Highly Sensitive Detection of MicroRNA-21 with ICP-MS via

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Highly Sensitive Detection of MicroRNA-21 with ICP-MS via Hybridization Accumulation of Upconversion Nanoparticles Xun Liu, Shang-Qing Zhang, Zi-Han Cheng, Xing Wei, Ting Yang, Yong-Liang Yu, Ming-Li Chen, and Jian-Hua Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03038 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 25, 2018

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Analytical Chemistry

Highly Sensitive Detection of MicroRNA-21 with ICP-MS via Hybridization Accumulation of Upconversion Nanoparticles

Xun Liu, Shang-Qing Zhang, Zi-Han Cheng, Xing Wei, Ting Yang, Yong-Liang Yu*, Ming-Li Chen, Jian-Hua Wang Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Box 332, Shenyang 110819, China

*E-mail: [email protected] (Y.-L. Yu). Tel: +86 24 83687659

ABSTRACT: A highly sensitive platform is developed for the determination of microRNA-21 with inductively coupled plasma mass spectrometry (ICP-MS). It includes the following operations: Hairpin structure DNA H1 and H2 are designed, and DNA H1 is bound to ultra-small lanthanide upconversion nanoparticles (UCNPs) to produce UCNPs@DNA conjugate probes. Target miRNA triggers a chain reaction for alternating hybridization between DNA H1 (bond on UCNPs@DNA probe) and DNA H2. This leads to UCNPs accumulation and serves as an efficient amplification strategy for UCNPs. The concentration of miRNA-21 is closely correlated to the number of UCNPs, thus the detection of 89Y by ICP-MS provides a promising approach for miRNA quantification. This protocol exhibits high sensitivity to 1

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miRNA-21 within 0.1-500 fM, along with a detection limit of 41 aM, which is among the hitherto reported most sensitive procedures. It is worth mentioning that rare earth elements are scarcely present in living systems, which minimizes the background for ICP-MS detection and excludes potential interferences from the coexisting species, which is most suited for biological assay.

KEYWORDS: MicroRNA-21, hybridization, accumulation/amplification, upconversion nanoparticles, inductively coupled plasma mass spectrometry (ICP-MS).

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INTRODUCTION MicroRNAs (miRNAs), a class of small non-coding RNA molecules, play significant functions in various biological processes, e.g., cell differentiation, apoptosis, and cancer development.1-4 Intracellular miRNAs are of concern as they are readily degraded by endogenous RNase in tissue fluid.5,6 The latest studies showed that miRNAs released from cancer cells could be present in human plasma in a stable form, which may become a new branch of biomarkers in early diagnosis and clinical management.7,8 Therefore, the accurate and sensitive detection of miRNAs in plasma is highly desired for early cancer diagnosis. MicroRNA-21 was reported as an oncogene and antiapoptotic indicator.9 While it is one of the greatest challenge to measure miRNAs in plasma because of their short sequences and ultra-low abundance.10 A number of assays were explored for miRNA inspection, e.g., fluorescence,11-13 electrochemistry,14 colorimetry,15 electrochemiluminescence,16-18 and Raman spectroscopy.19 Among these approaches, fluorescence detection is most widely adopted for monitoring miRNA and dynamic processes in real-time, providing direct visualization of biological species in their native state.20,21 However, the self-emission properties of fluorescent probes are sensitive to the change of environmental surrounding and light source, which hinders the quantitative measurement in biological systems.22 In addition, highly sensitive approaches are always required when considering the ultra-trace level of miRNA in real biological samples. In recent years, inductively coupled plasma mass spectrometry (ICP-MS) has 3



been focusing on biological studies due to its high sensitivity in the quantification of metal species within a wide dynamic range (generally 8-9 order of magnitudes).23,24 The unique advantages of ICP-MS, e.g., excellent mass resolution and low matrix effect for multiplex bioassay,25 were well demonstrated by its applications in multiplex protease assay,26 single cell cytometry27 and multiplex DNA assay.28 For such biological investigations, lanthanide based upconversion nanoparticles (UCNPs) are certainly among the choice,29 which are generally possessing high crystallinity, favorable stability as well as good dispersibility. These properties can efficiently prevent the degradation of UCNPs in complex biological sample matrixes.30 In addition, rare earth elements are rarely found in biological samples, which generates very low background for ICP-MS detection, and thus ensures high sensitivity.31 It is worth mentioning that lanthanides exhibit very strong binding strength to 5’ phosphoric acid group in DNA, and thus it is feasible to construct new probes by conjugating UCNPs and DNA for the purpose of performing detection.32 On the other hand, the favorable bio-safety of UCNPs with respect to organic dyes and semiconductor quantum dots should also be considered.33 A recent study on miRNA detection with ICP-MS derived a detection limit at the femtomol level by using lanthanide-labeled DNA probes.34 For the analysis of real biological samples, however, the improvement on the assay sensitivity is still highly desired. In the present work, we report an ultra-high sensitive protocol for miRNA detection by ICP-MS via the accumulation of ultra-small upconversion nanoparticles through a miRNA triggered hybridization chain reaction (HCR) with two 4


