Label-Free MicroRNA Detection Based on Fluorescence Quenching of

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Label-Free MicroRNA Detection Based on Fluorescence Quenching of Gold Nanoparticles with a Competitive Hybridization Wei Wang, Tao Kong, Dong Zhang, Jinan Zhang, and Guosheng Cheng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01930 • Publication Date (Web): 09 Oct 2015 Downloaded from http://pubs.acs.org on October 13, 2015

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Analytical Chemistry

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Label-Free MicroRNA Detection Based on Fluorescence Quenching of

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Gold Nanoparticles with a Competitive Hybridization

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Wei Wang,† Tao Kong,† Dong Zhang,† Jinan Zhang,‡ and Guosheng Cheng*,†

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†

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ABSTRACT

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MiRNAs, critical biomarkers of acute and chronic diseases, play key regulatory roles in many biological

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processes. As a result, there is great demand for robust assay platforms to enable an accurate and efficient

Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, 398 Ruoshui Road, Suzhou Industrial Park, Jiangsu 215123, China ‡ Bona Tianyuan Biotech LLC, 568 Longmian Avenue, Jiangning, Jiangsu 211100, China

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detection of low-level miRNAs in complex biological samples. In this work, a label-free and Au

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nanoparticles (NPs) quenching-based competition assay system was developed. In the designed system, Au

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NPs with diameter sizes of 10 and 20 nm displayed fluorescence quenching efficiencies of 84% and 82% for

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Cy3 dye on slide surface, whereas the quenching efficiency of commercial BHQ2 quencher was roughly

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50%. Assay conditions were optimized for miRNA-205 detection. A limit of detection of 3.8 pM and a

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detection range covering from 3.8 pM to 10 nM were achieved. Furthermore, the proposed system was

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capable of specifically discriminating miRNAs with slight variations in their nucleotide sequence, and was

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also qualified for assessing miRNA levels in human serum. Our strategy has the potential to provide new

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perspectives in profiling the pattern of miRNA expression and biomedical utilizations. 19 20

I

n vitro diagnostics on the basis of biomolecular analysis plays a significant role in modern healthcare due to increasing need for early detection of many diseases. As nucleic acids are regarded as important

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biomarkers, their detection has risen to the forefront of approaches to improve disease diagnosis.1

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MicroRNAs (miRNAs) are short non-coding RNAs (typically 19–24 nucleotides long) that exhibit pivotal

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regulatory functions in a diverse range of biological processes such as gene expression, cell differentiation,

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and disease development.2,3 Recently, miRNAs were demonstrated with greater stability than mRNAs and

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had been found in blood serum, urine, saliva, cell and tissue.4,5 Specifically, many studies have identified that

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aberrant expression of miRNA (fM to pM level) is closely associated with a variety of diseases including

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cancers, diabetes, and Alzheimer’s.6,7 For instance, high levels of miRNA-21 are correlated with breast

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cancer,8 and let-7 family members are often down-regulated in human cancers.9 Therefore, miRNAs have

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become increasingly important in determining disease diagnosis and prognosis, and are also useful in basic

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biomedical research. 1

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Accurate and efficient detection of miRNAs in biological samples is still challenging because of their

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short and highly homologous sequences and low concentrations. At present, primary technical platforms for

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miRNA profiling include northern blotting, sequencing, quantitative polymerase chain reaction (qPCR) and

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microarray,10,11 and each one has its own advantages and shortcomings. Being regarded as a gold standard

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method in early miRNA profiling studies, northern blotting suffers from intrinsic limitations of low

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throughput analysis, semi-quantitation, and requirement of a large amount of samples. Sequencing has the

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undoubtedly superiority of identifying unknown miRNAs, but it is not widely used owing to the high cost of

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implementation. Meanwhile, qPCR provides high sensitivity and large dynamic range. However, it requires

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complex and special laboratory skills, and highly purified samples. Its formidable capability is also impeded

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since the short length of miRNA limits the flexibility of primer design. Microarray technique is an efficient

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and economic tool for high-throughput miRNA profiling. The major concern of microarray is labeling

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procedure which is still inevitable to mainstream commercial miRNA microarrays currently.

