures with a

hold small-molecule Raman reporters adsorbed

stable SERS signal.

24-27

onto the AuNPs.

The nanostructure was

provided with a polyA DNA induced interior

In this work, we developed multicolor gold-silver

nanogap which contains non-fluorescent small

(Au-Ag) nano-mushrooms which could be used as

molecule Raman reporters. We demonstrated that

ready-to-use SERS probes for ultrasensitive and

such gold-gold nanostructures could be employed

multiplex

for

SERS

oligonucleotides assembled on the AuNPs were

nanoprobes are strongly suitable and show some

redesigned, facilitating the formation of the interior

great advantages compared to other optical

nanogap

multiplex

detection

for

which

DNA/RNA

and

detection.

hybridization

with

First,

DNA/RNA

probes28. However, all such nanostructures only

targets.

can act as SERS “tags”, not SERS probes.29-31 That

surface of the nano-mushrooms easily guided

is, only after bio-functionalization, these SERS

hybridization with complementary strands.43,44

nanostructures can have the ability to hybridize or

Thus, the oligonucleotides can act as both gap

bind to biomolecular targets. The other challenge

DNA and probe DNA. Second, we designed a new

remains in synthesizing silver-containing metal

strategy

nanostructures with interior nanogaps. Silver has a

nano-mushrooms. The common strategy is the

larger SERS effect than gold and can be excited

assembly of Raman molecular labeled alkanethiol

over much broader wavelengths (from UV to IR)

probe

than gold.25,32 Ren and colleagues33 recently

Raman-reporter molecules can’t be labeled to

synthesized Au-Ag core-shell nanoparticles with

oligonucleotides, we were the first establishing a

embedded

internal

The oligonucleotides exposed on the

for

the

DNA

generation

on

AuNPs.

of

multicolor

Since

many

for

reliable

new protocol in which thiol containing Raman

After

further

reporters were co-assembled with the probe DNA.

bio-functionalization, the nanoparticles can be

Third, we demonstrated successful simultaneous

involved in biological applications. We developed

detection of the three virus DNA targets in one

a

synthesizing

sample, as each Raman reporter has its own unique

silver-containing bimetallic nano-mushrooms with

spectroscopic fingerprint. Also, the design of the

interior nanogaps.34 The oriented growth of silver

SERS nanoprobes could be effectively adapted for

on the surface of gold nanoparticles (AuNPs)

the detection of RNA.

quantitative

SERS

DNA-mediated

standards

40-42

analysis.

approach

for

modified with DNA leads to the formation of

EXPERIMENTAL SECTION

interior nanogaps between the silver cap and the

Reagents and materials

gold head in a size range of 1-2 nm. We could

AgNO3,

reach enhancement factors of about 109 orders of

modified with streptavidin (Dynabeads® My

respect to SERS enhancing effects34-37. Especially, different

nano-mushrooms

Raman

molecules

decorated could

and

Sigma-Aldrich (St. Louis, MO, USA). MMPs

introduction of silver which outperforms gold with Au-Ag

(ABT),

4-Nitrothiophenol (NBT) were purchased from

magnitude. This could also be contributed to the

the

4-Aminothiophenol

OneTM Streptavidin C1) were purchased from

with

Invitrogen (Carlsbad,CA, USA). Other reagents

display

including polyvinylpyrrolidone (K-30), (+)-sodium

well-defined Raman fingerprint peaks, thus they

L-ascorbate,

can serve as multicolor probes.38,39 However,

HAuCl4,

were

purchased

from

Sinopharm Chemical Reagent Co. Ltd (China).

multicolor nano-mushrooms are not easily obtained

HPLC-purified ssDNAs and RNAs were purchased

because the interior nanogap is partially opened,

from TaKaRa Biotechnology Co. Ltd (DaLian, 2


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China). NANO pure water (>18.0 MΩ, Milli-Q)

with vigorous vortexing for 1 min. Then the

was used in all experiments.

