High-Throughput and Sensitive Fluorimetric Strategy for MicroRNAs in

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Biological and Medical Applications of Materials and Interfaces

A High-Throughput and Sensitive Fluorimetric Strategy for MicroRNAs in Blood Using Wettable Microwells Array and Silver Nanoclusters with Red Fluorescence Enhanced by Metal Organic Frameworks Luping Feng, Min Liu, Huan Liu, Chuan Fan, Yuanyuan Cai, Lijun Chen, Mingliang Zhao, Su Chu, and Hua Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07137 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on June 27, 2018

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ACS Applied Materials & Interfaces

A High-Throughput and Sensitive Fluorimetric Strategy for MicroRNAs in Blood Using Wettable Microwells Array and Silver Nanoclusters with Red Fluorescence Enhanced by Metal Organic Frameworks Luping Feng†‡, Min Liu†, Huan Liu†, Chuan Fan†, Yuanyuan Cai†, Lijun Chen†, Mingliang Zhao†, Su Chu†, and Hua Wang*†‡§ †

Institute of Medicine and Materials Applied Technologies, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, Shandong 273165, P. R. China. ‡

School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, Heilongjiang 150090, P. R. China.

§

School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai, Shandong 264209, P. R. China

* Corresponding Author: E-mail addresses: [email protected]; Tel: +86 537 4456306; Fax: +86 537 4456306; Web: http://wang.qfnu.edu.cn. ABSTRACT: A high-throughput and sensitive fluorimetric analysis method has been developed with wettable microwells array for probing short-chain microRNAs (miRNAs) in blood using silver nanoclusters (AgNCs) with red fluorescence amplified and stabilized by metal organic frameworks (MOFs). Glass slides were first spotted with polyacrylic acid to form hydrophilic microdots and then patterned with hydrophobic hexadecyltrimethoxysilane, followed by etching the microdots to yield the microwells array. Furthermore, DNA capture probes with silver-binding G sequences were covalently bound onto the functionalized microwells to hybridize with targeting miRNAs. Exonuclease I-catalytic digestion was then conducted to remove any single-strand DNA probes unhybridized. Eventually, AgNCs were applied to specifically recognize the G sequences of DNA probes survived to be further coated with MOFs of ZIF-8. Unexpectedly, the red fluorescence of AgNCs probes could be dramatically enhanced due to the “electron-donor effect” of nitrogen-containing ligands of ZIF-8 coatings, together with improved sensing stability. High detection sensitivity and reproducibility could thereby be expected for detecting miRNAs in blood with the concentrations linearly ranging from 0.175 to 500 pM. Markedly different from the common sandwiched DNA detection methods, the developed fluorimetric strategy using AgNCs coated with MOFs and DNA probes designed with silver-recognizing sequences would be tailored for quantifying various kinds of short-chain miRNAs with low levels in the complicated blood media. KEYWORDS: Silver nanoclusters, Metal organic frameworks, Microwells array, Fluorimetric strategy, MicroRNAs

INTRODUCTION MicroRNAs (miRNAs), a kind of small non-coding RNAs with the length of 18-25 nucleotides, play the important roles in physiological expression of genes in lives.1-5 They are also the meaningful diagnostic and prognostic biomarkers for many serious diseases like cancers.6-9 Especially, the miRNAs levels in human fluids are closely related to the metastasis of cancers, thus making the monitoring of miRNAs a hot topic in the clinical laboratories. Up to date, a variety of detection methods have been developed to quantify miRNAs,10-15 nevertheless, the quantitative evaluation of miRNAs may be challenged by their short-chain profiles and low-level expressions in human body.10,11 Remarkably, many analysis methods have

commonly been performed through the sandwiched DNA hybridizations, which might risk the unwound or detached issues of the hybridized double chains because of the short-chain targeting miRNAs, thus showing the poor analysis reproducibility and / or low detection sensitivity. Moreover, the current detection methods may generally encounter with low detection throughput for multiple biological analysis. As a result, increasing efforts have been devoted to the aplications of high-throughput ones like the fluorimetric microarrays for the analysis of various biomarkers including miRNAs.16-21 However, the common microarrays might still be trapped by the formidable crossover contaminations of sample droplets in-between the arrayed testing microdots or microwells, which should challenge the analysis of multiple samples within a single experiment. Therefore, exploring some simple, sensitive, high-throughput, and reproducible analysis candidates for