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Labeling procedures are usually laborious, cumbersome and error-prone. Moreover, 2’-O-methylation of

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3’-termini of miRNAs can raise serious challenges for labeling prior to microarray analysis.12,13 Therefore,

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developing a non-labeling detection method for miRNAs comes into prominence. Recently a variety of

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strategies have been developed for direct miRNA analysis, including electrochemical devices,14,15 nanopore

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sensor,16 surface plasmon resonance,17 silicon nanowire,18,19 surface-enhanced Raman spectroscopy,20,21

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molecular beacons,22,23 and stacking-hybridized universal tag-based microarrays,24 etc. In addition,

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competitive binding assay, being widely used to detect small molecules in environmental and food safety

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monitoring,25 also provides label-free analysis. Wang et al. reported amplified voltammetric detection of

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trace-level miRNA-182 using a competitive hybridization.26 Yao et al. developed an exchange-induced

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remnant magnetization technique for let-7a assay with single-base specificity based on sequence-specific

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exchange reactions.27

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On the other hand, biomolecular analysis using fluorescence quenching with nanomaterials as quenchers

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have been developed considerably. Compared to organic quenchers, nanomaterial quenchers including Au

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nanoparticles (NPs),28,29 carbon nanotubes,30 graphene,31,32 silicon nanowires,33 and carbon nitride

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nanosheets,34 possess prominent properties of high stability and super-quenching efficiency. Moreover, the

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virtue of high efficiency over a wide range of wavelengths allows them to quench several fluorophores

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simultaneously, while generally organic quencher specifically works well for appropriate fluorophore.

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Among these nanomaterial quenchers, Au NPs with superior plasmonic and biocompatible properties can be

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reliable and readily conjugated with various biomolecules and targeting agents.35 Owning to an establishment 2


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of synthesis techniques, size- and shape-tunable Au NPs have been well accomplished.36, 37 Besides

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fluorescence quenching, in terms of biochemical assays Au NPs have also been widely utilized in

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colorimetry, lateral flow assays, surface-enhanced Raman scattering, surface plasmon resonances, and

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scanometry.38-41

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In this paper, we report a label-free and competition assay based on fluorescence quenching of gold NPs

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for quantitative analysis of miRNA in buffer solutions and biological samples. MiRNA-205 was chosen as

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the target miRNA analyte. Previous reports indicated that miRNA-205 was extensively involved in cancer

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pathogenesis and aberrantly expressed in lung cancer development and progression.42,43 Experimental

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conditions were optimized for this miRNA-205 assay with a detection limit of 3.8 pM and a specificity

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ability of single-base discrimination. Notably the proposed bioassay technique could also be used to analyze

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other biomolecules and provides new perspectives in diagnostics.

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EXPERIMENTAL SECTION

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Materials and Reagents. Sodium dodecyl sulfate (SDS), sodium borohydride (NaBH4),

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tris-(2-carboxyethyl)phosphine hydrochloride (TCEP), trisodium citrate dihydrate, tannic acid,

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DNase/RNase-free distilled water, phosphate buffered saline (PBS, 1×, pH 7.4), and tris acetate-EDTA (TAE)

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buffer (10×, pH 8.2) were purchased from Sigma-Aldrich (St. Louis, MO). Saline sodium citrate (SSC)

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buffer (20×, pH 7.0), bovine serum album (BSA), and acetate buffer (3 M, pH 5.2) were provided by

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Sunshine Biotechnology (Nanjing) Co., Ltd (China). Ethylenediaminetetraacetic acid (EDTA) and

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hydroxylamine (NH2OH) were purchased from Tokyo Chemical Industry (TCI) Co., Ltd (Japan). Urea was

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acquired from Alfa Aesar (China) Chemical Co., Ltd.