solutions were incubated for 0.5h. The particles

Preparation of DNA-modified AuNPs

were washed three times with 10 mM PB (pH 7.4) the

using centrifugation (7500 rpm, 7 min, 4 °C) and

oligonucleotide-coated AuNPs based on literature

re-suspended in 10mM PB buffer. The Au-Ag

We

modified

and

characterized

procedures. For the preparation of DNA-modified

nano-mushrooms were characterized with a TEM

AuNPs, HPLC purified SH decorated DNA was

(Tecnai G2F20S-TWIN, FEI, USA) and a UV-vis

mixed with 50nm AuNPs solutions. In detail, 10

spectrophotometer

µL of a 100µM DNA solution was mixed with a

Japan). The TEM samples were prepared by

50nm AuNPs solution (0.1 nM, 600µL). The

dropping the above solution (10 µL) onto a

resulting solution was placed on an orbital shaker

copper-coated

overnight at room temperature. The mixtures were

temperature, the samples were imaged using TEM.

adjusted to obtain a final phosphate concentration

SERS detection of DNA/RNA targets

of 10 mM (pH 7.4) with a 100mM phosphate

Capture-DNA-coated

buffer (PB). Next, the mixtures were adjusted to a

according to the manufacturer’s instructions.

final concentration of 0.1 M NaNO3 after the

Briefly, the MMPs modified with streptavidin were

addition of salting buffer (2 M NaNO3) every 1

first washed three times with washing buffer (10

hour for three times. Then the resulting solutions

mM sodium phosphate buffer with 0.15 M NaCl

were placed on an orbital shaker overnight at room

and 0.05% (v/v) Tween 20, pH 7.4) and

temperature. The solutions were then centrifuged at

re-suspended in 2 × B&W buffer (10 mM

7500 rpm for 7 min at 4 °C. The supernatants were

Tris-HCl, 1mM EDTA, 2 M NaCl, pH 7.5) to a

poured carefully to remove the unmodified DNA.

final concentration of 2µg/µL. For every 40µL

The precipitate was dispersed with 10 mM PB

2µg/ml beads, 2µL 100µM capture DNA was

solution (pH 7.4). These procedures were repeated

added. For the multiplex DNA/RNA markers

three times. Finally, the precipitate was dispersed

analysis, the mixture of 6µL biotinylated capture

with 600µL 10 mM PB (pH 7.4).

DNA/RNA (equal volume of the three kinds of

Preparation of DNA and Raman molecule co-modified AuNPs

DNA/RNA) was added. Similarly, the equal

The DNA-modified AuNPs (600µL) were mixed

the multiplex analysis. The solution was shaked

with 6µL of non-fluorescent small molecular

gently for 15 minutes at room temperature. Extra

Raman report solution (100mM, NBT, ABT) and

DNA/RNA was removed by washing three times

the mixture was incubated for 2 hours at room

with PBST and then magnetic beads were

temperature with gentle shaking. The particles

dispersed in PBS to the concentration of 0.2mg/ml.

were washed three times with 10 mM PB (pH 7.4)

For DNA/RNA detection assays, 10µL target

using centrifugation (7500 rpm, 7 min, 4 °C) to

DNA/RNA

remove excess Raman reporters. Then they were

incubated with MMPs at the same volume at 37°C

re-suspended in 600µL of 10 mM PB buffer (pH

for 2 hours. The solution was shaked gently in

7.4).

order to get the MMPs dispersed. Then extra

Synthesis and characterization of mushroom-like nanostructures

DNA/RNA target was removed by washing with

The modified AuNPs solution (100 µL, 0.1 nM)

functionalized Au-Ag SERS probes were added to

was first mixed with PVP (500 µL, 1%). Sodium

the solution and incubated for 2 hours at 37°C, and

ascorbate (30 µL, 20 mg/ml) was added, followed

the shaking state should be kept to avoid the

by the rapid addition of AgNO3 (30 µL, 0.001 M)

uncontrollable aggregation in the liquid phase.

45

(U-3010,

grid.

After

MMPs

Hitachi,

drying

were

Tokyo,

at

room

prepared

volume of targets of DNA/RNA was added during

with varying concentrations

was

PBST for three times. 0.2 nM of probe DNA

3



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After the reaction, the MMPs were washed with

mW. Raman scattering was collected at a spectral

PBST to remove extra SERS probes. MMPs were

resolution of 4 cm-1 with a charged-coupled device

collected and dispersed onto the silicon wafer.

