Gold Nanoparticle Couples with Entropy-Driven Toehold-Mediated

Subscriber access provided by Kaohsiung Medical University

Article

Gold Nanoparticle Couples with Entropy-Driven ToeholdMediated DNA Strand Displacement Reaction on Magnetic Beads: Toward Ultrasensitive Energy-Transfer-Based Photoelectrochemical Detection of miRNA-141 in Real Blood Sample Nan Zhang, Xiao-Mei Shi, Hong-Qian Guo, Xiaozhi Zhao, Wei-Wei Zhao, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01966 • Publication Date (Web): 19 Sep 2018 Downloaded from http://pubs.acs.org on September 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 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

Analytical Chemistry

Gold Nanoparticle Couples with Entropy-Driven Toehold-Mediated DNA Strand Displacement Reaction on Magnetic Beads: Toward Ultrasensitive Energy-Transfer-Based Photoelectrochemical Detection of miRNA-141 in Real Blood Sample Nan Zhang1a, Xiao-Mei Shi1a, Hong-Qian Guo2, Xiao-Zhi Zhao2, Wei-Wei Zhao1,3*, Jing-Juan Xu1 and Hong-Yuan Chen1* 1

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China. 2 Department of Urology, Nanjing Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, China. 3 Department of Materials Science and Engineering, Stanford University, Stanford, California 94305, United States. ABSTRACT: Highly stable circulating microRNAs (miRNAs) are currently recognized as a novel potential biomarker for clinical cancer diagnosis in the early stage. However, limited by its low concentration, high sequence similarity as well as the numerous interferences in body fluids, detection of miRNA in whole blood with sufficient selectivity and sensitivity is still challenging. Herein, we reported the integration of entropy-driven toehold-mediated DNA strand displacement (ETSD) reaction with magnetic beads (MB) toward the energy-transfer-based photoelectrochemical (PEC) detection of the prostate carcinoma (PCa) biomarker miRNA-141 in real blood sample. In this protocol, ETSD reaction was divided into two steps, and cooperated with magnetic separation, target extraction and amplification could be realized in a single test and ultrasensitive detection of miRNA-141 could be achieved in undiluted whole blood sample. This work proposed a new solution for sensitive biomolecular detection in complex biological milieu and exhibited great promise for future clinical cancer diagnosis.

One of the major issue in the current state of art cancer research is developing new methods for accurate diagnosis, especially before distant metastasis occurring.1,2 Highly stable circulating, blood-based microRNAs (miRNAs) nowadays serve as promising and minimally invasive cancer biomarker candidates,3-5 which are short noncoding RNA molecules (~22 nt) that function as regulators at the post-transcription level by repressing or degrading messenger RNAs (mRNAs) through complementary binding, and their aberrant expression is frequently observed in diseases such as Parkinson’s disease,6 major depression,7 strokes,8 and various cancers.9-11 Prostate carcinoma (PCa) is now the third leading cause of cancer-related death,12 however, the current gold-standard biomarker prostate-specific antigen (PSA) suffers from the lack of specificity to malignant prostatic diseases and high rate of false-negative results. In this case, a series of miRNAs including miRNA-141, miRNA-200b and miRNA-375 etc. have been recognized as potential biomarkers to identify PCa patients especially in previously undetectable micrometastases.13-15. Currently, the quantification of miRNA is commonly achieved by real time reverse transcription quantitative PCR (RT-qPCR)16 and some other protocols including northern blotting and microarray-based hybridization.17-19 However, these methods suffer many limitations such as insufficient specificity and sensitivity caused by cross-hybridization and contamination, complex and time-consuming operating

processes. In contrast, isothermal amplifications of nucleic acids are simple processes that rapidly and efficiently accumulate nucleic acid sequences at one reaction temperature, which require no complex thermocycling to mediate denaturation, annealing, and subsequent extension, and have been developed as alternatives of PCR in biosensing since early 1990s.20 Some of them are based on enzyme catalytic chemistry in replication, prolongation or digestion of nucleic acids,21,22 and others are based on enzyme-free nucleic acid assembly, such as hybridization chain reaction (HCR) and catalyzed hairpin assembly (CHA).23,24 Significantly, entropy-driven toehold-mediated DNA strand displacement (ETSD) is a newly developed DNA isothermal amplification strategy by Yurke’s group in 2007.25 This reaction is actuated by a short catalysis strand, driven by increases in the configurational entropy of the system instead of the enthalpy released from new base-pair formation, converting a ternary DNA complex into a duplex as a result. This strategy demonstrated great potential in the application of nucleic acid analysis in complex biological milieu, since unusual secondary structures like loops and pseudoknots were of no requirement and undesired interactions thus avoided. In 2015, utilizing this strategy, Tan’s group and Ma’s group further achieved DNA detection and cellular miRNA imaging, respectively.26,27 However, there is no report on cell-free miRNA analysis using this reaction so far. This is probably because the extraction and quantification are still challenging 1