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the evalutations of multiple low-level miRNAs has been an attractive target to pursue. It is well recognized that the fluorimetric strategies as the very popular sensitive detection tools can present the outstanding analysis performances, which may largely depend on the optical and physiochemical properties of fluorescent probes such as the emission intensities, environmental stability, and recognition selectivity. In recent years, noble metal nanoclusters (NCs) with strong luminescence, good photostability, low toxicity, and high biocompatibility, have been widely introduced as the probes in the fluorimetric assays.22-24 As the most interesting representatives, Au or Ag NCs have been preferentially employed as the fluorimetric probes for sensing various nucleic acids.25-29 For example, Yang’s group proposed a AgNCs-based fluorescent biosensor for the determination of miRNAs based on the photoinduced electron transfer.25 Ye and co-workers have reported the DNA-scaffolded AgNCs for the detection of miRNAs.29 Nevertheless, a formidable challenge may be generally encountered regarding the non-specific adsorption of signal probes or labels of noble metal NCs onto the oppositely-charged phosphodiester backbones of nucleic acids hybridized. Alternatively, some non-ionic nucleic acid analogs, such as charge-neutral peptide nucleic acids and morpholinos, have been applied for probing nucleic acids.30-32 For example, Zhang et al. reported an electrochemical DNA analysis strategy by using neutral peptide nucleic acids as the capture probes and Ag nanoparticles as the electroactive labels, achieving greatly reduced signal background and high analysis sensitivity.30 Tercero and colleagues developed a label-free analysis strategy using morpholinos for the recognition of DNAs with the minimized non-specific adsorption.32 An ultrasensitive electrochemical strategy has also been proposed in Gao’s group for sensing miRNAs by use of exonuclease to catalytically digest the single-strain DNA probes unhybridized.33 Furthermore, fluorescent AgNCs as the probes can feature the intriguing aesthetic structures and exotic luminescence properties, however, they are notorious for their inherent instability, which may hamper their applications on a large scale.34-38 It is also recognized that the environmental stability and photophysical properties of noble metal NCs like AgNCs can depend on their cores, surface ligands, and media properties.38,39 For example, the synthetic modifications of some ligands (S, P and N) have been demonstrated to be one of the efficient ways to achieve the improved stability and especially enhanced luminescence properties of noble metal clusters.40 In recent years, metal-organic frameworks (MOFs) have been increasingly applied for chemical sensing, catalysis, and optoelectronic design, due to they possess ultrahigh porosity, high surface areas, tunable metrics, good mechanical stability, and desirable photophysical properties.34,41-48 For instance, Yang et al. developed a sensitive electrochemical immunoassay for probing C-reaction protein by using Cu-MOF-labeled antibody.46 Zang’s group synthesized silver chalcogenide clusters embedded into porous MOFs showing the hypersensitive switching of dual-function luminescence.34 In particular, MOFs were employed as the carriers or supports for loading

enzymes and other catalysts with enhanced catalysis efficiencies and stabilities.47,48 Additionally, MOFs were utilized to encapsulate metal nanomaterials to effectively improve the formidable agglomeration and thermodynamic stability against some extreme conditions.49-51 Inspired by these pioneering works above, in the present work, a high-throughput and sensitive fluorimetric strategy has been developed for the evaluation of miRNAs using wettable microwells array and AgNCs coated with MOFs of zeolitic imidazolate frameworks (ZIF-8). Herein, the microwells array was fabricated on glass slides with the hydrophobic-hydrophilic interfaces, where polyacrylic acid (PAA) was first spotted on the slides to produce microdots and then patterned with hydrophobic hexadecyltrimethoxysilane (HDS) (Scheme 1). Afterwards, the PAA microdots were etched to yield the hydrophilic microwells array further derivatized with amine groups. Moreover, the capture probes of single-strand DNAs (ssDNAs) were covalently immobilized on the microwells to hybridize with targeting miRNAs (Scheme 2). Furthermore, exonuclease I (Exo I) was employed to catalytically digest the ssDNA probes unhybridized. AgNCs were then introduced to recognize the special G sequences of DNA probes survived, followed by being coated with ZIF-8 MOFs. To our surprise, dramatically amplified red fluorescence could be obtained for AgNCs probes together with improved sensing stability, so that highly sensitive and reproducible fluorimetric analysis for miRNAs could be expected. Remarkably, the wettability profile of the microwells arrays would aid to depress any crossover contamination of testing sample droplets so as to expect the high-throughput and selective analysis of targeting miRNAs. To the best of our knowledge, this is the first successful design of wettable microwells array in combination with AgNCs probes with MOF-enhanced red fluorescence toward the high-throughput and ultrasensitive fluorimetric assay to be tailored for sensing short-chain miRNAs. The practical application feasibilty of the microwells array-based fluorimetric method was subsequently demonstrated in probing miRNAs with low levels in blood.