CCD) camera. The MMPs sandwich complexes

Surface-Enhanced Raman scattering detection

samples were prepared by dropping 5µL of

SERS

using

complexes solutions on the silicon base. After

XPLORA (HORIBA, Jobin Yvon, France) Raman

drying at room temperature, the Raman spectra

microscope system. A Melles Griot He-Ne laser

reported here were collected for 10 exposure times

operating at λ=638nm was used as the excitation

in the range of 1000-2000 cm-1.

measurements

were

obtained

source with a laser power of approximately 10

Figure 1. a) TEM images of Au-Ag nano-mushrooms using AuNP seeds with the diameter of 50nm. b) The Au-Ag nano-mushrooms used as ready-to-use SERS nanoprobes for ”sandwich-type” DNA detection. c) SEM images of sandwich MMP complexes. Left: In the presence of target DNA (10 nM), a large number of 100 nm Au-Ag nano-mushrooms were coupled with the MMPs due to DNA hybridization. Right: The blank control experiment in which no DNA target was added. 4


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RESULTS AND DISCUSSION

(>90%). All of the Au-Ag nano-mushrooms have

Ready-to-use nano-mushrooms

an average size of 100 nm in diameter and a

Bimetallic nano-mushrooms with DNA-mediated

uniform interior nanogap (Figure 1a). In addition,

interior nanogap for high-efficiency SERS signal

the

amplification were previously developed in our

nano-mushrooms was characterized (Figure S1a),

UV-vis

spectrum

of

the

Au-Ag

Au-Ag

indicating that uniform nano-mushrooms were

nano-mushrooms increases with the size of gold

formed. Time-dependent Raman spectra indicated

seeds. The bigger the mushroom, the more DNA it

that the signal of the Au-Ag nano-mushrooms was

can load. Here we synthesized the Au-Ag

highly uniform and reproducible (Figure S1b),

nano-mushrooms

which arises from their highly uniform and stable

group.

34

The

SERS

with

signal

50nm

of

AuNPs.

The

structures of the Au-Ag nano-mushrooms were

nanogap-based

confirmed by transmission electron microscopy

nano-mushrooms dispersed in PB buffer should be

structures.

Note,

the

Au-Ag

(TEM) and UV-vis spectroscopy. For all of the

stored at 4 °C for three months without aggregation.

nano-mushroom types, highly uniform Au-Ag

Thus, compared with the SERS substrates made of

nanostructures at high yields were obtained. For

metal nanoparticles on the solid support,46-48 the

example, the X-rhodamine (ROX)-encoded Au-Ag

Au-Ag nano-mushrooms could show similar

nano-mushrooms were synthesized at a high yield

stability in the real application.

Figure 2. a) Dose–response SERS spectra for HAV target detection. b) Plot of SERS intensity at peak 1504 cm-1 as a function of logarithm of HAV DNA concentration. The concentration of HAV DNA ranged from 100 fM to 1 nM. c) SERS spectra of specific analysis for single base mismatch and non-cognate DNA. d) SERS intensity from HAV wild type DNA, HAV DNA with T mismatch, C mismatch, A mismatch, non-cognatic DNA and blank sample. Error bars indicate the standard deviations from 3 measurements. Error bars indicate the standard deviations from at least 3 measurements (Intensities shown were after background subtraction).

Besides

strong

SERS

signals,

the

DNA/RNA hybridization. As gold heads are

Au-Ag

nano-mushrooms have the inherent ability of

partially

covered

5


by

silver

hats

in

the


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nano-mushrooms, exposed oligonucleotides on the