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Table 1. Oligonucleotide sequences for miRNA detection by using Au NPs quenching with a competitive hybridization. Oligonucleotide

Sequence (5’ to 3’)

Probe DNA

/NH2-(CH2)6/TTTTTC AGA CTC CGG TGG AAT GAA GGA/Cy3/

miRNA-205

UCC UUC AUU CCA CCG GAG UCU G

Cy5-miRNA

/Cy5/UCC UUC AUU CCA CCG GAG UCU G

BHQ2-miRNA

/BHQ2/UCC UUC AUU CCA CCG GAG UCU G

SH-DNA1

/SH-(CH2)6/TTTTTTTT TCC TTC ATT CCA CCG GAG TCT G

SH-DNA2

/SH-(CH2)6/TTTTTTTTTTTTTTTTTTTTTTTTT

One-base mismatched miRNA

UCC UUC AUU ACA CCG GAG UCU G

Three-base mismatched miRNA

UCC UUG AUU ACA CCG GAA UCU G

Totally non-matched miRNA

AUA CCA UAC UGU AUA ACU CAA A

24 3



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Hydrogen tetrachloroaurate(III) tetrahydrate (HAuCl4⋅4H2O) was obtained from Sinopharm Chemical

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Reagent Co., Ltd (China). Aldehyde-functionalized glass slides and microarray printing solution were

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received from CapitalBio Co (China). Ultrapure water obtained from a Milli-Q Plus system (Millipore) was

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used throughout the experiments. MiRNA-205 target sequence and its complementary probe DNA

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oligonucleotide were synthesized by Integrated DNA Technologies (USA). Other DNA/miRNA

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oligonucleotides used in this paper were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. The

7

sequences of oligonucleotides are listed in Table 1. DNA/miRNA stock solutions were prepared by

8

dissolving oligonucleotides in DNase/RNase-free distilled water.

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Preparation of Au NPs and AuNP-DNA Conjugates. All glassware used was soaked in chromic acid

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for 24 h, and then thoroughly rinsed with water before use. HAuCl4 was used as the gold source in

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preparation of Au NPs. 10 nm Au NPs were synthesized by using tannic acid/trisodium citrate according to

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previous developed protocol.44 Meanwhile, 20 nm Au NPs were prepared by sodium citrate reduction. 36,45

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The preparation of AuNP-DNA conjugates was performed according to previous report with revision.46 In

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detail, 30 µL of 1 mM TCEP and 5 µL of 0.5 M acetate buffer were added into a microcentrifuge tube

15

containing a thiol DNA mixture of 0.5 nmol SH-DNA1 and 5.5 nmol SH-DNA2. After being incubated at

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room temperature for 1 h, the sample was transferred into a glass vial with 5 mL of 10 nm Au NPs (10 nM).

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Protect the solution from light, and rotate it gently for 17 h at room temperature on an orbital shaker. 50 µL

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of TAE buffer (10×), 25 µL of 2% SDS, and 125 µL of 2 M NaCl were added into the vial, and continued to

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shake for 1 h. Then an appropriate amount of NaCl was added to adjust its concentration from 0.05 M to 0.1

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M and shook for another 1 h. This process was repeated for every 0.1 M NaCl increment until a final 0.5 M

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NaCl concentration was reached, and incubated the solution for 18 h. After that, the AuNP-DNA conjugates

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were separated by centrifugation and washed with 1× TAE buffer (containing 0.1 M NaCl), and repeated this

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process for at least three times. Finally, the AuNP-DNA were dissolved in 1× TAE buffer with 0.3 M NaCl,

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and stored at 4 °C. Conjugation of thiol DNA to 20 nm Au NPs was carried out under similar conditions.

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Immobilization of probe DNA. Amine and Cy3 linked DNA oligonucleotide probes (probe DNA) were

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dissolved in a mixture solution of purchased printing solution and water with a volume ratio of 1/1. Probe

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DNA (1 µM) was spotted on an aldehyde-activated glass slide at room temperature under ~60% humidity

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with an aid of a commercial arrayer (PersonalArrayer 16, CapitalBio Co., China). Protected from light, the

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printed slide was incubated for 24 h at room temperature under a relative humidity of ~80%. After being

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rigorously rinsed with a washing solution (3× SSC and 0.1% SDS) twice to remove unbound probe DNA,

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the slide was further washed with water. For inactivating surplus aldehyde groups and reducing non-specific 4


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binding, the probe DNA functionalized slide was soaked in 50 mL of fresh NaBH4 solution (dissolving 0.1 g

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of NaBH4 in 40 mL of PBS (1×) and 10 mL of ethanol) for approximately 5 min. After three washes with

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water, the slide was immersed in hot water for several minutes to denature any annealed DNA, and blocked

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in 0.2% BSA solution for 30 min. Finally the slide was washed with water and dried by nitrogen gas.