ACS Paragon Plus Environment

Analytical Chemistry 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

due their innate low concentrations and extensive interferences existing in blood. Scheme 1. Schematic Illustration for Entropy-Driven Toehold-mediated DNA Strand Displacement Reaction on Magnetic Beads Coupled with Gold NPs

new way for time-saving, easy operating and ultrasensitive early diagnosis of cancers. EXPERIEMENTAL SECTION Materials and Apparatus. Photoactive polymer poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1,3}-thi adazole)] (PFBT), tris(2-carboxyethyl) phosphinehydrochloride (TCEP), poly(diallyldimethylammonium chloride) (PDDA; 20%, w/w in water, MW = 200000~350000), NaBH4, 100× Tris-EDTA (TE, containing 1 M Tris-HCl and 100 mM EDTA) buffer were obtained from Sigma-Aldrich. Poly(styrene-co-maleic anhydride) (PSMA) was obtained from Tianjin Heowns Biochem LLC (China). HAuCl4 was obtained from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). NaCl, MgCl2, monoethanolamine (MEA), tris(hydroxymethyl) aminomethane (Tris) were from Sinopharm Chemical Reagent Co., Ltd. Magnetic beads (Dynabeads MyOne Streptavidin C1, Cat No. 65001) was from Invitrogen. The ITO slices (type N-STN-S1-10, China Southern Glass Holding Co., Ltd.) were used as the working electrode. Non-denaturing polyacrylamide gel (4-20%) was from BBI Life Sciences. DEPC water was from Sangon Biotechnology Company, Ltd. All the experimental water used related to ETSD reaction on MB was DEPC water, and the tubes and tips were DNase and RNase free. DNA oligonucleotides were acquired from Genescript Company, and the sequence and stocking solutions were listed in Table S1. SEM images were recorded by a Hitachi S4800 scanning electron microscope (Hitachi Co., Japan). Transmission electron microscopy (TEM) was conducted using a JEM-2100 microscope (JEOL, Japan). XPS was obtained from PHI 5000 VersaProbe (UlVAC-PHI Co., Japan). Photoluminescence (PL) was measured using an F-7000 fluorescence spectrometer (Hitachi Co., Japan) equipped with a xenon lamp or a Varioskan® Flash multimode reader (Thermo Fisher Scientific Inc.). UV-vis absorption spectra were obtained using a NanoDrop 2000 full-spectrum microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific Inc.). Polyacrylamide gel electrophoresis (PAGE) images were scanned with Bio-Rad Molecular Imager. PEC measurements were performed with a homemade PEC system: a 5 W LED lamp with monochromatic emitting at 450 nm was used as irradiation source to produce the monochromatic illuminating light on the front of the electrode. Photocurrents were measured on a CHI 660C electrochemical workstation (Chenhua, China) with a three-electrode system: a modified Pdots/ITO photocathode with a geometric circular area (0.5 cm in diameter) as the working electrode, a Pt wire as the counter electrode, a saturated Ag/AgCl electrode as the reference electrode, and PBS buffer (pH 7.4) as the electrolyte. Native PAGE Analysis on ETSD reaction mechanism. Samples were run in precast 4-20% non-denaturing PAGE at 140 V for 40 min. For each lane, the final concentration of each single strand was 200 nM and the total volume was 10 µL. Before loading the samples on a gel, reactions were run for 2 h at 37 °C. Synthesis of Au NPs-labelled DNA 1. Au NPs with diameter ca. 5 nm were synthesized through the reduction of HAuCl4 by sodium NaBH4. Specifically, 1.2 mL of 0.1 M ice cold NaBH4 was added to 40 mL of ice cold aqueous solution containing 2.5×10-4 M HAuCl4 under stirring, and the solution

Herein, aiming at early diagnosis of PCa through a blood test, we report the novel detection of circulating miRNA-141 based on the integration of ETSD reaction with gold nanoparticles (Au NPs), magnetic beads (MB) as well as the photoelectrochemical (PEC) technique. As a newly emerged methodology, PEC bioanalysis possessed desirable properties and attractive potential in future bioassay. On the basis of various photoactive materials, substantial strategies have been developed toward miRNA determination, and satisfactory analytical performance has been achieved with the detection limit down to femtomole level. 28-37 However, their application in real sample analysis is limited by the potential electrode contamination cause by various electroactive interferences in body fluids. In this protocol, magnetic separation was used to solve this problem. As illustrated in Scheme 1, on the basis of magnetic separation, we divided the ETSD reaction into two independent steps and assigned each step different functions of extraction and amplification respectively, aiming at achieving sensitive detection in complex matrix. In the first step, the target miRNA (T) was extracted from blood sample through complementary binding starting from toehold 1 on the ternary DNA substrate (S), forming intermediate 1 (I1), meanwhile equivalent amount of strand 2 (DNA 2) was displaced and abandoned, and toehold 2 was exposed; Then magnetic separation was carried out to help remove the complex interferent in whole blood, and when dispersed in buffer solution, the following reactions was able to proceed in simple matrix. Subsequently, sufficient fuel strand (F) was added into the reaction system, Au NPs-coupled strand 1 (Au NPs-DNA 1) was thus leased free into liquid phase, as well as the previously extracted target miRNA. The released miRNA would participate in multiple turnovers and achieved its quantitative amplification through a translation to Au NPs-DNA 1, which was finally steered to bind with complementary sequences on a photoactive polymer dots (Pdots)/indium tin oxide (ITO) electrode.38 By measuring the photocurrent decrement caused by the energy transfer between Au NPs and Pdots, the ultrasensitive detection of circulating miRNA-141 in blood sample could be achieved. This study proposed a novel protocol for cell-free miRNA extraction, amplification and quantitative measurement, and opened up a 2


Page 2 of 8

Page 3 of 8 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

Analytical Chemistry immediately turned to a red-brown color, indicating the formation of Au NPs. Then the solution was kept under stirring in the ice bath for 10 min and for another 3 h at room temperature with the color changing to wine red. The synthesized Au NPs were stored at 4 °C for further use and the concentration was calculated to be 6×10-8 M. Thiolated DNA 1 was used for the modification with Au NPs through Au-SH bond. Experimentally, 10 µL of 10 mM TCEP was added to 50 µL 10 µM thoilated DNA 1, and incubated at 37 °C for 1 h to reduce the disulfide bond. Then the mixed solution was added to 1 mL Au NPs, and further incubated for 16 h at room temperature with gentle shaking under dark environment. Within the above incubation step, 300 µL of 0.5 M NaCl was smoothly added into the Au NPs-DNA 1 solution. The resultant solution was ultra-filtrated for 10 min at 14000 ×g and 4 °C with 30 kDa Millipore to remove the unbinded DNA 1. After removal of the supernatant, the precipitate was resuspended in 900 µL 10 mM PBS of pH 7.4 containing 0.1 M NaCl.