decreased with HAV DNA concentration changed

gold heads are free and can readily hybridize to

from 100fM to 1nM.The SERS intensity versus

their complementary sequences, enabling the

HAV DNA concentration was plotted as a

Au-Ag

dose-response curve at selected peak (1504 cm-1

nano-mushrooms

to

be

ready-made

SERS-active probes for DNA detection. To

for ROX). It is obvious that the SERS signal could

evaluate the hybridization properties of Au-Ag

be observed at DNA target concentration as low as

nano-mushrooms,

universal

100 fM, which corresponds to 1 amol of DNA

analytical strategy for virus DNA marker (Figure

molecules in 10 µL sample (see Figure 2a,b). Also,

1b). We designed a specific oligonucleotide

we challenged the hybridization specificity of the

labelled with ROX which can act as both a

Au-Ag nano-mushrooms by designing different

mediated DNA for the synthesis of Au-Ag

types of mutation oligonucleotide sequences and a

nano-mushrooms and a probe DNA to recognize

noncognatic sequence (see Table S1 in Supporting

DNA fragments of hepatitis A virus (HAV) Vall7

Information for detailed sequences). As can be

polyprotein gene. After hybridizing to the Au-Ag

observed in Figure 2 (c, d), only the wild type

nano-mushrooms,

were

DNA target reached the highest SERS signal, while

micro-magnetic

the signals resulted from 1 µM of mutation

nanoparticles (MMPs) modified with capture DNA

oligonucleotide sequences and the noncognatic

via a streptavidin–biotin bridge which can also

sequence showed no significant differences to the

hybridize to HAV DNA targets. Then we measured

blank control sample. The results indicated that

separated

from

we

developed

HAV

DNA

solutions

by

a

targets

Au-Ag

HAV DNA targets could be detected even in the

nano-mushrooms. As shown in Figure 1c, a large

presence of interfering DNA sequences present

number of 100nm nano-mushrooms were coupled

with a more than 103-fold greater concentration in

with MMPs due to DNA hybridization in the

the sample.

the

SERS

intensity

of

separated

presence of the DNA target, while SERS intensity

Scheme 1. a) Schematic illustration of synthesis of mushroom-like Au-Ag SERS probes in two different ways. (1) Fluorescence Raman reports are labeled on the probe DNA, (2) Thiol-containing Raman reports are adsorbed on the AuNPs. b) Multiplex analytical strategy for virus gene DNA/RNA. Three types of SERS nanoprobes were prepared via the functionalization of probe DNA on Au-Ag mushrooms encoded by Raman reports. Each type of DNA/RNA target was captured by the corresponding capture DNA/RNA followed by hybridization with its own SERS nanoprobes, leading to the formation of concomitant multiple sandwich complexes.

6


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Multicolor nano-mushrooms

is assembling Raman molecular-labeled alkanethiol

Compared with fluorescent tags which often suffer

DNA on gold nanoparticles. The other is that

from spectral overlap of different fluorophores and

thiol-containing Raman reporters and DNA are

needing excitation light with different wavelength,

co-assembled on the surface of AuNPs. As a

SERS nanoprobes can produce molecular specific

demonstration, we selected ROX-labeled DNA,

vibrational spectra that can easily differentiate

CY3-labeled DNA, 4- Aminothiophenol (ABT)

between mixtures of Raman labels with single-laser

and 4-Nitrothiophenol (NBT) as Raman reporters

excitation. However, we had difficulties with

to obtain four kinds of nano-mushrooms according

obtaining multicolor nano-mushrooms based on the

to the two strategies. Figure S2 presents the SERS

above-mentioned protocol because quite few

spectra of all of the Au-Ag nano-mushrooms and

Raman dyes can be labeled to oligonucleotides and

demonstrates that every specific SERS mushrooms

there are fewer Raman-labeled DNA commercially

can be clearly identified based on its unique

available. Taking advantage of thiol-containing

spectroscopic fingerprint, which is suitable for

Raman reporters, we designed a new protocol to

multiplex biodetection. Predictably, combining the

synthesize SERS-active Au-Ag nano-mushrooms.

Raman-labeling model and the co-assembling

Thus, we had developed two strategies to obtain

model, much more Au-Ag nano-mushrooms can be

Au-Ag

designed and synthesized, having their own SERS

nano-mushrooms

containing

different

spectroscopic fingerprints.

Raman reporters, acting as multicolor SERS-active nanoprobes. As shown in scheme 1(a), one strategy

Figure 3. a) Concentration dependent SERS spectra at different Raman peaks (1590 cm-1 for HIV, 1504 cm-1 for HAV, 1338 cm-1 for HBV ) for HIV, HAV, and HBV DNA detection. The DNA target concentration ranged from 1pM to 10 nM. b) Plot of SERS intensity at selected Raman peaks (1590 cm-1 for HIV) at a function of HIV DNA concentration. c) Plot of SERS intensity at selected Raman peaks (1504cm-1 for HAV) at a function of HAV DNA concentration. d) Plot of SERS intensity at different Raman peaks (1338cm-1 for HBV) at a function of HBV DNA concentration. Error bars indicate the standard deviations from at least 3 measurements (Intensities shown were after background subtraction). 7



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Multiplex SERS detection of DNA