EXPERIMENTAL SECTION Reagents. Silver nitrates (AgNO3), glutathione (GSH), α-lipoic acid (LA), and sodium borohydride (NaBH4) were purchased from Sigma-Aldrich (Beijing, China). Poly acrylic acid (PAA) and dithiothreitol (DTT) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Hexadecyltrimethoxysilane (HDS), dimethylsulfoxide (DMSO), ethylenediaminetetra acetic acid (EDTA), aminopropyltriethoxysilane (APTES), and tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) were bought from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). Zinc acetate (Zn(Ac)2), succinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carbox ylate (SMCC), dimethylimidazole, and cetyltrimethyl ammoniumbromide (CTAB) were obtained from Dibai Reagents (Shanghai, China). Blood samples were kindly provided by the local university hospital. All other reagents


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were of analytical grade. Sterile water from deionized water (>18 MΩ, RNase-free) was supplied from an Ultra-pure water system (Pall, USA) for high pressure sterilizer. Exonuclease I (Exo I), DNA capture probe of thiolated single-stranded DNA (ssDNA), and wild miRNAs were obtained from Sangon Biotech Co., Ltd. (Shanghai, China), of which the base sequences of oligonucleotides include: 1. DNA capture probe with silver-binding sequence: 5'-SH-TGGGTAGGGCGGGTTGGGAAAAACTATACAA CCTACTACCTCA-3'; 2. DNA capture probe without silver-binding sequence: 5'-SH-TAAATAAAACAAATTAAAAAAAACTATACAA CCTACTACCTCA-3'; 3. Wild miRNA: 5'-UGA GGU AGU AGG UUG UAU AGU U-3'. Buffer solutions include: the conjugation buffer (pH 7.2) containing 100 mM phosphate buffered saline (PBS) and 150 mM NaCl; hybridization buffer (pH 7.4) consisting of 10 mM Tris-HCl, 1.0 mM EDTA, 1.0 mM CTAB, and 0.50 M NaCl; DNA rinsing buffer (pH 7.4) composing of 100 mM NaCl, 10 mM Tris-HCl; Exo I buffer containing 67 mM glycine-KOH, 6.7 mM MgCl2, and 10 mM DTT. Apparatus. The measurements of contact angles (CAs) were conducted to monitor the changing hydrophobic and hydrophilic properties of the HDS-patterned slides and spotted microwells during the step-by-step fabrication procedure using the contact-angle measurement machine (Jinhe, Jiangsu, China). Their surface structures were also

CA = 62°

observed using inverted fluorescence microscope (Olympus, IX73-DP80, Japan). Moreover, the fluorimetric measurements of miRNAs with the wettable microwells array were performed using the fluorescence spectrometer (Horiba, FluoroMax-4, Japan). The elemental mapping measurements were conducted for the products using a scanning electron microscope (SEM, Hitachi E-1010, Horiba Ex-250) with a microanalysis system (EDAX, USA). High-resolution transmission electron microscopy (HRTEM, Tecnai G20, FEI, USA) and X-ray photoelectron spectrometer (Thermo ESCALAB 250XI) were utilized for the characterization of AgNCs in absence and presence of ZIF-8. Besides, the photographs of the products were taken under the UV lamp at the excitation wavelength of 365 nm. Fabrication of amine-derivatized wettable microwells array. The fabrication process for the wettable microwells array with amine derivatization on the hydrophobic patterns is schematically illustrated in Scheme 1. The glass slides (25 mm x 76 mm) were first cleaned with the Piranha solution (H2SO4 : H2O2 = 7 : 3), followed by being rinsed with deionized water, and then dried in nitrogen gas. Furthermore, a 2-µL aliquot of PAA (10 mg/mL) was spotted separately onto the cleaned slides to be dried forming the PAA testing microdots. Afterwards, the substrates were dipped into the hydrophobic mixture containing HDS, isopropanol, and concentrated sulfuric acid (mass ratios of 10 : 100 : 1) for 5 min. Then, they were took out to be further deposited for