concentrated by rotary evaporation at 55 °C. Then the Pdots/ITO electrode was fabricated using the layer by layer electrostatic self-assembly interaction for 4 layers. 38 Modification of Pdots/ITO electrode and PEC assay. Capture DNA was first immobilized on the Pdots/ ITO electrode via amide bond. Specifically, the electrode was immersed into 1 mL aqueous solution containing 20 mg EDC and 10 mg NHS at room temperature for 1 h, and washed with 10 mM PBS solution (pH 7.4) for three times. Then 25 µL of 1 µM capture DNA was dropped onto the working area of the electrode and kept at 4 °C overnight in a moisture atmosphere. The capture DNA modified electrode was blocked by 1 mM MEA solution at 4 °C for 1 h and washed with 10 mM PBS (pH 7.4) for three times. The collected Au NPs-DNA 1 containing supernatant was immobilized on the electrode and let react at 37 °C for 2 h in a moisture atmosphere. The photocurrent was measured before and after immobilization of the supernatant in an air saturated 10 mM PBS solution (pH 7.4). Real sample analysis. The fresh whole blood samples of healthy human were from Nanjing Drum Tower Hospital and kept at 4 °C for further use. When used, the blood sample was blended gently to homogenous liquid and divided into several tubes for 85 µL each, then 5µL RNase inhibitor (20 U/µL) and 10 µL target miRNA-141 with various concentrations were mixed uniformly with the samples, and the final target concentration was a tenth of the original one. B&W buffer used in real sample analysis was without tween 20. After incubating with whole blood sample, the MB was washed for eight times with 1× B&W buffer until no obvious absorption peak was observed. The rest of the procedure was same with the one in buffer. RESULTS AND DISSCUSSION Native PAGE Analysis of the ETSD reaction. The circulation pathway of the ETSD reaction was illustrated in Figure 1A, which was validated through PAGE with the results and annotations shown in Figure 1B. The ternary S was prepared by annealing three partially complementary strands: linker (L, 71 nt), DNA 1 (34 nt) and DNA 2 (35 nt), which resulted in a clean and sharp band in PAGE analysis (lane 4) at an obviously different position with either L+DNA 1 (lane 2) and L+DNA 2 (lane 3), indicating its successful and complete formation. By reacting S with the miRNA-141 cognation DNA strand (T’) in the absence of Fuel strand (F), almost all of S converted to intermediate 1 (I1) (lane 5), which shared the same position with L+DNA 1+T’ in lane 7, and displaced DNA 2 at the same time. This implied that the first strand migration mediated by toehold 1 was fast and independent of F, and the product I1 was rather stable. Subsequently, F reacted with I1 by binding with the exposed toehold 2, forming intermediate 2 (I2), and then liberating DNA 1 and T’ through strand displacement (lane 6). The new product of this step and the whole net reaction was the double-stranded waste strand (W), which was composed by complementary sequences of L and F. Figure S1 showed the net equation of the ETSD reaction, followed by corresponding thermodynamic calculation. Specifically, the standard Gibbs free energy change of the reaction was -1339.5 J/mol, and the enthalpy change was approximately equal to zero, thus the entropy change was positive, acting as the driving force of the reaction. On the other hand, the conversion efficiency was calculated to be greater than 99.9%.

Au NPs-coupled ETSD reaction on MB. 50 µL 10 µM DNA 2 and 50 µL 10 µM biotin-labeled L were mixed with 1 mL Au NPs-DNA 1 solution, annealed at 95 °C for 3 min, and let slowly cool down to room temperature in 2 h, forming the ternary Au NPs labeled S, then the Au NPs-S was diluted for 10 times to a DNA complex concentration of 50 nM. Simultaneously, 100 µL MB was washed for three times and resuspended in 1 mL 2× B&W buffer (recipe listed in Table S1) according to the operation manual. Then 1 mL 50 nM Au NPs-S was added in 1 mL MB and incubated at room temperature with rotation for 30 min. Then the part of Au NPs-S unbounded with MB was separated with a magnet for 3 min, and MB was washed for three times with 1× B&W buffer, resuspended in 500 µL 1× B&W buffer and divided evenly in 20 tubes with 25 µL for each one. Subsequently, 25 µL target with different concentration was added in 25 µL MB, followed by incubating in 37 °C for 2 h with rotation, discarding the supernatant, washing the MB with 1× B&W buffer after magnetic separation and resuspending in 25 µL 1× B&W buffer. 25 µL 200 nM F was then incubated with MB in 37 °C for 3 h with rotation, followed by magnetic separation, and differently, collected the supernatant for the next step. When incubating with F, another 100 µL fresh MB was washed and bound with 60 µL 1 µM separation DNA (Sep) and divided evenly in 20 tubes with 15 µL for each one. Then the collected supernatant was incubated with the Sep-labeled MB in 37 °C for 1 h to eliminate existing F, and after magnetic separation, the supernatant was collected and kept for further use. Fabrication of the Pdots/ITO electrode. The Pdots with an average diameter of 5 nm was prepared with reprecipitation method according to the previous report with a slightly modification.38 Specifically, the photoactive PFBT polymer (1 mg/mL) and functional polymer PSMA (1 mg/mL) were dissolved and mixed in THF with a PFBT concentration of 100 µg/mL and a PSMA concentration of 20 µg/mL. Then, the mixed solution was sonicated to form a homogeneous solution. A total of 10 mL of deionized water was sonicated in a bath sonicator, while 2 mL of the mixed solution was injected into deionized water quickly. After that, THF was removed by nitrogen stripping, and the solution was filtered through a 0.22 µm filter. Finally, the solution was 3