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Assay protocol. In AuNP-DNA based competition assay, the AuNP-DNA and target miRNA were

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prepared in hybridization buffer (1× TAE, 0.6 M NaCl, and 0.2% SDS), and competitive hybridization

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between AuNP-DNA and target miRNA for pre-immobilized probe DNA on slide was performed. 60 µL of

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the mixture comprised of AuNP-DNA with a fixed concentration and target miRNA of varying

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concentrations was heated to 50 °C for 5 min, and the solution was cast into one well of a SureHyb gasket

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slide (G2534-60014, Agilent Technologies, Inc., USA), then the slide bound with probe DNA was placed

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down and clamped by a SureHyb Hybridization Chamber (Agilent G2534A). The chamber was incubated at

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42 °C for 7 h in a heating shaking drybath (i-MIXCOOL 100, Shanghai Utrao Medical Instrument, China)

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with a constant shaking speed of 400 rpm. After hybridization, the slide was washed in 2× SSC and 0.1%

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SDS at hybridization temperature for 5 min, and then washed in 1× SSC and 0.05% SDS for 1 min three

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times at room temperature. The slide was gently dried by nitrogen gas, and then stored at -20 °C before

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microarray scanning. For Cy5-miRNA based competition assay, the process was similar to that of the

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AuNP-DNA based assay except the hybridization buffer (4× SSC and 0.2% SDS).

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Serum assay. Serum sample from normal healthy adult was from Thermo Fisher Scientific Inc (USA).

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To denature background proteins, appropriate amounts of SDS was added to the serum and heated to 90 °C

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for 5 min, then cooled to room temperature immediately in ice water. 30 µL of TAE buffer with appropriate

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amounts of NaCl was added to 30 µL of the serum. Assuming standard NaCl concentration of 0.15 M in

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human serum, the final solutions contained 1× TAE, 0.6 M NaCl, and 0.2% SDS in 60 µL reaction volume.

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The AuNP-DNA and target miRNA were then added, and the assay of the sample was identical to preceding

24

procedure.

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Image scanning. Slides were scanned by a two-color microarray scanner (LuxScan 10K, CapitalBio Co.,

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China). Before each scanning experiment, a scan of fluorescence calibration slide (CalSlide, CapitalBio Co.,

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China) was performed to ensure the stability of microarray scanner. Fluorescence images for Cy3 and Cy5

28

dyes were acquired through green (532 nm wavelength) and red (635 nm wavelength) channels, respectively.

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Artifact-associated spots were eliminated by both visual inspection and software-guided flags. Raw

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fluorescence intensities of microarray spots were acquired using the GenePix Pro 6.0 software (Molecular

31

Devices, USA), and median intensity values were obtained after background subtraction. 5



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RESULTS AND DISCUSSION

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Scheme 1. Schematic diagram of the competition assay based on fluorescence quenching of Au NPs.

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MiRNA detection strategy. The principle of miRNA detection in our system bases on competition

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hybridization for probe DNA between AuNP-DNA and target miRNA. The process is illustrated in Scheme 1.

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Herein, complementary probe DNA to target miRNA of interest was immobilized on glass slide. The

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oligonucleotides of AuNP-DNA conjugate contain DNA1 and DNA2 (Table 1) whose sequences are

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complementary and non-complementary to probe DNA, respectively. DNA1 competes with target miRNA

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for probe DNA, while DNA2 used as a blocking agent strengthens the stability of colloidal Au NPs in

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hybridization buffer. Since Au NP and Cy3 are respectively attached at 5’ end of DNA1 and 3’ end of probe

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DNA, the Cy3 would be in close proximity to the Au NP through hybridization of probe DNA and

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AuNP-DNA. Therefore the fluorescence of Cy3 can be efficiently quenched by Au NPs. In the assay,

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AuNP-DNA and target miRNA are mixed and applied onto the slide surface. The added amount of

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AuNP-DNA is fixed with varied amounts of target miRNA. A large number of AuNP-DNA would hybridize

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with surface-bound probe DNA under a low concentration of target miRNA, which leads to high

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fluorescence quenching efficiency and low Cy3 fluorescence signal. On the contrary, high fluorescence

20

signal can be detected for high miRNA concentration sample. In our research, another competition-based

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miRNA assay is also explored. Cy5-miRNA with the same sequence to target miRNA replaces AuNP-DNA

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to compete with target miRNA. The detection process is similar to the assay using AuNP-DNA. Its final

23

detection signal is acquired through Cy5 fluorescence.