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Scheme 1. Schematic illustration of the fabrication process of wettable microwells array with the hydrophobic-hydrophilic interfaces by PAA spotting, HDS patterning, NaOH etching of PAA microdots, and amine derivatization, showing the interface wettabilities manifested by CAs and images, of which the fluorimetric and colorimetric microwells arrays were exemplified by rhodamine B droplets separately under the white (top) and UV (bottom) lamps.



in buffer or spkied in blood were separately added to hybridize with the DNA capture probes immobilized there at 37 °C for 40 min. Then, they were washed twice using the hybridization buffer to remove any non-specifically adsorbed targets. After that, the microwells array was dried in nitrogen. Furthermore, an aliquot of Exo I (0.50 U/mL) was introduced into the microwells to be incubated at 37 °C for 30 min, followed by being flushed twice separately with the Exo I buffer and PBS buffer. After that, an aliquot of AgNCs (0.40 mM), which were fabricated using glutathione by following the procedure described elsewhere,52 was separately dropped into each of microwells and incubated for 1.0 h to complete the Ag-G sequence binding. After being washed twice with the PBS buffer, 80 mM Zn(Ac)2 and 320 mM dimethylimidazole were subsequently introduced into the microwells for 30 min to form the coatings of ZIF-8. The fluorescence signals of the microwells array were measured with the instrument-incidental holder at λex = 425 nm.

30 min at room temperature. Furthermore, a 2-µL aliquot of 1.0 M NaOH was introduced to etch the spotted PAA microdots by the acid-base neutralization reaction, then rinsed twice with water, yielding the microwells array. Eventually, a certain amount of APTES was dropped into the microwells to generate the NH2-derivatized surfaces. The changing wettabilities of the interfaces of the yielding microwells arrays were monitored by the contact angle measurements. The microwells array-based fluorimetric assays for miRNAs. The microwells array-based fluorimetric analysis for miRNAs was performed as illustrated in Scheme 2. Typically, thiol-derivatized ssDNA capture probes containing the silver-binding sequences were conjugated onto the amine-derivatized surfaces of microwells using the SMCC cross-linker according to the instruction of the reagent kit. Targeting miRNAs with different concentrations

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Scheme 2. Schematic illustration of the detection procedure and principle of the microwells array-based fluorimetric assays for miRNAs including the fabrication of amine-derivatized microwells, immobilization of DNA capture probes containing silver-binding sequence, hybridization of targeting miRNAs, Exo I digestion of unhybridized ssDNA probes, AgNCs binding with the G sequence of DNA probes, and MOF coatings, showing the changing fluorescence of the products from rose to bright red.

RESULTS AND DISCUSSION Fabrication and characterization of wettable microwells arrays. The wettable microwells arrays were fabricated with the hydrophobic-hydrophilic interfaces by following the procedure schematically illustrated in Scheme 1. An aliquot of PAA solution was first spotted onto the cleaned glass slides to form the PAA microdots. The resulting slides were then dipped into the HDS-containing

mixture to yield the hydrophobic pattern. Furthermore, NaOH was introduced to etch the PAA microdots to produce the microwells. Finally, APTES was added into the microwells to generate the amine-derivatized substrates for binding DNA capture probes afterwards. The step-by-step fabrication process of microwells array was manifested by monitoring the wettabilities of the interfaces with changing contact angles (CAs). One can note from Scheme 1 that the average CA of the cleared glass slides is about 62°. After being spotted with hydrophilic PAA, it could be decreased to about 38o. Once the glass slide was further patterned with


hydrophobic HDS, the maximal CA of about 123° would be achieved, which would endow the arrays of anti-fouling effect.53,54 Moreover, the etching of PAA microdots with NaOH would reduce the average CA of the microwells substrates down to about 54°. Interestingly, once the microwells were deposited with APTES, the resulted hydrophilic substrates could display a minimum CA of about 31°. The so yielded microwells arrays were further characterized by spotting rhodamine B droplets with different concentrations, showing the well-defined arraying droplets promising for colorimetric and fluorimetric assays. The so obtained wettable microwells arrays were characterized by inverted fluorescence microscope imaging with light field (Figure 1). It can be seen that the microwells were formed with pretty high density on the hydrophobic pattern (top), of which the substrates could be furnitured with numerous particles as revealed in the topological magnified image (bottom). Notably, such a wettable profile of the microwells arrays could provide a self-cleaning interface between the testing microdots so as to avoid any in-between fouling or crossing contamination of sample droplets.53,54 As a result, the construction of denser microwells array could be expected for the high-throughput and selective analysis of targeting miRNAs within a single experiment.