Figure 1. (A) Illustration and (B) PAGE analysis of the reaction mechanism, where lane 1 is 20bp marker and 1 or 2 referred to DNA 1 or DNA 2. Tentative Fluorescent-labeled ETSD Reaction on MB. In order to improve the anti-interference capability of the ETSD circulation and achieve ultrasensitive analysis in complex biological environment, MB were introduced to the system. The binding specificity and efficiency of MB with biotinylated DNA was tested tentatively with a fluorescent-labeled single strand nucleic acid and the results were shown in Figure S2 and Figure S3. Then the ternary substrate S was assembled on the MB through biotin-streptavidin interaction, where a ROX (6-Carboxy-X-rhodamine)-labeled DNA 1 (ROX-DNA 1) was used to verify the feasibility of circulation occurrence on MB and to monitor the binding and reaction process, results shown in Figure S4. The binding efficiency of MB and S was 95.7%, close to the data of single-stranded DNA 96.4%, and the circulation was confirmed to be completed on MB accurately and successfully. Au NPs-labeled ETSD Reaction on MB. Au NPs, combined with DNA 1 through Au-SH bond, were further used as a substitute of ROX. As demonstrated in Figure 2A, the as-fabricated Au NPs appeared as quasi-spherical particles with a diameter of around 5 nm and had a maximum plasma absorption peak at ca. 515 nm as shown in Figure 2B. After linking with thiolated DNA 1 followed by ultrafiltration, a new peak at the characteristic absorption region of ca. 260 nm nucleic acids appeared, indicating the successful modification of oligonucleotides on Au NPs, and the slight red shift to around 522 nm could be attributed to the change of Au NPs surface charges caused by the DNA. By measuring the absorption spectra of the supernatant after ultrafiltration, the ratio of Au NPs and attached DNA 1 was calculated to be 1: 8.

Figure 2. (A) TEM image of 5 nm Au NPs. (B) UV-vis absorption spectra of Au NPs before and after modification of DNA 1; (C) Illustration of the experimental process of Au NPs-labeled TSD circulation on MB. Step 1: solution of Au NPs-S; step 2: the supernatant after Au NPs-S combing with MB and the following magnetic separation; step 3: the supernatant after adding 100 nM target, reacted for 2 h and followed by magnetic separation; step 4: the supernatant after adding 200 nM strand F, reacted for 3 h and followed by magnetic separation. (D) UV-vis absorption spectra of each step and (E) the corresponding digital graph.

Subsequently, UV-vis absorption spectrometer was used to reveal the circulation process, illustrated in Figure 2C and the results recorded in Figure 2D. By annealing with L and DNA 2, the Au NPs-S was prepared (step 1) and allowed to bind with MB at room temperature. After magnetic separation, the supernatant containing free Au NPs-S was collected and measured (step 2). By comparing the peak values at 522 nm before and after combination, the binding efficiency of MB and Au NPs-S was calculated to be 91.7%, which is a bit lower than the result of ROX-S on the account of steric hindrance caused by the Au NPs. The addition of 100 nM target miRNA-141 left no obvious peaks expect for the one at 260 nm, which was attributed to the displaced DNA 2 by the target, while Au NPs-DNA 1 still stick on L (step 3). The exposed toehold 2 facilitated the binding of L and the newly added F, resulting in the release of T for multiple turnovers, and the liberation of Au NPs-DNA 1 to the supernatant, which could be detected by UV-vis spectrometer (step 4). In the end, the peak value at 522 nm recovered to 91.0% after reacting with F, which not only demonstrating that the Au NPs remained colloidal and stable after a series of reactions close to MB, but also the total completion of the DNA circulation. 4


Page 4 of 8

Page 5 of 8 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

Analytical Chemistry In addition, in order to avoid the influence of F to the following assembly of Au NPs-DNA 1 with the capture DNA on electrode, further magnetic separation procedure was carried out to remove abundant F.

547 nm. Their further immobilization on ITO glass electrode resulted in a uniform film as depicted in Figure 4C, and under the irradiation of a monochromatic light of 450 nm, the Pdots/ITO electrode exhibited a stable cathodic photocurrent of about 80 nA, as shown in Figure 4D.

Figure 3. The dependence of disassembly of Au NPs-DNA 1 on the catalytic target concentration, recorded by (A) digital graph and (B) UV-vis absorption spectra; (C) the absorbance variation with the target concentration at 522 nm. The dependence of disassembly of Au NPs-DNA 1 on the catalytic target concentration was also investigated. Since the displaced Au NPs-DNA 1 had a pink color, the completion of the circulation could be observed directly by naked eye, depicted in Figure 2E; As the concentration of target miRNA-141 decreased, the color of the obtained supernatant lightened, and finally faded at 4 nM, as depicted in Figure 3A. Correspondingly, as demonstrated in Figure 3B, the peak value at 522 nm decreased with the target concentration, with a linear range of 10 nM to 5 nM, which is narrower to the one measured by fluorescence, due to the insufficient detection limit of the instrument, as shown in Figure 3C. It’s worth mentioning that the peak value of 10 nM target was the same as the one of 100 nM, indicating a substochiometric quantity (0.1×) of target enables the reaction to proceed rapidly to near-completion, and a 10-fold amplification was found in the ETSD reaction. PEC Performance Based on Pdots-Au NPs Interaction. The content of circulating miRNA-141 in prostate cancer patients is at femtomole level, which was beyond the detection range of the above-mentioned absorption-measuring methodology. To break this bottleneck, a sensitive energy-transfer based PEC bioassay was applied to futher amplify the signal. Among the numerous exploited photosensitive materials,39-50 Pdots, with extraordinary properties such as visible-light-responsivity, high photostability, minimal toxicity and good biocompatibility,51,52 has recently been found capable of affording sensitive PEC signaling via the semiconductor-noble metal interaction.38 In present case, the Pdots was prepared via reprecipitation method with photoactive conjugated polymer PFBT and functional polymer PSMA, which showed quasi-spherical morphology and good dispersity with a diameter of ca. 5 nm, as shown in Figure 4A. The black line and blue line in Figure 4B revealed their light-harvesting and emitting properties. Two absorption peaks at 340 nm and 455 nm were showed in the absorption spectra, and the emission value maximized at