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Figure 1. (A) Fluorescence images and (B) fluorescence intensities of probe DNA with various printing concentrations

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immobilized on functionalized glass slide. The distance between the centers of adjacent spots is 300 µm. Fluorescence

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signals were acquired through green channel (532 nm wavelength). The fluorescence intensity of microarray spots

5

reflects relative immobilized amount of probe DNA (labeled with Cy3) on slide surface. Thus, by changing the printing

6

concentration, the bound probe DNA amount can be adjusted. Theoretically, the bound probe DNA amount would

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determine the saturated concentration of Cy5-miRNA or AuNP-DNA which is designed to compete with target miRNA,

8

and hence the competition assay performance could be altered.

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Cy5-miRNA competition based assay. In surface-based DNA/RNA detection, coupling efficiency of

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probe DNA is regarded as a fundamental and influential factor. As seen from Figure 1A and B, fluorescence

12

intensities of microarray spots were getting stronger as printing concentration of probe DNA went up from

13

0.1 to 5 µM, indicating increased loading amount of bound probe DNA. Furthermore, the microarray spots

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were patterned with high-quality morphology, and background signal was very low. High loading density is

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usually desired for typical sandwich assay, since more target DNA/RNA would be bound if more probe DNA

16

is immobilized on surface. However, unlike sandwich assay, competition assay does not require high loading

17

density which may hinder detection sensitivity. On the other hand, too low amount of probe DNA cannot

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provide sufficient Cy3 fluorescence to be quenched by subsequently bound Au NPs. Accordingly, probe

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DNA with a printing concentration of 1 µM was employed in this assay. Figure S2 illustrates that amount of 7



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hybridized Cy5-miRNA obviously increased with elevation of hybridization time from 1 to 7 h, and

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hybridization of Cy5-miRNA and surface-bound probe DNA reached equilibrium while further increasing

3

the time. Thus optimal hybridization time was determined to be 7 h for the planar surface-based binding

4

process.

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Figure 2. Comparison of probe DNA printing concentration for miRNA detection (A/B: 1 µM, C/D: 0.2 µM). (A, C)

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Various concentrations of Cy5-miRNA were hybridized to probe DNA functionalized surface. Calibration curves for

9

miRNA detection with fixed Cy5-miRNA concentrations of (B) 100 pM and (D) 30 pM. Fluorescence signals were

10

acquired through red channel (635 nm wavelength). The error bars represent the standard deviation in triplicate.

11 12

To demonstrate feasibility of competitive hybridization, Cy5-miRNA competition based assay was first

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explored. Effect of surface loading amount of probe DNA on miRNA detection was shown in Figure 2. For

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the microarray with a probe DNA printing concentration of 1 µM, fluorescence signal became saturated as

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added concentration of Cy5-miRNA reached 100 pM (Figure 2A). This saturated concentration of

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Cy5-miRNA is a key role and needs to be determined before following competition assay, in which target

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miRNA with various amounts competes with Cy5-miRNA at a constant amount. As indicated in Figure 2C,

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the saturated concentration of Cy5-miRNA reduced to 30 pM while decreasing probe DNA printing

19

concentration from 1 to 0.2 µM, probably as a result of decreased bound amount of probe DNA. By

20

calculating signals of Cy5 fluorescence to various concentrations of target miRNA, a calibration curve was

21

plotted as shown in Figure 2B. Limit of detection of the assay was 6 pM (signal/noise ratio of 3), and a

22

detection range of target miRNA from 6 pM to 1 nM was observed. Significantly, a lower detection limit of 8


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1.5 pM was measured as the printing concentration of probe DNA reduced to 0.2 µM (Figure 2D). It is

2

verified that sensitivity and detection range of the competition assay can be tuned by changing the bound

3

amount of probe DNA.