Figure 1. The photographs of wettable microwells array with the hydrophobic-hydrophilic interfaces created on the glass slides (top), with the magnified image of a mcirowell so created under fluorescence microscopy (bottom).

Analysis protocol of microwells array-based fluorimetric assays for miRNAs. The microwells array-based fluorimetric assays for miRNAs were carried out using DNA capture probes of ssDNAs containing silver-binding G sequences and AgNCs with fluorescence and sensing stability enhanced by MOFs coatings of ZIF-8 (Scheme 2). Herein, targeting miRNAs were captured by the ssDNA probes immobilized in microwells to form the double-stranded DNAs (dsDNAs). Furthermore, the ssDNA probes un-hybridized were selectively removed through the Exo I-catalytic digestion,18,55 which might help to depress any non-specific adsorption between DNA probes and AgNCs signaler oppositely charged, so as to increase the

detection selectivity for targeting miRNAs. Afterwards, AgNCs were introduced to interact with the special G sequences of ssDNA probes survived,56 so as to produce the fluorescent signals. Ultimately, the MOF precursors were introduced into the microwells to form ZIF-8 coatings onto AgNCs, which was thought to be mediated by the surface peptide ligands of AgNCs (i.e., glutathione) and DNA hybrids there.57 Herein, imidazole-containing ZIF-8 would also possibly conduct a covalent reaction with AgNCs, which might aid to form ZIF-8 coatings on AgNCs. Importantly, the fluorescence of AgNCs would thus be dramatically enhanced, which would change from the rose to the bright red, as visually illustrated by the fluorescent images (insert), because the nitrogen-containing ligands (i.e., imidazole) of MOFs might conduct the “electron-donor effect”, as reported elsewhere for other noble metals clusters.34 Figure 2 shows the comparison of UV-vis absorbance and fluorescence spectra between AgNCs with and without MOF coatings. It can be noted from Figure 2A that MOF-coated AgNCs could present an obvious difference in the absorbance spectra profile. Herein, the characteristic absorption peaks of AgNCs at 330 nm and 490 nm would disappear after being coated with MOFs, although there is no significant color change of the testing solutions as disclosed in the corresponding photographs (Figure 2A, insert). Moreover, as shown in Figure 2B, AgNCs could display the fluorescence emission peak at 640 nm. However, once coated with ZIF-8 MOFs, dramatically enhanced fluorescence could be obtained for AgNCs at 660 nm, showing a slight red shift. Also, a significant change in the fluorescence colors was witnessed from the rose to the bright red, as visually shown in corresponding photographs (Figure 2B, insert). Herein, the MOF-enhanced fluorescence of AgNCs might result from the “electron-donor effects” of nitrogen-containing ligands (i.e., imidazole) of ZIF-8 coatings aforementioned. Therefore, the introduction of MOFs could greatly enhance the luminescence intensities of AgNCs probes, so as to expect the improved fluorimetric sensitivity for probing the targeting miRNAs thereafter. 2.0

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AgNCs in the absence and presence of ZIF-8 were characterized by HRTEM images (Figure 3). It was found that AgNCs could enjoy the uniform mono-dispersion,



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showing a nearly spherical shape with the size of 5.0 nm (Figure 3A). After they were coated with ZIF-8, a drastic change in the topological structure and shape was witnessed (Figure 3B). Furthermore, the products were analyzed by X-ray photoelectron spectroscopy (XPS) (Figure 4). As expected, the key elements of Zn, C, N, Ag, and O could be included, thus demonstrating AgNCs coated with ZIF-8.