Page 8 of 13

targets varied from 1pM to 10nM, and the SERS

Having established that Au-Ag nano-mushrooms

spectra changed with the increasing concentration

have

specific

of the three DNA targets (see Figure 3a). It is

hybridization properties and that multicolor Au-Ag

obvious that each SERS spectrum only displays the

nano-mushrooms can be synthesized via different

unique

strategies, we employed them as ready-to-use

corresponding Au-Ag nano-mushroom, and the

SERS nanoprobes for multiplex DNA detection.

specific DNA target can be easily and distinctly

Here, three kinds of Au-Ag nano-mushrooms

identified

fingerprinted with NBT, ROX and ABT were

fingerprint of the nanoprobes. With the decreasing

designed to recognize and hybridize to HAV DNA,

concentration of DNA targets, the SERS peaks

hepatitis

human

from 1338cm-1, 1504cm-1 and 1590cm-1 decreased

immunodeficiency virus (HIV) DNA, respectively.

gradually and blank control sample displayed very

As shown in scheme1 (b), common MMPs

weak SERS signal. As shown in the Figure 3(b-d),

functionalized by three capture DNAs were used to

the SERS signal could be observed as low as 1pM

separate all hybridized Au-Ag nano-mushrooms for

for each of the DNA target, demonstrating the

SERS

multiplexing

strong

B

SERS

signals

virus (HBV)

measurements.

and

DNA

We

and

challenged

the

spectroscopic

according

to

signature

the

capability

of

visually

of

the

the

unique

Au-Ag

nano-mushrooms.

simultaneous detection of three DNA targets in the same sample. The concentration of the DNA

Figure 4. a) Dose–response SERS spectra for miRNA-21 detection. b) Plot of SERS intensity at peak 1504 cm-1 as a function of logarithm of miRNA-21 concentration. The concentration of miRNA-21 ranged from 10fM to 100pM. Error bars indicate the standard deviations from 3 measurements. c) SERS spectra of specific analysis for random RNA. d) SERS intensity from random RNA and blank sample. Error bars indicate the standard deviations from at least 3 measurements (Intensities shown were after background subtraction).

Multiplex SERS detection of microRNA

and

hybridize

microRNA

(miRNA),

Au-Ag

nano-mushrooms can also have the potential for

As the probe DNA can be designed to recognize

multiplex detection of miRNA. For a quantitative 8


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investigation,

we

Au-Ag

1µM random RNA and the blank sample,

nano-mushroom SERS probes with a series of

indicating that the Au-Ag nano-mushroom probe

concentrations of the synthetic miRNA-21 in a

could recognize and hybridize with the RNA target

range from 10 fM to100 pM. As shown in Figure 4

very specifically. Subsequently, we challenged the

(a, b), the characteristic SERS peak of ROX

Au-Ag nano-mushroom probes for multiplex

(1504cm-1) increased monotonically with the

miRNA

logarithm concentration of miRNA-21, which is

nano-mushroom SERS probes were designed via

similar to the DNA detection. In the absence of

the modification of three types of probe DNA on

miRNA-21, there was no significant SERS signal

Au-Ag nano-mushrooms encoded by ROX, 4-ABT

observed,

control

and CY3, while capture DNA for the three targets

experiment indicated no binding of the Au-Ag

were co-assembled on the MMPs. Each type of

nano-mushroom

lowest

miRNA target was captured by the corresponding

concentration of miRNA-21 target detected was 10

capture DNA followed by hybridization with its

fM (see Figure S3 of Supporting Information for

own SERS probes, leading to the formation of

details). The reason may be that the hybridization

concomitant multiple sandwich complexes. Thus,

which

challenged

means to

the

the

blank

MMPs.

The

between DNA and RNA is more stable. investigated

the

specificity

of

49

the

detection.

Three

types

of

Au-Ag

three miRNA targets in a single sample would be

We also

detected simultaneously.

