Oligonucleotide Cross-Linked Hydrogel for Recognition and

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Biological and Medical Applications of Materials and Interfaces

Oligonucleotide Cross-Linked Hydrogel for Recognition and Quantitation of MicroRNA Based on Portable Glucometer Readout Yanmei Si, Lulu Li, Ningning Wang, Jing Zheng, Ronghua Yang, and Jishan Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21727 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 9, 2019

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ACS Applied Materials & Interfaces

Oligonucleotide Cross-Linked Hydrogel for Recognition and Quantitation of MicroRNA Based on Portable Glucometer Readout

Yanmei Si,† Lulu Li,† Ningning Wang,† Jing Zheng,† Ronghua Yang,‡ and Jishan Li*,†

†State

Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and

Chemical Engineering, Hunan University, Changsha 410082, P. R. China ‡School

of Chemistry and Biological Engineering, Changsha University of Science and Technology,

Changsha 410114, P. R. China

*Corresponding author. E-mail: [email protected].

Fax: +86-731-8882 1848

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ABSTRACT A novel sensing platform for recognition and quantification of target miRNAs was developed by combining an amylase-trapped DNA hydrogel, multi-component nucleic acid enzymes (MNAzymes) and a portable glucometer (PGM) readout. First, the amylase was encapsulated inside the DNA hydrogel and physically separated from its substrate of amylose, which was in a solution outside the hydrogel. After addition of the target miRNA, the activity of the MNAzyme was restored, which cuts off the substrate linker strand. The active MNAzyme can catalytically act upon multiple substrate strands through diffusion, leading to the collapse of the hydrogel and the release of amylase, which catalyzes the hydrolysis of amylose to produce a large amount of glucose and generate a high PGM signal. The smart usage of the PGM enables simple portable detection of miR-21, with a detection limit as low as 0.325 fmol. Additionally, through the simple rational design of the target-binding sensor arms, the amylase-trapped DNA hydrogel sensing platform was successfully applied in the detection of multiple endogenous miRNAs (including miR-21, miR-335, miR-155, miR-122) extracted from HeLa cells, HepG2 cells, MCF-7 cells, and L02 cells.

KEYWORDS: DNA hydrogel, DNAzymes, microRNA, biomarker, signal amplification, personal glucometer.


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INTRODUCTION MicroRNAs (miRNAs) are endogenous small RNAs, of about 20-24 nucleotides in length, which perform many important regulatory functions in life processes, including early development, cell proliferation, apoptosis, and cell death.1-3 Recent, studies have suggested that the expression level of a miRNA is also closely related to the occurrence of cancer, metastasis and postoperative recovery of tumors.4,5 Thus, a miRNA would be an ideal potential biomarker for the corresponding kind of cancer in which it is involved. In general, a miRNA is expressed not only in cancer cells or tumor tissues, but also in peripheral blood.6 Usually, the expression levels of miRNAs in peripheral blood have a certain correlation with miRNAs levels in cancer cells or tumor tissues, and most of the miRNAs with abnormal expression in cell/tissue have the same change trend in peripheral blood.7-10 Moreover, peripheral blood miRNAs have been found to remain stable after overnight incubation at room temperature, boiling, repeated freeze-thaw cycles, and peracid treatment.11 A peripheral blood test has the advantages of being non-invasive, repeatable and amenable to simultaneous multi-index testing. Therefore, methodologies for the recognition and quantitation of miRNA in cancer cells or tumor tissues, especially in peripheral blood, will be of great value for early diagnosis and treatment of cancer, as well as of prognostic values for patients with cancer. The sensitive and simple detection of miRNAs still often involves many challenges due to their unique characteristics of short length, similar nucleotide sequences, and low abundance in human total RNAs. A series of conventional techniques for miRNA detection have been developed, such as Northern blotting, microarray, reverse transcription polymerase chain reaction (RT-PCR), flow cytometry, and enzyme-catalytic amplification technology,12-17 and some of them are used routinely in medical centers and research laboratories for the detection of miRNA. Nevertheless, they are still limited by their shortcomings, such as the need for accurate temperature controls, cumbersome ACS Paragon Plus Environment