Figure 4. (A) TEM image of Pdots; (B) Light absorption and emission properties of Pdots; (C) SEM image of Pdots/ITO electrode; (D) the stability of the electrode. The photocurrent was measured in 10 mM PBS solution (pH 7.4) with a monochromic irradiation light of 450 nm. Due to the high colloidal stability, feasibility of functionalization and unique optical-electronic properties, Au NPs have been extensively studied and utilized in various sensing fields, especially as an efficient quencher. In the previous study, we revealed that when in close proximity of Au NPs, the photocurrent of TPP-PFBT Pdots would be quenched significantly by ~60%, due to the interparticle resonance energy transfer.38,53 In this case, PFBT Pdots without TPP doping was utilized instead, the emission spectra of which had a better overlap with the plasma absorption of Au NPs (red line in Figure 4B), indicating possibly higher quenching efficiency. This conjecture was confirmed by further photocurrent measurement before and after anchoring Au NPs-DNA 1 on the Pdots/ITO electrode. As shown in Figure 5A, after immobilizing capture DNA (cDNA) via amide bond and then blocked by monoethanolamine (MEA), the photocurrent decreased slightly for the steric hindrance brought by the oligonucleotides; after assembling with 100 nM Au NPs-DNA1, however, the photocurrent value showed a sharp decrement down to zero, with a PEC quenching efficiency of nearly 100%. The intense photocurrent diminution was caused by two factors: i) the recombination of charge pair in the excited Pdots was enhanced by the collective oscillation of the conduction electrons of Au NPs under irradiation due to the above mentioned proper spectra overlap, leading to the changed equilibrium between charge recombination and spatial electron transfer to the electrode; ii) the plasma absorption of Au NPs partly hindered the absorption of Pdots, resulting in attenuated excitation energy. In addition, the energy transfer effect between Pdots and Au NPs was confirmed by the fluorescent image before and after Au NPs anchoring, shown in Figure S5. The successful 5



assemble of Au NPs on Pdots/ITO electrode was also characterized by XPS, with the results demonstrated in Figure S6 and Table S2. Finally, as a result of ETSD-MB circulation, Au NPs-DNA 1 containing supernatant was then collected and steered to bind on the Pdots/ITO electrode. As expected and shown in Figure 5B, the photocurrent signal varied in a positive correlation with the concentration of the initial miRNA-141 concentration. When the target concentration was beyond 10 pM, the photocurrent would be completely quenched, and the linear relationship was found to be 1 fM~10 pM, as shown in Figure 5C. The linear equation was (I-I0)/I0= -0.25 lgC (fM)-0.01, with a detection limit of 0.5 fM, which showed a significant improvement in analytical performance compared with the absorption and photoluminescence method. Note that the linear range was similar to the one measured by immobilizing pure Au NPs-DNA 1. That’s because there were around 8 DNA complexes on each Au NPs, and the Au NPs would be set free after an average of 8 cycles, which would counteract part of the amplification effect of the ETSD reaction. In addition, a comparison in analytical performance between this protocol and other miRNA detection strategies, including traditional methods such as microarray assay, and dynamically developing optical, magnetic, electrochemical and photoelectrochemical methods, was listed in Table S3. The selectivity of the protocol was investigated by detecting various oligonuleiotides at the same concentration of 100 nM, including complementary (C), mono-base mismatched (M), double-base mismatched (D) and non-complementary (NC) ones, results demonstrated in the inset of Figure 5C.

Real Blood Sample Analysis. The feasibility of the proposed protocol for real application was tested in undiluted whole blood. Since the clinical reference quantity of the target miRNA-141 was lacked, standard additional method was applied to healthy human blood samples. As known to all, whole blood testing is a tricky task in biosensing field due to the existence of numerous interfering species in micromole even millimole scale. In this work, these species were efficiently excluded by the integration of specially designed probe sequence and magnetic separation process. As recorded in Figure 5D, after reaction with the ternary S modified on MB and the following washing procedure, the successful matrix separation was proofed by the disappearance of characteristic absorption spectra of the blood. Figure 5D inset of the photographs further convincingly certified this since the color of the supernatant turned from bloody red to colorless. As shown in Table S4, for 5 blood samples from different individuals, the average recovery of spiked miRNA-141 was between 96%-108%, and the relative standard deviation was between 6.0-9.0%, indicating acceptable accuracy and reproducibility of the protocol for real sample analysis. CONCLUSIONS Aimed at determining PCa-related miRNA-141 in real blood sample, this work designed an Au NPs-coupled ETSD reaction on MB, which was divided into two independent steps that was capable of different functions of target extraction and amplification, respectively. This design allowed removal of complex interferent in whole blood, and subsequent target analysis in simple matrix. As a result, the Au NPs-DNA 1 were released and finally steered to bind with complementary sequences on the Pdots/ITO electrode. Further interaction between the free Au NPs and Pdots could greatly affect the photocurrent signal of the electrode, leading to significant improvement in analytical performance and thus achieving the sensitive detection of circulating miRNA-141 in whole blood sample. This work held great promise for future clinical application and provided a new option for sensitive biomolecular assay in complex biological fluids.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.xxxxx. Supporting Information. Sequences and stocking solutions of ETSD reaction; The fluorescent intensity of biotinylated DNA before and after binding with MB; The fluorescent intensity of un-biotinylated DNA before and after binding with MB; Illustration of the experimental process of ROX-labeled ETSD circulation on MB; Results of ROX-labeled ETSD circulation on MB; Element analysis of the Pdots/ITO electrode via XPS before and after assembly of Au NPs; Element proportion change before and after assembly of Au NPs; Recovery results for the assay of miRNA-141 in whole blood; Results of whole blood analysis using standard additional method. (PDF)