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Figure 3. Characterizations of Au NPs and AuNP-DNA. TEM images of Au NPs with sizes of (A) 10 nm and (B) 20

7

nm. UV-vis spectra of (C) 10 nm and (D) 20 nm sized Au NPs and their AuNP-DNA conjugates. DLS size distributions

8

of (E) 10 nm and (F) 20 nm sized Au NPs and their AuNP-DNA conjugates. The morphology was characterized by a

9

transmission electron microscopy (Tecnai G2 F20 S-Twin, FEI, USA). The UV-vis spectra were recorded on a UV-2550

10

spectrophotometer (lambda 25, PerkinElmer, USA). The DLS size distributions were examined on a Zetasizer Nano S

11

system (Malvern Instruments, UK).

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Competition assay based on fluorescence quenching of Au NPs. Metallic NPs and their surfaces

14

exhibit diverse and complex optical properties. As one of the most intensely studied metallic NPs, Au NPs

15

with a wide size range (1-50 nm) have been reported for efficiently quenching of various fluorescence

16

dyes.28,29,47-50 It is also worthwhile to note that Au film, assembled with about 100 nm sized gold NPs and

17

strictly controlled NPs separation distance, led to fluorescence enhancement rather than quenching.51 9



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Fluorescence dye placed in the vicinity of a metal surface could be quenched due to a greater enhancement

2

of the non-radiative decay rate than radiative decay rate.47,52 The absorption of Au NPs is responsible for

3

increased non-radiative decay, while raised radiative decay is created by the scattering (re-radiating) of

4

plasmonic extinction. According to Mie theory,52,53 the absorption and scattering abilities of metal NPs are

5

determined by both size and shape of the metal structures. Small colloids are expected to quench

6

fluorescence since absorption is dominant over scattering, whereas larger ones are supposed to enhance

7

fluorescence. Accordingly, small Au NPs with size below 20 nm were used and investigated herein.

8

Characterizations of Au NPs and AuNP-DNA were presented in Figure 3. TEM images (Figure 3A and B)

9

show that colloidal Au NPs with spherical morphology and average sizes of 10 and 20 nm were successfully

10

prepared. The two kinds of AuNPs displayed characteristic UV-vis absorption spectra with plasmon bands at

11

518 and 523 nm respectively (Figure 3C and D), which were ascribed to surface plasmon resonance of the

12

AuNPs. The conjugation of DNA sequences on surface of Au NPs caused a 4 nm red-shift of the absorbance

13

peak for both 10 and 20 nm, since the DNA presence could generate an environment with a different

14

dielectric constant. Dynamic light scattering (DLS) was used to measure the hydrodynamic size of Au NPs

15

and AuNP-DNA. The results (Figure 3E and F) reveal that after DNA conjugations the mean hydrodynamic

16

sizes of 10 and 20 nm Au NPs greatly increased to 18 and 30 nm, respectively. To some extent it verifies that

17

the preparation of AuNP-DNA conjugates was accomplished. Additionally, salt stability is of particular

18

importance for AuNP-DNA, since high salt concentration is usually required in hybridization buffer. As

19

shown in Figure S3, both Au NPs and AuNP-DNA formed a stable red colloidal suspension prior to salt

20

addition. No obvious change for the color of AuNP-DNA solution was observed with increasing salt

21

concentration from 0 to 1 M. This is in striking contrast to the Au NPs solution, whose color was apparently

22

changed from red to grayish blue while increasing salt concentration to 0.2 M. Aggregation of Au NPs in salt

23

solutions is caused by salt-induced reduction of electrostatic repulsion between Au NPs. Due to

24

electrostatic/steric stabilization of surface DNA, AuNP-DNA possessed robust salt stability in aqueous

25

solution even with a salt concentration as high as 1 M.