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experimental procedures, expensive equipment, and professional operators. As a result, few of them are available for the public to use at home or in the field. Moreover, considerable time and cost are required for the public to take the samples to the professional institutes and wait for the result delivery, which not only delay the diagnosis of early cancer but also prevents the effective monitoring of post-operative recovery. Therefore, a simple, rapid and sensitive detection method that can be directly used by the public for the miRNA test is still urgently needed. In the past few years, stimuli-responsive hydrogels have attracted particular attention in the development of biosensors by virtue of their simplicity, sensitivity, and portability, as well as ease of preparation and storage.18 In particular, DNA hydrogels with synthetic polymers as backbone and functional DNA as cross-linker have been widely used for the detection of various targets, such as small molecules, ions, and proteins.19-21 DNAzymes are DNA molecules with catalytic activity and have been used in DNA, protein, and metal ion detection.22-24 Additionally, DNAzymes can be split into multi-component nucleic acid enzymes (MNAzymes), which have more flexible application value in biodetection.25 In addition, the glucometer (PGM) has become one of the most successful examples of patient self-testing, due to their portability, easy operation, low cost, and reliable quantitative results.26 However, the traditional PGMs only can be used to monitor blood glucose. Recently, pioneering work using PGMs to detect other targets including organic molecules, proteins, and metal ions have been reported based on the signal switching strategy developed by Lu’s group.27,28 Subsequently, this concept was further extended for the detection of different kinds of targets.29,30 Accordingly, motivated by the encapsulation and release capability of hydrogels, the exceptional performance of DNAzyme, and the easy operation of PGM, herein, we developed a miRNA-responsive hydrogel sensing platform for the quantitative detection of miRNA based on the catalytic performance of DNAzyme and quantitative ability of PGM.


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In this work, polyacrylamide I and II (PA-I and PA-II), each containing a pendant DNA sequence (strands I and II), were synthesized by copolymerization of acrylic DNA and acrylamide monomers. Strands I and II are complementary to partial sequences of the substrate linker strand which contains the DNAzyme cleavage site. The DNAzyme was split into MNAzymes (EA, EB and target), and thus the enzyme activity occurs only when a three-part hybridization forms an enzyme structure with a cavity. EA and EB are complementary to the two segments of the DNAzymes cleavage site of the substrate linker sequence forming an inactive DNA motif. Upon the addition of the substrate linker strand which has been hybridized with EA and EB, strand I of PA-I and strand II of PA-II hybridize with the linker strand to form hydrogel with amylase encapsulated. Initially, amylase is stably trapped inside the hydrogel and separated from its substrate, amylose, which is in a solution outside the hydrogel. After the addition of a target miRNA, the conformation and activity of MNAzymes will be restored, leading to the cleavage of the substrate linker strand in the presence of Pb2+.31 The collapsed hydrogel releases amylase, which catalyzes the hydrolysis of amylose to produce a large amount of glucose and generate the high PGM signal (Scheme 1). In addition, each activated MNAzyme catalytically cleaves many crosslinks, resulting in signal amplification. The strategy is simple, rapid, and achieves quantitative analysis of the target miRNA.



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Scheme 1. Schematic illustration of the oligonucleotide cross-linking hydrogel sensing platform for detection of target miRNA via portable glucometer (PGM) readout.

EXPERIMENTAL SECTION Materials and Reagents. Glucometer (Johnson & Johnson, New Brunswick, NJ, USA) was obtained from Johnson & Johnson medical equipment Co. Ltd. (Shanghai, China). Amylase, amylose, N, N, N´, N´-tetramethylethylenediamine (TEMED), ammonium persulfate (APS) and acrylamide were purchased from J&K Scientific Ltd (Beijing, China). Acrylic-DNA and all other oligonucleotides used in this work (Table S1 in Supporting Information (SI)) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Other reagents were purchased from Sinopharm Chemical Reagent (Shanghai, China). The buffer solution (pH 7.3) contains 72.9 mM Na2HPO4, 27.1 mM NaH2PO4, 140 mM NaCl, 5 mM MgCl2. Synthesis of the DNA Side-chain Polymer. An 8-μL volume of 25% acrylamide was first mixed with 24 μL of buffer solution, and then added into a 16-μL volume of 3 mM Acrydite-DNA