Figure 5. (A) Photocurrent change during modification process of Au NPs-DNA 1 on Pdots/ITO electrode; (B) Photocurrent dependency on target concentration; (C) The corresponding linear range. I0 and I referred to the photocurrent before and after Au NPs-DNA1 immobilization, respectively; and inset the selectivity investigation at the same concentration of 100 nM; (D) UV-vis absorption spectra of whole blood sample before and after extraction-washing process; and inset the corresponding digital photograph. The photocurrents were all measured in 10 mM PBS solution (pH 7.4) with a monochromic irradiation light of 450 nm.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected] *E-mail: [email protected]

Author Contributions 6


Page 6 of 8

Page 7 of 8 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

Analytical Chemistry MicroRNA-466 inhibits tumor growth and bone metastasis in prostate cancer by direct regulation of osteogenic transcription factor RUNX2. Cell Death Dis. 2017, 8, e2572. (16) Wu, H.; Wang, J.; Ma, H.; Xiao, Z.; Dong, X. MicroRNA-21 inhibits mitochondria-mediated apoptosis in keloid. Oncotarget 2017, 8, 92914-92925. (17) Varallyay, E.; Burgyan, J.; Havelda, Z. MicroRNA detection by northern blotting using locked nucleic acid probes. Nat. Protoc. 2008, 3, 190-196. (18) Choi, Y. S.; Edwards, L. O.; DiBello, A.; Jose, A. M. Removing bias against short sequences enables northern blotting to better complement RNA-seq for the study of small RNAs. Nucleic Acids Res. 2017, 45, e87-e87. (19) Schwarzkopf, M.; Pierce, N. A. Multiplexed miRNA northern blots via hybridization chain reaction. Nucleic Acids Res. 2016, 44, e129-e129. (20) Zhao, Y.; Chen, F.; Li, Q.; Wang, L.; Fan, C. Isothermal Amplification of Nucleic Acids. Chem. Rev. 2015, 115, 12491-12545. (21) Jung, C.; Ellington, A. D. Diagnostic applications of nucleic acid circuits. Acc. Chem. Res. 2014, 47, 1825-1835. (22) Min, X.; Zhang, M.; Huang, F.; Lou, X.; Xia, F. Live Cell MicroRNA Imaging Using Exonuclease III-Aided Recycling Amplification Based on Aggregation-Induced Emission Luminogens. ACS Appl. Mater. Inter. 2016, 8, 8998-9003. (23) Huang, Y.; Liu, X.; Huang, H.; Qin, J.; Zhang, L.; Zhao, S.; Chen, Z. F.; Liang, H. Attomolar detection of proteins via cascade strand-displacement amplification and polystyrene nanoparticle enhancement in fluorescence polarization aptasensors. Anal. Chem. 2015, 87, 8107-8114. (24) Li, B.; Jiang, Y.; Chen, X.; Ellington, A. D. Probing spatial organization of DNA strands using enzyme-free hairpin assembly circuits. J. Am. Chem. Soc. 2012, 134, 13918-13921. (25) Zhang, D. Y.; Turberfield, A. J.; Yurke, B.; Winfree, E. Engineering entropy-driven reactions and networks catalyzed by DNA. Science 2007, 318, 1121-1125. (26) Lv, Y.; Cui, L.; Peng, R.; Zhao, Z.; Qiu, L.; Chen, H.; Jin, C.; Zhang, X. B.; Tan, W. Entropy Beacon: A Hairpin-Free DNA Amplification Strategy for Efficient Detection of Nucleic Acids. Anal. Chem. 2015, 87, 11714-11720. (27) He, X.; Zeng, T.; Li, Z.; Wang, G.; Ma, N. Catalytic Molecular Imaging of MicroRNA in Living Cells by DNA-Programmed Nanoparticle Disassembly. Angew. Chem. Int. Ed. 2016, 55, 3073-3076. (28) Zhou, H.; Liu, J.; Zhang, S. Quantum dot-based photoelectric conversion for biosensing applications. TrAC, Trends Anal. Chem. 2015, 67, 56-73. (29) Zhao, W. W.; Xu, J. J.; Chen, H. Y. Photoelectrochemical DNA biosensors. Chem. Rev. 2014, 114, 7421-7441. (30) Zhang, N.; Zhang, L.; Ruan, Y. F.; Zhao, W. W.; Xu, J. J.; Chen, H. Y. Quantum-dots-based photoelectrochemical bioanalysis highlighted with recent examples. Biosens. Bioelectron. 2017, 94, 207-218. (31) Haddour, N.; Chauvin, J.; Gondran, C.; Cosnier, S. Photoelectrochemical Immunosensor for Label-Free Detection and Quantification of Anti-cholera Toxin Antibody. J. Am. Chem. Soc. 2006, 128, 9693-9698. (32) Gill, R.; Zayats, M.; Willner, I. Semiconductor quantum dots for bioanalysis. Angew. Chem. Int. Ed. 2008, 47, 7602-7625. (33) Tang, J.; Zhang, Y.; Kong, B.; Wang, Y.; Da, P.; Li, J.; Elzatahry, A. A.; Zhao, D.; Gong, X.; Zheng, G. Solar-driven photoelectrochemical probing of nanodot/nanowire/cell interface. Nano Lett. 2014, 14, 2702-2708. (34) Guo, L.; Li, Z.; Marcus, K.; Navarro, S.; Liang, K.; Zhou, L.; Mani, P. D.; Florczyk, S. J.; Coffey, K. R.; Orlovskaya, N.; Sohn, Y. H.; Yang, Y. Periodically Patterned Au-TiO2 Heterostructures for Photoelectrochemical Sensor. ACS Sens. 2017, 2, 621-625. (35) Zhang, H.; Zhao, L.; Geng, F.; Guo, L.-H.; Wan, B.; Yang, Y. Carbon dots decorated graphitic carbon nitride as an efficient metal-free photocatalyst for phenol degradation. Appl. Catal. B-Environ. 2016, 180, 656-662.