26

As a dark quencher, Black Hole Quencher-2 (BHQ-2) is extensively used in commercial assays. As

27

illustrated in Figure S4, BHQ-2 has an absorbance peak of 590 nm, with an effective absorbance range of

28

550-650 nm. Spectral overlap of BHQ-2 absorbance and Cy3 emission led to fluorescence quenching of Cy3,

29

as BHQ2-miRNA hybridized with probe DNA in solution (Figure S4).

10


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Figure 4. Fluorescence quenching of (A) BHQ2-miRNA, (B) 20 nm AuNP-DNA, and (C) 10 nm AuNP-DNA for

3

probe DNA functionalized surface. (D) Calibration curves for miRNA detection with a fixed AuNP-DNA (10 nm)

4

concentration of 100 pM. The AuNP-DNA concentration is equivalent to Au NPs concentration. The printing

5

concentration of probe DNA was set as 1 µM. Fluorescence signals were acquired through green channel (532 nm

6

wavelength). The error bars represent the standard deviation in triplicate.

7 8

For the tests on slide surface (Figure 4A), fluorescence intensity gradually decreased with increasing the

9

BHQ2-miRNA concentration from 0 to 100 pM, since more Cy3 fluorescence were quenched as more

10

BHQ2-miRNA were bound to probe DNA on surface. No distinct change in fluorescence intensity was

11

noticed with a further increase in BHQ2-miRNA concentration, indicating that hybridization of

12

BHQ2-miRNA and surface-bound probe DNA approached equilibrium. The fluorescence quenching

13

efficiency by BHQ2 was found to be roughly 50%. It is observed that AuNP-DNA showed excellent

14

quenching capability for Cy3 in solution (Figure S5). For slide-based detection, much higher quenching

15

efficiencies by Au NPs were measured (Figure 4B and C) in comparison with BHQ2. Quenching efficiencies

16

of 82% and 84% for 20 and 10 nm Au NPs, respectively, were achieved as both of the two AuNP-DNA

17

reached a concentration of 100 pM. Therefore, 10 nm AuNP-DNA with a fixed concentration of 100 pM was

18

utilized to detect target miRNA. Compared to the BHQ2 quencher, Au NPs may also quench adjacent Cy3

19

dyes, due to their larger size and the ability to quench those with relatively large separation distance from

20

AuNPs.49 Moreover, multiple DNAs to one Au NP allows one AuNP-DNA conjugate to bind to multiple

21

probe DNAs on slide surface. This could also help to quench Cy3 fluorescence. A calibration curve for this 11



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1

miRNA assay was obtained (Figure 4D), in which fluorescence intensity was plotted as a function of the

2

logarithm of miRNA concentration. The limit of detection was estimated to be 3.8 pM at three times the

3

standard deviation of the control (free of target miRNA), and the detection range covered from 3.8 pM to 10

4

nM for this miRNA assay was obtained. It indicates that the proposed competition assay based on

5

fluorescence quenching of Au NPs, was sensitive and reliable for the determination of miRNA

6

concentrations. Furthermore, the light scattering signal of surface-bound Au NPs after gold enhancing may

7

also be used for miRNA detection (Figure S6 and S7). SEM results (Figure S8) indicate that nonspecific

8

binding of AuNP-DNA was pretty low in this assay.

9

10 11

Figure 5. (A) Specificity tests for detection of 3 nM complementary target and mismatched strands. (B) MiRNA

12

detections in healthy human serum. The concentrations of AuNP-DNA (10 nm) and probe DNA were fixed at 100 pM

13

and 1 µM, respectively. The AuNP-DNA concentration is equivalent to Au NPs concentration. The error bars represent

14

the standard deviation in triplicate.

15 16

The recyclability of the assay. To further examine the recyclability of this competition assay,

17

dehybridization and subsequent rehybridization of Cy5-miRNA on probe DNA bound surface by a

18

continuous mode were surveyed. Three aspects of Cy3 fluorescence stability in probe DNA, binding activity

19

of probe DNA, and dehybridization efficiency should be taken into account. As illustrated in Figure S9A,

20

urea treatment introduced effective dehybridization and removed Cy5-miRNA from the surface. The 12


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1

fluorescence signal from subsequent Cy5-miRNA hybridization showed no obvious change for four cycles,

2

suggesting that the surface-bound probe DNA was quite stable. Thus the Cy5-miRNA competition based

3

assay could be used repeatedly. However, the intensity of Cy3 fluorescence in probe DNA displayed a

4

distinct decrease after urea treatment (Figure S9B). It indicates that the competition assay based on

5

fluorescence quenching of Au NPs was not eligible for repetitive use. To replace the fluorescence label (Cy3)

6

in probe DNA with more stable dyes, such as quantum dots, might resuscitate the recyclability of this assay.