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(Strands-I or Strands-II in Table S1) solution. The mixture was kept in a vacuum drier for 10 min to remove the air. Then, 2 μL of 5% APS and 2 μL of 5% TEMED were added into the above solution. Next, the solution was immediately incubated in the vacuum drier for 18 min, and then the polymer strands (PA-I and PA-II) were obtained and stored at 4 oC for further usage. Preparation of the Amylase-encapsulated DNA Hydrogel. First, the

substrate linker

(Substrate-link in Table S1) was mixed with EA and EB (Table S1) at a ratio of 10:1, and incubated at 37 oC for 30 min. Then, a certain volume of the above solution was added into a 0.5-ml tube containing the mixture solution of PA-I, PA-II and amylase (The molar ratio of Strands-I, Strands-II and Substrate-link is 1:1:1, and the final concentration of amylase is 0.4 µg/µL.) at room temperature, and then the amylase-trapped hydrogel was immediately formed. MiRNA Extraction from Cells. HeLa cells, HepG2 cells, MCF-7 cells, and L02 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin, and incubated in an incubator containing 95% air and 5% CO2 at 37 °C. After the cells were all over the bottom of the bottle (~ 107 cells), the total RNA containing the target miRNA was extracted using TRIzol® solution according to the instructions provided by the manufacturer (Sangon Biotech, Shanghai, China). Measurement of miRNA. For the target miRNA assay, 5 μL of the sample solution with 5 μM of Pb2+ and different concentration of miRNA was first added into the hydrogel-containing tube, and incubated at 37 °C for 4 h to ensure the complete cleavage reaction. Then, 20 μL of 14 μg/μL amylose was added and incubated at 37 °C for another 2 h. Subsequently, a 5-μL supernatant was used to detect the glucose content with the PGM.

RESULTS AND DISCUSSION ACS Paragon Plus Environment


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Formation and Dissolution of the Amylase-trapped DNA Hydrogel. As we all know, the color of the amylose solution can turn dark blue after mixing with a small amount of KI/I2, while the dark blue will turn colorless if the amylose is broken down into glucose by amylase. Therefore, an amylose/I2 mixture system was used to investigate the formation and hydrolysis of the amylase-encapsulated DNA hydrogel. The amylase-encapsulated hydrogel was prepared by adding the substrate linker into a well-mixed solution containing PA-I, PA-II, and amylase. The image in Figure 1A shows that the dark blue color of the amylose/I2 solution turned colorless only for the amylase-encapsulated hydrogel containing the DNAzyme sequence (c), while both the blank hydrogel (a) and the amylase-encapsulated hydrogel without the DNAzyme (b) all maintain the dark blue color of the amylose/I2 mixture in the top solution. These results indicate that the amylase-trapped DNA hydrogel can be formed successfully, and both amylose and amylase can be separated by the hydrogel with amylase trapped inside the hydrogel and amylose outside the hydrogel. Additionally, in the presence of the DNAzyme sequence, the substrate linker can be cleaved, leading to the dissolution of the hydrogel and subsequent release of the amylase, which digests the amylose into glucose. These procedures are exactly as expected.

Figure 1. (A) Validation of the formation and dissolution of the DNA hydrogel platform by using the amylose/I2 mixture solution. a) Hydrogel without amylase and DNAzyme; b) Hydrogel with


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amylase but without DNAzyme; c) Hydrogel with amylase and DNAzyme. (B) Validation of the hydrogel sensing platform for miR-21 by using the amylose/I2 mixture solution. a) Without amylase, EA-21&EB-21 and miR-21DNA; b) With amylase but without EA-21&EB-21 and miR-21DNA; c) With amylase and EA-21&EB-21 but without miR-21DNA; d) With amylase and miR-21DNA but without EA-21&EB-21; e) With amylase, EA-21&EB-21 and miR-21DNA.