a. N.Z. and X.-M.S. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT Financial support from the Science and Technology Ministry of China (Grant No. 2016YFA0201200), National Natural Science Foundation of China (Grant nos. 21327902 and 21675080), and the Natural Science Foundation of Jiangsu Province (Grant BK20170073). Authors also thank Tian Tian, Tingting Zhang, Fan Yang, Nina Wang and Jielin Chen for their kind help and suggestions.

REFERENCES (1) Cohen, J. D.; Li, L.; Wang, Y.; Thoburn, C.; Afsari, B.; Danilova, L.; Douville, C.; Javed, A. A.; Wong, F.; Mattox, A.; Hruban, R. H.; Wolfgang, C. L.; Goggins, M. G.; Dal Molin, M.; Wang, T.-L.; Roden, R.; Klein, A. P.; Ptak, J.; Dobbyn, L.; Schaefer, J. Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science 2018, 359, 926-930. (2) Labib, M.; Mohamadi, R. M.; Poudineh, M.; Ahmed, S. U.; Ivanov, I.; Huang, C. L.; Moosavi, M.; Sargent, E. H.; Kelley, S. O. Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping. Nat Chem 2018, 10, 489-495. (3) Calin, G. A.; Croce, C. M. MicroRNA signatures in human cancers. Nat. Rev. Cancer 2006, 6, 857-866. (4) Croce, C. M. Causes and consequences of microRNA dysregulation in cancer. Nat. Rev. Genetics 2009, 10, 704-714. (5) Sierzega, M.; Kaczor, M.; Kolodziejczyk, P.; Kulig, J.; Sanak, M.; Richter, P. Evaluation of serum microRNA biomarkers for gastric cancer based on blood and tissue pools profiling: the importance of miR-21 and miR-331. Br. J. Cancer 2017, 117, 266-273. (6) Thome, A. D.; Harms, A. S.; Volpicelli-Daley, L. A.; Standaert, D. G. microRNA-155 Regulates Alpha-Synuclein-Induced Inflammatory Responses in Models of Parkinson Disease. J. Neurosci. 2016, 36, 2383-2390. (7) McLoughlin, D.; Kolshus, E.; Ryan, K. 759. Whole Blood MicroRNA Expression Following ECT: A Role for VEGF in Psychotic Depression. Biol. Psychiatry 2017, 81, S308-S309. (8) Bam, M.; Yang, X.; Sen, S.; Zumbrun, E. E.; Dennis, L.; Zhang, J.; Nagarkatti, P. S.; Nagarkatti, M. Characterization of Dysregulated miRNA in Peripheral Blood Mononuclear Cells from Ischemic Stroke Patients. Mol. Neurobiol. 2018, 55, 1419-1429. (9) Ma, L.; Teruya-Feldstein, J.; Weinberg, R. A. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 2007, 449, 682-688. (10) Heller, G.; Altenberger, C.; Topakian, T.; Ziegler, B.; Lang, G.; End-Pfützenreuter, A.; Steiner, I.; Zehetmayer, S.; Döme, B.; Klepetko, W.; Posch, M.; Zielinski, C. C.; Zöchbauer-Müller, S. Abstract 2766: Genome-wide miRNA methylation analyses in non-small cell lung cancer patients. Cancer Res. 2016, 76, 2766-2766. (11) Rupaimoole, R.; Calin, G. A.; Lopez-Berestein, G.; Sood, A. K. miRNA Deregulation in Cancer Cells and the Tumor Microenvironment. Cancer Discov. 2016, 6, 235-246. (12) Sita-Lumsden, A.; Dart, D. A.; Waxman, J.; Bevan, C. L. Circulating microRNAs as potential new biomarkers for prostate cancer. Br. J. Cancer 2013, 108, 1925-1930. (13) Liu, C.; Kelnar, K.; Liu, B.; Chen, X.; Calhoun-Davis, T.; Li, H.; Patrawala, L.; Yan, H.; Jeter, C.; Honorio, S.; Wiggins, J. F.; Bader, A. G.; Fagin, R.; Brown, D.; Tang, D. G. The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44. Nat. Med. 2011, 17, 211-215. (14) Bonci, D.; Coppola, V.; Patrizii, M.; Addario, A.; Cannistraci, A.; Francescangeli, F.; Pecci, R.; Muto, G.; Collura, D.; Bedini, R.; Zeuner, A.; Valtieri, M.; Sentinelli, S.; Benassi, M. S.; Gallucci, M.; Carlini, P.; Piccolo, S.; De Maria, R. A microRNA code for prostate cancer metastasis. Oncogene 2016, 35, 1180-1192. (15) Colden, M.; Dar, A. A.; Saini, S.; Dahiya, P. V.; Shahryari, V.; Yamamura, S.; Tanaka, Y.; Stein, G.; Dahiya, R.; Majid, S.