Au-Ag

Then we investigated

nano-mushroom probes which was used for

the sensitivity of the multiplex RNA assay. As

miRNA detection. Significantly, it can effectively

shown in Figure 5a, in a serial dilution of the

differentiate miRNA-21 from random RNA at pM

samples,

level (Figure 4c.d). Fully complementary 100 pM

miRNA-31 and miRNA-141 from 1 pM to 10 nM

the

concentrations

of

miRNA-21,

miRNA target could be clearly differentiated from

Figure 5. a) Concentration dependent SERS spectra at different Raman peaks (1504 cm-1 for miRNA-21, 1078 cm-1 for miRNA-31, 1590cm-1 for miRNA-141) for miRNA detection. The miRNA target concentration ranged from 1pM to 10 nM. b) Plot of SERS intensity at selected Raman peak (1504 cm-1 for miRNA-21) at a function of miRNA-21 concentration. c) Plot of SERS intensity at selected Raman peak (1078 cm-1 for miRNA-31) at a function of miRNA-31 concentration. d) Plot of SERS intensity at different Raman peak 9



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Page 10 of 13

(1590cm-1 for miRNA-141) at a function of miRNA-141 concentration. Error bars indicate the standard deviations from at least 3 measurements (Intensities shown were after background subtraction).

were detected. The signal intensity of the

SERS detection of serum samples

characteristic SERS peaks of ROX, 4-ABT and CY3 increased with increasing concentration of the

To test the feasibility of the practical application of

miRNA-21, miRNA-31 and miRNA-141 targets.

the Au-Ag nano-mushroom probes, we conducted

As low as 1pM miRNA target could be clearly

analyses of miRNA-21 in biological fluids. As

distinguished

The

demonstrated in Figure 6, we found that there was

quantitative results for the detection of miRNA-21,

no significantly difference between SERS signal

miRNA-31 and miRNA-141 were shown in Figure

from buffer and diluted serum (20%) sample, thus

5(b–d). Also, the multiplex assay for these

the

miRNAs exhibited a good dynamic range of 5

anti-interference ability. Combined with the result

orders.

As the ready-to-use nano-mushrooms can

that the Au-Ag nano-mushroom SERS probe could

be easily synthesized, and two strategies have been

specifically recognize the RNA target in the

developed to obtain Au-Ag nano-mushrooms

presence of 104 fold of the random RNA in the

containing various Raman reporters, the probe

buffer (Figure 4d). This clearly demonstrated that

DNA can also be designed to recognize and

our SERS probes could selectively identify RNA

hybridize other kinds of bioactive molecules

target even in serum. Since serum is the most

through assembling the aptamer sequence on

complicated biological fluid in in-vitro diagnostics,

AuNPs, thus the Au-Ag nano-mushroom probes

other body fluidics are relatively clean mediums

have the great potential for multiplex detection of

and contain few interfering proteins. Accordingly,

different biomarkers and the mixture of various

the Au-Ag nano-mushroom probes could also be

bioactive molecules, including cell, protein and

used in other body fluidics such as saliva, urine and

small

from

the

molecules.24,50-51,

blank

control.

Therefore,

Au-Ag

nano-mushroom

had

strong

ascites, which may greatly broaden the application

Au-Ag bio-

range of the SERS probes, and facilitate the

functionalization could be developed to be a

molecular diagnosis of diseases in an early stage

universal multi-analysis platform. 24, 52

which is necessary for effective treatment.44

nano-mushroom

probe

without

further

CONCLUSION In summary, we developed multicolor Au-Ag nano-mushrooms that can act as ready-to-use SERS nanoprobes

for

ultrasensitive

and

multiplex

DNA/RNA detection. We demonstrated that the Au-Ag nano-mushroom probes have specific hybridization properties because of the inherent ability of DNA/RNA hybridization with exposed oligonucleotides on AuNPs. Multicolor Au-Ag nano-mushrooms

become

available

via

two

strategies for the assembly of Raman reporters, enabling them to recognize and distinguish specific

Figure 6. Detection of 100 pM miRNA-21 target in

DNA/RNA

buffer and in diluted (20%) serum. Data were

compared

collected from at least three independent sets of experiments.

Intensities

shown

were

targets with

from other

others.

Especially,

silver-containing

nanostructures, the Au-Ag mushroom SERS probes

after

are easy to synthesize and

background subtraction.

could be used without

further bio-functionalization. Therefore, Au-Ag 10


Page 11 of 13

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nano-mushrooms

hold

great

promise

for

(3) Wang, Y.; Yan, B.; Chen, L. Chem. Rev. 2012, 113,

developing multiplex bio-sensing strategies for

1391-1428.

molecular

(4) Zhang, P.; Zhang, R.; Gao, M.; Zhang, X. ACS Appl.

diagnostics

and

cellular

imaging.