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hairpin-structured DNA probes H1 and H2. DNA H1 is bound to UCNPs at 5’ phosphoric acid group, and miRNA initializes its hybridization with DNA H2 to produce a single stranded tail in H1, which facilitates its further hybridization with H2 and restores a single-strand tail in H2 with the same sequence of the target miRNA. In this way, a chain reaction is triggered for alternating hybridization between H1 and H2, producing a chain-like assembly of H1 (UCNPs@DNA conjugate) and H2. The content of yttrium is quantified with detection by ICP-MS, and the concentration of miRNA is derived indirectly with its close correlation to the amount of yttrium. In this study, it is highly important to adopt ultra-small UCNPs in order to avoid the steric hindrance and facilitate the HCR process for achieving a sufficient level of amplification.

EXPERIMENTAL SECTION Chemicals and Materials. Yttrium chloride hexahydrate (YCl3·6H2O, 99.9%), ytterbium chloride hexahydrate (YbCl3·6H2O, 99.9%), erbium chloride (ErCl3, 99.9%) and albumin from bovine serum (98%, BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-octadecene (ODE, 98%) was obtained from Aladdin Reagent Co., Ltd., China. Oleic acid (C17H33COOH), ammonium fluoride (NH4F) and sodium hydroxide (NaOH) were obtained from National Medicines Co. (Shanghai, China). Methanol (CH3OH), ethanol (CH3CH2OH), cyclohexane (C6H12) and hydrochloric acid (HCl) were purchased from Tianjin Damao Chemical Reagent Factory (Tianjin, China). Reduced glutathione (98%, GSH) was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). DNA (DNA H1: 5’-AAT CAA CAT TAG TCT GAT AAG 5



CTA CTA AGT TAG CTT ATC AGA CTA ATC-3’, DNA H2: 5’-TAG CTT ATC AGA CTA ATC TTG TTA CAT TAG TCT GAT AAG CTA ATC TAG-3’), microRNA-21 (5’-UAG CUU AUC AGA CUA AUG UUG A-3’), microRNA-582 (5’-UAA CUG GUU GAA CAA CUG A-3’), microRNA-193b (5’-CGG GGT TTT GAG GGC GAG-3’) were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China). All reagents were of analytical reagent grade and used without further purification unless otherwise specified. Ultra Pure DEPC-treated water was obtained from Sangon Biotech Co., Ltd. (Shanghai, China), and used throughout the experiments. Apparatus. The size and morphology of the as-prepared nanoparticles were observed on a JEM-ARM 200F transmission electron microscope (JEOL, Japan) using an accelerating voltage of 200 kV. UV-vis absorption spectra were recorded with a UH5300 spectrophotometer (Hitachi, Japan) with a 1.0 cm quartz cell. The DNAs after HCR process were characterized by a Bruker Dimension icon atomic force microscopy (Bruker, Germany) using tapping mode with a tip type of Scanasystair. ZS90 Nano Zetasizer (Malvern, UK) was used to analyze the surface charge properties of the related materials. Upconversion fluorescent spectra were measured on an F-7000 fluorescence spectrophotometer (Hitachi Ltd., Japan) attached with an external 980 nm semiconductor laser (Changchun New Industries Optoelectronics Technology Co., Ltd., China) instead of internal excitation source. Inductively coupled plasma mass spectrometer (7500a, Agilent Technologies, USA) was used for the quantification of yttrium by detecting the isotope of 89Y. 6