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Comparable investigations of the fluorimetric responses to miRNAs based on Exo I digestion and MOF-enabled signal amplification. A comparison was conducted between the fluorimetric miRNA responses without and with using Exo I for the digestion of ssDNA capture probes after miRNA hybridization (Figure 5A),

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Figure 5. (A) Fluorimetric responses to miRNAs (a) without and (b) with Exo I digestion for DNA capture probes after the miRNA hybridization, taking (c) DNA capture probes un-hybridized for Exo I digestion as the control. (B) Fluormetric responses to miRNAs using AgNCs (a) with and (b) without the MOFs coating, comparing to (c) the ones using DNA capture probes but without silver-binding sequence.

As expected, considerably low fluorescent response was observed, indicating that the Exo I digestion would ideally aid to depress any background interference for the fluorimetric miRNA detections. Moreover, comparable investigations were carried out on the fluorimetric assays for targeting miRNAs using the capture ssDNA probes with and without the MOF-enabled amplification for AgNCs signals (Figure 5B). Obviously, the MOF coatings could dramatically enhance the fluorescence intensities of AgNCs probes (curve a) comparing to the ones without MOFs coatings (curve b). Additionally, the use of the ssDNA probes containing the silver-binding G sequences could exhibit much larger fluorescence intensity (curve b) than the ones without the silver-binding G sequences (curve c). Accordingly, AgNCs were confirmed to interact with the G sequences by the Ag-G binding, so as to generate the fluorescence signals for the fluorimetric analysis of miRNAs. Figure 6 discloses the results of energy dispersive spectroscopy (EDS) and elemental mapping for the products of MOF-coated AgNCs yielded in the microwells arraybased fluorimetric assays for miRNAs. Both results reveal that the main elements of C, N, O, Ag, and Zn were uniformly dispersed throughout the products by a discretely mixed way, indicating the successful formation of MOF-coated AgNCs for amplifying AgNCs signals towards the sensitive fluorimetric detections of low-level miRNAs. Optimization of the main fluorimetric detection conditions. The dosages of DNA capture probes were first optimized by measuring the fluorimetric responses to miRNAs (Figure 7A). It was discovered that the fluorimetric responses could increase with the increasing concentrations of DNA probes till 1.0 µM, which was thereby selected for the fluorimetric miRNA assays. Moreover, the hybridization time was explored (Figure 7B), showing that the hybridization reactions tended to be steady after 40 min,


which is chosen as the optimal hybridization time. Also, experimental investigations were carried out on the amounts

Detection performances of the microwells array-based fluorimetric assay for miRNAs. The storage stability of MOFs-coated AgNCs and the detection reproducibility of the fluorimetric miRNA method were investigated by comparing with AgNCs without MOFs coatings (Figure 8). One can find from Figure 8A that in contrast to bare AgNCs (curve a), the MOFs-coated AgNCs displayed no significant change in the fluorescence intensities when stored up to seven months, showing the high environmental stability (curve b). Also, Figure 8B shows that the developed fluorimetric method can exhibit the better detection reproducibility and higher responses to miRNAs. Accordingly, MOF coatings could not only stabilize but also amplify the fluorescent signals of AgNCs, so as to endow the fluorimetric system the greatly improved detection performances.

Figure 6. Characterization of the typical reaction products formed in the arraying microwells in the fluorimetric assays for miRNAs with (A) EDS spectra with image, and (B) the element mapping of (a) overlapped image of (b) C, (c) N, (d) Ag, (e) O, and (f) Zn elements.

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Under the optimized conditions, the wettable microwells array-based fluorimetric strategy was employed to probe miRNAs with different concentrations in buffer (Figure 9A). The fluorescence spectra reveal that the fluorescence intensities could increase as miRNAs concentrations increased (Figure 9A, insert). A detection calibration curve was thus obtained for miRNAs with the fluorescence intensities versus the logarithms of miRNAs concentrations linearly ranging from 10 fM to 100 nM (R2 = 0.9941), with a limit of detection (LOD) of 5.0 fM, estimated by the 3σ rule.

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of AgNCs probes, with the data manifested in Figure 7C. Apparently, the fluorimetric responses could depend on the AgNCs dosages, showing that 0.40 mM AgNCs is suitable for the fluorimetric signal outputs. In addition, the precursor amounts for forming MOF coatings were investigated (Figure 7D), indicating that 45.71 mM Zn(Ac)2 should be selected as the optimal conditions.


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1.2

1.5

10

1.8

20

30

40

50

60

Hybridization Time / min

Probe Concentration / µM

8.0


9.0


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


FL Intensity x 10 5 / a.u.