7



(36) Li, L.; Zhang, Y.; Zhang, L.; Ge, S.; Liu, H.; Ren, N.; Yan, M.; Yu, J. Paper-Based Device for Colorimetric and Photoelectrochemical Quantification of the Flux of H2O2 Releasing from MCF-7 Cancer Cells. Anal. Chem. 2016, 88, 5369-5377. (37) Li, X.; Zhu, L.; Zhou, Y.; Yin, H.; Ai, S. Enhanced Photoelectrochemical Method for Sensitive Detection of Protein Kinase A Activity Using TiO2/g-C3N4, PAMAM Dendrimer, and Alkaline Phosphatase. Anal. Chem. 2017, 89, 2369-2376. (38) Shi, X. M.; Mei, L. P.; Wang, Q.; Zhao, W. W.; Xu, J. J.; Chen, H. Y. Energy Transfer between Semiconducting Polymer Dots and Gold Nanoparticles in a Photoelectrochemical System: A Case Application for Cathodic Bioanalysis. Anal. Chem. 2018, 90, 4277-4281. (39) Kang, Q.; Yang, L.; Chen, Y.; Luo, S.; Wen, L.; Cai, Q.; Yao, S. Photoelectrochemical detection of pentachlorophenol with a Multiple Hybrid CdSexTe1−x/TiO2 Nanotube Structure-Based Label-Free Immunosensor. Anal. Chem. 2010, 82, 9749-9754. (40) Long, Y.-T.; Kong, C.; Li, D.-W.; Li, Y.; Chowdhury, S.; Tian, H. Ultrasensitive Determination of Cysteine Based on the Photocurrent of Nafion-Functionalized CdS–MV Quantum Dots on an ITO Electrode. Small 2011, 7, 1624-1628. (41) Zhuang, J.; Tang, D.; Lai, W.; Xu, M.; Tang, D. Target-induced nano-enzyme reactor mediated hole-trapping for high-throughput immunoassay based on a split-type photoelectrochemical detection strategy. Anal. Chem. 2015, 87, 9473-9480. (42) Chen, D.; Zhang, H.; Li, X.; Li, J. Biofunctional Titania Nanotubes for Visible-Light-Activated Photoelectrochemical Biosensing. Anal. Chem. 2010, 82, 2253-2261. (43) Li, M.; Zheng, Y.; Liang, W.; Yuan, Y.; Chai, Y.; Yuan, R. An ultrasensitive "on-off-on" photoelectrochemical aptasensor based on signal amplification of a fullerene/CdTe quantum dots sensitized structure and efficient quenching by manganese porphyrin. Chem. Commun. 2016, 52, 8138-8141. (44) Hao, N.; Zhang, X.; Zhou, Z.; Qian, J.; Liu, Q.; Chen, S.; Zhang, Y.; Wang, K. Three-dimensional nitrogen-doped graphene porous hydrogel fabricated biosensing platform with enhanced

photoelectrochemical performance. Sensor. Actuat. B-Chem. 2017, 250, 476-483. (45) Yan, K.; Yang, Y.; Okoth, O. K.; Cheng, L.; Zhang, J. Visible-Light Induced Self-Powered Sensing Platform Based on a Photofuel Cell. Anal. Chem. 2016, 88, 6140-6144. (46) Dai, H.; Zhang, S.; Hong, Z.; Lin, Y. A Potentiometric Addressable Photoelectrochemical Biosensor for Sensitive Detection of Two Biomarkers. Anal. Chem. 2016, 88, 9532-9538. (47) Wang, J.; Long, J.; Liu, Z.; Wu, W.; Hu, C. Label-free and high-throughput biosensing of multiple tumor markers on a single light-addressable photoelectrochemical sensor. Biosens. Bioelectron. 2017, 91, 53-59. (48) Wang, L.; Liu, Z.; Wang, D.; Ni, S.; Han, D.; Wang, W.; Niu, L. Tailoring heterostructured Bi2MoO6/Bi2S3 nanobelts for highly selective photoelectrochemical analysis of gallic acid at drug level. Biosens. Bioelectron. 2017, 94, 107-114. (49) Xin, Y.; Zhang, Z. Photoelectrochemical Stripping Analysis. Anal. Chem. 2018, 90, 1068-1071. (50) Liu, S.; Zhao, S.; Tu, W.; Wang, X.; Wang, X.; Bao, J.; Wang, Y.; Han, M.; Dai, Z. A “ Signal On ” Photoelectrochemical Biosensor Based on Bismuth@ N, O-Codoped-Carbon Core-Shell Nanohybrids for Ultrasensitive Detection of Telomerase in HeLa Cells. Chemistry–A European Journal 2018, 24, 3677-3682. (51) Wang, L.; Fernandez-Teran, R.; Zhang, L.; Fernandes, D. L.; Tian, L.; Chen, H.; Tian, H. Organic Polymer Dots as Photocatalysts for Visible Light-Driven Hydrogen Generation. Angew. Chem. Int. Ed. 2016, 55, 12306-12310. (52) Liu, H. Y.; Wu, P. J.; Kuo, S. Y.; Chen, C. P.; Chang, E. H.; Wu, C. Y.; Chan, Y. H. Quinoxaline-Based Polymer Dots with Ultrabright Red to Near-Infrared Fluorescence for In Vivo Biological Imaging. J. Am. Chem. Soc. 2015, 137, 10420-10429. (53) Fan, C.; Wang, S.; Hong, J. W.; Bazan, G. C.; Plaxco, K. W.; Heeger, A. J. Beyond superquenching: hyper-efficient energy transfer from conjugated polymers to gold nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 6297-6301.

For TOC only

8


Page 8 of 8

Gold Nanoparticle Couples with Entropy-Driven Toehold-Mediated

Recommend Documents