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Preparation of upconversion nanoparticles NaYF4:Yb3+,Er3+ (UCNPs) and conjugation with DNA H1. Ultra-small sized UCNPs, NaYF4:Yb3+,Er3+ (Yb 20%, Er 2%), are prepared according to the literature approach with minor modification by high-temperature coprecipitation in oleic acid and 1-octadecane.35 Shortly, 236.6 mg of YCl3·6H2O (0.78 mmol), 77.5 mg of YbCl3·6H2O (0.20 mmol), and 5.5 mg of ErCl3 (0.02 mmol) are mixed with 9 mL of oleic acid and 15 mL of 1-octadecene in a 250-mL flask under protection by argon and heated to 180°C at a rate of 20°C min-1, and the reaction mixture is continuously stirred with magnetic stirring for 1 h. A standard 2-cm Teflon-coated stirring bar is employed with a stirring rate of ca.450 rpm. After cooling down to room temperature, 10 mL of methanol containing 2.5 mmol NaOH and 4 mmol NH4F is added into the flask. The mixture is stirred for 0.5 h and then heated to 90°C and stirred for 30 min, followed by further heating to 270°C with stirring for another 0.5 h. The mixture is allowed to stand for cooling to room temperature, adding 20 mL ethanol to produce UCNPs precipitate. The product UCNPs is washed with ethanol for three times and separated via centrifugation at 8000 rpm for 5 min, followed by drying at 60°C for 12 h in a drying oven. Ligand-free UCNPs are prepared by following the literature method.34 1.0 mg of the UCNPs are dispersed in 1.0 mL of 0.1 mM HCl solution, which is then sonicated for 1 h to remove the ligand, i.e., oleic acid. The ligand-free nanoparticles are obtained after centrifugation at 12000 rpm for 0.5 h followed by washing with deionized water for three times. These UCNPs are re-dispersed in 1.0 mL of water for the ensuing studies. 7



100 µL of DNA H1 solution (6.0 µM) is added into 0.5 mL of the above ligand-free UCNPs solution followed by magnetic stirring for 12 h. The UCNPs@DNA conjugate is achieved by centrifugation at 8000 rpm for 5 min with washing by water for three times. The conjugate is then re-dispersed into 300 µL of deionized water. The preparation process for UCNPs@DNA is schematically illustrated in Scheme 1.

UCNPs

New ligand

Ligand removal

UCNPs

0.1 mM HCl

:

DNA

Ln3+

Ln3+

Oleic Acid

O

:O

P

: :

O

: :

: O:

: : : :

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O

DNA

:O:

Scheme 1. Schematic illustration for the preparation of the UCNPs@DNA conjugate probe.

Detection of miRNA-21 by ICP-MS. 1.0 µL of UCNPs@DNA conjugate solution and 1.0 µL of hairpin DNA H2 solution (2 µM) are introduced into a series of miRNA-21 solutions (98 µL, with various concentrations within 0.1-5000 fM). The mixture is incubated at 37°C with gentle shaking at 200 rpm for 4 h. Afterwards, the mixture is placed in centrifugal filter units, and purified via centrifugation at 5000 g for 15 min (M.W. 100 KDa, Merck Millipore, Germany). The supernatant is diluted to 1.0 mL for facilitating ICP-MS detection. The mechanisms for the accumulation/amplification of UCNPs and the ensuing quantification of target miRNA-21 with detection by ICP-MS are illustrated in Figure 1. The operating parameters are listed in Table S1. 8


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For the purpose of quantifying miRNA in human plasma, 4.0 mL of human blood is obtained from healthy, lung cancer and breast cancer volunteers (provided by the Hospital of Northeastern University and the first Affiliated Hospital of Dalian Medical University), and 0.5 mL of sodium citrate solution (3.8%, m/m) is added. After centrifuging at 3000 g and 4°C for 10 min, the supernatant plasma is collected. Afterwards, the same procedure as mentioned above is followed for performing ICP-MS detection. H1 UCNPs

Purify

miRNA-21

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n

ICP-MS

H2

Figure 1. Schematic diagram illustrating the mechanisms for the accumulation/amplification of UCNPs and the ensuing quantification of target miRNA-21 with detection by ICP-MS.

RESULTS AND DISCUSSION Characterizations. As described above that oleic acid-coated NaYF4:Yb3+,Er3+ nanoparticles are obtained by following a typical coprecipitation procedure. High resolution transmission electron microscopy (HRTEM) images (Figure S1) demonstrate that the UCNPs are uniform spheres with good dispersion and high crystallinity, exhibiting a narrow diameter distribution of ~2.8 nm. The growth of UCNPs can be readily regulated by controlling the temperature and the reaction time, which regulates the size/diameter of the UCNPs. In the presence of NaOH and NH4F, 9



the reaction mixture is controlled at

Highly Sensitive Detection of MicroRNA-21 with ICP-MS via

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