Page 7 of 11

C

7.0

6.0

5.0 0.25

0.30

0.35

0.40

0.45

[AgNCs] / mM

0.50

8.0

D

7.0 6.0 5.0 4.0 10

20

30

40

50

60

70

[Zn Precursor] / mM

Figure 7. Optimization of main detection conditions for the microwells array-based fluorimetric assays for miRNAs, including (A) DNA capture probe concentrations, (B) hybridization time, (C) AgNCs dosages, and (D) Zn precursor amounts for coating of MOFs.

Preliminary applications of the developed fluorimetric assays for miRNAs spiked in blood. The application feasibility of the wettable microwells array-based fluorimetric method was investigated in identifying miRNAs spiked in blood with various concentrations (Figure 9B), of which the corresponding photographs were taken under the UV lamp (insert). A linear relationship was thereby obtained for the fluorescence intensities vernus the logarithms of miRNAs concentrations spiked in blood ranging from 0.175 to 500 pM (R2 = 0.9864). Therefore, the developed fluorimetric method with microwells array could determine the low-level miRNAs in blood with the favorable detection sensitivity, thus promising the potential applications for the quantification of miRNAs in the practical samples.



AUTHOR INFORMATION 10.0

8.0


A

8.0

Y = 1.128 X + 3.333 R2 = 0.9941

6.0

FL Intensity / a.u.


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

4.0 2.0 0.0

560

-3

-2

-1

0

1

2

640 720 Wavelength / nm

3

4

Log[miRNA] / pM

5

6

Corresponding Author * E-mail address: [email protected]; Tel: +86 537 4456306; Fax: +86 537 4456306; Web: http://wang.qfnu.edu.cn.

B

6.0

Y = 2.8352X + 2.5339 R2 = 0.9864

4.0

2.0

0.0 -1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

Log[miRNA] in blood / pM

Figure 9. (A) Fluorescence intensities of the microwells array-based fluorimetric method depending on different concentrations of miRNAs (insert: corresponding fluorescence spectra for sensing miRNAs); (B) the detection calibration curve of the fluorescence intensities versus the concenttration logarithms of miRNAs spiked in blood (insert: the photograph of corresponding microwells array taken under UV lamp).

Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundations of China (Nos. 21675099 and 21375075), Science and Technology Development Project of Weihai City (2015DXGJZD002), Shandong Province, and the Taishan Scholar Foundation of Shandong Province, China.

CONCLUSIONS In summary, a high-throughput and sensitive fluorimetric strategy has been constructed for miRNAs in blood using microwells arrays with the wettability profile and AgNCs with MOFs-enhanced fluorescence and sensing stability. The so developed fluorimetric assay can possess some advantages over the common sandwiched analysis methods for sensing short-chain miRNAs. First, the use of MOFs coatings could greatly enhance the fluorescence and sensing stability of AgNCs towards highly sensitive and reproducible fluorimetric assays for miRNAs in blood. Increased stability of environmental storage would also be expected for MOF-coated AgNCs to ensure the extensive applications. Second, highly selective analysis could be achieved for miRNAs through the specific Exo I-catalytic digestion of ssDNA capture probes un-hybridized. Third, the so fabricated microwells array with the wettability profile could not only accumulate more targets, but also depress any crossover contamination of sample droplets. Importantly, highly dense microwells array could thus be obtained towards the high-throughput analysis of multiple biomarkers. Finally, the design of DNA capture probes with the G sequences could enable the specific AgNCs binding for the output of their fluorescence signals. In particular, such a special design route of DNA capture probes with silver-recognizing G sequences should be generalized for probing different kinds of targeting miRNAs or DNAs on purpose simply through changing the sequences of DNA probes. Therefore, the developed microwells array-based fluorimetric strategy should hold great promise for the extensive applications for sensing short-chain miRNAs for the clinical diagnosis of various cancers and warning of potential cancer metastasis.

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For TOC Only

G

G G G G G

G

G G G G G

G

AgNC

G G G G G

G

G G G G G

G

G G G G G

G

G G G G G

4 oC, overnight

R.T, 60 min Enzyme digestion

ZIF8 precursors

Ag-G binding


MOF coating

High-Throughput and Sensitive Fluorimetric Strategy for MicroRNAs in

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