Because of their bimetallic and nanobio-based

Mater. Interfaces.2013, 6, 370-376.

properties, these Au-Ag nano-mushrooms also hold

(5) Song, C.; Yang, Y.; Yang, B.; Sun, Y.; Zhao, Y.; Wang,

great potential for other applications in chemistry

L. Nanoscale. 2016, 8, 17365-17373.

and life science.

(6) Saha, K.; Agasti, S. S.; Kim, C.; Li, X.; Rotello, V. M. Chem. Rev. 2012, 112, 2739-2779. (7) Lei, J.; Ju, H. Chem. Soc. Rev. 2012, 41, 2122-2134.

ASSOCIATED CONTENT

(8) Baker, B. R.; Lai, R. Y.; Wood, M. S.; Doctor, E. H.;

Supporting Information

Heeger, A. J.; Plaxco, K. W. J. Am. Chem. Soc. 2006, 128,

Figure

S1

showed

time-dependent

the

Raman

UV-vis

results

of

spectra the

3138-3139.

and

(9) Anker, J. N.; Hall, W. P.; Lyandres, O.; Shah, N. C.;

Au-Ag

Zhao, J.; Van Duyne, R. P. Nat. Mater. 2008, 7, 442-453.

nano-mushrooms. Figure S2 showed multi-color

(10) Alvarez-Puebla, R. A.; Liz-Marzan, L. M. Chem. Soc.

Au-Ag nano-mushrooms. FigureS3 showed the the

Rev. 2012, 41, 43-51.

LOD of miRNA-141 assay. Table S1 showed blank

(11) Driskell, J. D.; Tripp, R. A. Chem. Commun. 2010, 46,

SERS signals in the detection of DNA and miRNA

3298-3300.

targets. TableS2 showed the sequences of the DNA

(12) Alvarez‐Puebla, R. A.; Liz‐Marzán, L. M. Angew.

and miRNA used in the paper.

Chem., Int. Ed. 2012, 51, 11214-11223.

The Supporting Information is available free of charge

(13) Cho, W. J.; Kim, Y.; Kim, J. K. ACS Nano. 2011, 6,

on the ACS Publications website.

249-255.

AUTHOR INFORMATION

(14) Chung, A. J.; Huh, Y. S.; Erickson, D. Nanoscale. 2011, 3, 2903-2908.

Corresponding Author

(15) Li, W.; Camargo, P. H. C.; Lu, X.; Xia, Y. Nano Lett.

*E-mail: [email protected] (S.S.). 2009, 9, 485-490.

[email protected] (S.M.) (16) Qian, X.-M.; Nie, S. Chem. Soc. Rev. 2008, 37,

Author Contributions

912-920.

‖J.S. and D.W. contributed equally.

(17) Fang, Y.; Seong, N.-H.; Dlott, D. D. Science. 2008, 321,

Notes

388-392.

The authors declare no competing ﬁnancial interest.

(18) Hwang, J. H.; Singhal, N. K.; Lim, D. K.; Nam, J. M. Bull. Korean Chem. Soc. 2015, 36, 882-886.

ACKNOWLEDGMENT

(19) Oh, J.-W.; Lim, D.-K.; Kim, G.-H.; Suh, Y. D.; Nam,

We thank the National Basic Research Program of

J.M. J. Am. Chem. Soc. 2014, 136, 14052-14059.

China (2013CB932800 and 2016YFA02012), the

(20) Kang, J. W.; So, P. T.; Dasari, R. R.; Lim, D.-K. Nano

National Science Foundation of China (21373260,

Lett.2015, 15, 1766-1772.

31571014 and 21390414), Chinese Academy of

(21) Song, J.; Duan, B.; Wang, C.; Zhou, J.; Pu, L.; Fang, Z.;

Sciences (QYZDJ-SSW-SLH031) and Alexander von

Wang, P.; Lim, T. T.; Duan, H. J. Am. Chem. Soc. 2014, 136,

Humboldt Foundation for the financial support.

6838-6841. (22) Osberg, K. D.; Rycenga, M.; Harris, N.; Schmucker, A.

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Multicolor GoldâSilver Nano-Mushrooms as Ready-to-Use SERS

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