7

Specificity of the assay and serum test. The existence of homologous miRNAs and small size of

8

miRNAs pose a great challenge to carry out highly specific assay. The specificity of the proposed system in

9

this study was investigated by three kinds of miRNA sequences with artificially introduced mismatches

10

under the same condition. The results (Figure 5A) clearly suggest that the assay was capable of

11

discriminating complementary target and mismatched strands. The signal of complementary target was

12

approximately 2.4-fold higher than that of single-base mismatched strand, and three-base mismatched strand

13

with a signal of only 29% of that for the complementary target was measured. Furthermore, the signal for

14

totally non-matched strand was almost identical to the blank control which generated a signal of only 18.5%

15

of that for the complementary target. The observed high specificity of this assay allows it to be potential

16

applied in single nucleotide polymorphism analysis. To investigate the applicable possibility of the proposed

17

system for real samples, healthy human serum was used for spiking target miRNA standards with varied

18

concentrations. The analytical results for the spiked miRNA were shown in Figure 5B and Table S1. The

19

signal for the healthy human serum was close to the background buffer signal. The recoveries (between 84 %

20

and 130 %) for the miRNA ranging from 50 pM to 5 nM were acceptable. It is clearly demonstrated that the

21

developed assay which was not compromised in serum could provide a potential analytical tool in real

22

biological samples.

23

CONCLUSIONS

24

In conclusion, this work presented a label-free competition assay system by using Au NPs as

25

high-performance fluorescence quencher. Au NPs with sizes of 10 and 20 nm were successfully prepared and

26

conjugated with the nucleic acids. Fluorescence quenching efficiencies of 84% and 82% were obtained for

27

10 and 20 nm Au NPs while commercial BHQ2 quencher exhibited a much lower quenching efficiency

28

(50%). Furthermore, influences of printing concentration of probe DNA, hybridization time, and AuNP-DNA

29

concentration were evaluated. This miRNA assay achieved a detection limit of 3.8 pM and a detection range

30

from 3.8 pM to 10 nM, and could even discriminate single-nucleotide differences with a high specificity. The 13



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1

proposed assay method was also capable of monitoring miRNA levels in human serum. To summarize, the

2

experimental results demonstrated that this label-free and Au NPs quenching based competition assay system

3

shows promising application prospect in biomolecular analysis for biomedical research and clinical

4

diagnosis.

5

ASSOCIATED CONTENT

6

Supporting Information

7

Additional information as noted in text. This material is available free of charge via the Internet at

8

http://pubs.acs.org.

9

AUTHOR INFORMATION

10

Corresponding Authors

11

*E-mail address: [email protected]. Tel.: +86 512 62872595.

12

Notes

13

The authors declare no competing financial interest.

14

ACKNOWLEDGMENTS

15

This work was financially supported by Nanjing 321 Strategy Project, Ministry of Science and Technology

16

of China (973 Grant No. 2014CB965003), National Natural Science Foundation of China (NSFC Grant No.

17

51361130033), and Suzhou Science and Technology Program (SYG201212). We are grateful for the

18

professional services of Platforms of Characterization and Test and the Nanofabrication Facility at the

19

Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences. We also thank Prof. Jiong

20

Li for his assistance in microarray printing and scanning.

21

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1 2

For TOC only

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4 5

18


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Fig. 1 77x99mm (300 x 300 DPI)



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Fig. 2 84x75mm (300 x 300 DPI)


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Fig. 3 83x105mm (300 x 300 DPI)



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Fig. 4 83x77mm (300 x 300 DPI)


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Fig. 5 71x90mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1 165x59mm (300 x 300 DPI)


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Label-Free MicroRNA Detection Based on Fluorescence Quenching of

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