Validation of the Design Scheme. The amylose/I2 mixture system was also employed to demonstrate the sensing principle of the enzyme–hydrogel platform to detect a miRNA by visually observing the color change. As shown in Scheme S1, we have split DNAzyme sequence into MNAzymes, including partzymes A and B (EA-21, EB-21), and each of which contains a target-binding sensor arm, a partial catalytic core, a substrate-binding arm. EA-21 and EB-21 are complementary to the sequences at both ends of the cleavage site of the substrate linker, forming a stable inactive MNAzyme motif. Both exposed ends of the sequence of the substrate linker are complementary to Strands-I and Stands-II, to form the hydrogel system. The addition of miR-21 DNA (a sequence of DNA, serving as a model) restores the enzymatic activity of MNAzyme, leading to the break of the substrate linker and subsequent release of the amylose encapsulated in the hydrogel. The image in Figure 1B shows that when no amylase and EA-21&EB-21 were trapped in the hydrogel and no miR-21DNA was introduced (a), the tube has an obvious layer of hydrogel at the bottom and a blue solution of the amylose/I2 mixture at the top. In other words, the hydrogel is very stable under the normal conditions. Similarly, for the amylase-encapsulated hydrogel without EA-21&EB-21 and miR-21DNA (b), or with EA-21&EB-21 but without miR-21DNA (c), or with miR-21DNA but without EA-21&EB-21 (d), all show no color change that can be observed, which indicates that amylase, EA-21&EB-21 and miR-21DNA are all



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indispensable for the enzyme-hydrogel response. After miR-21DNA was added into the amylase-encapsulated hydrogel with EA-21&EB-21 (e), a significant change of the blue solution to a colorless solution can be observed, indicating that the hydrogel was partially disintegrated in the presence of a target oligonucleotide sequence, and released enough amylase to the amylose/I2 solution, which caused the hydrolysis of amylose. All the above results further proved the feasibility of the experimental procedure. Optimization of the Hydrogel Sensing Platform. On the one hand, an increased amount of amylase will result in an increased glucose production to guarantee higher sensitivity of the sensing device. On the other hand, the hydrogel may be overloaded, such that even without the target miRNA, the excess amylase will digest amylose, giving false-positive PGM signals. Accordingly, the loading of amylase should be precisely controlled to minimize amylase leakage. Therefore, an amylose/I2 solution was used to investigate the optimal amount of amylase. From the image in Figure 2A reveals, no observable color change for the hydrogels with amylase concentration below 0.4 μg/μL even after 6 h. In contrast, obvious color fading of the blue amylose/I2 solution can be observed when the concentration of amylase increased to 0.6 μg/μL, indicating that the amylase leakage will become a serious problem if too large amount of amylase is used. Therefore, we chose 0.4 μg/μL of amylase to prepare the amylase-entrapping hydrogel. Commonly, with the help of amylase, the glucose production resulting from amylose will be initially increased with the increase of the incubation time, and then tends to reach a plateau level. Accordingly, the incubation time of the amylase-catalyzed hydrolysis reaction was investigated by using the PGM (Figure 2B). The result indicates the PGM signal was increased with the increasing hydrolysis time and become steady after 2 h. Additionally, the concentration of amylose in the hydrolysis reaction was also investigated to ensure the higher sensitivity of the PGM signal. As


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shown in Figure 2C, the PGM signal increased with the increasing concentration of amylose until 14 μg/μL was reached, indicating the saturation of the amylose concentration under the fixed concentration of amylase. Therefore, 2 h and 14 μg/μL were selected as the incubation time and amylose concentration for the amylase hydrolysis reaction, respectively. Moreover, Lead ions (Pb2+) are a necessary cofactor for the DNAzyme used in this work, but the activity of proteases is generally susceptible to heavy metal ions. Thus, the influence of Pb2+ on the amylase activity should be examined. As shown in Figure S1, the dark blue amylose/I2 becomes colorless and the PGM signal shows no significant difference even at a high concentration of Pb2+ (e.g. 10 μM), indicating that the presence of Pb2+ has no significant effect on the amylase-catalyzed hydrolysis of amylose under a certain concentration range of Pb2+. Considering that the activated MNAzymes can autonomously move to additional cleavage sites to perform further cutting reactions and generate an enzymatic signal amplification, so the amount of EA-21&EB-21 may affect the degree of the hydrogel dissolution. Therefore, the amount of EA-21&EB-21 was optimized by observing the content of glucose in the upper amylose solution (Figure 2D). The results shown in Figure 2D indicate that the PGM signal increased with the increasing mole ratio of EA-21&EB-21/linker strand. However, a too high mole ratio of EA-21&EB-21/linker strand lead to the gradually decreased PGM signals. This phenomenon is presumably due to the too high density of EA-21&EB-21, which might limit the autonomous move of the activated MNAzymes to the neighboring substrates to perform further cleavage and thus signal amplification. Thus, the mole ratio of 1/10 of the EA&EB/linker strand was chosen for the construction of the hydrogel sensing platform.



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Figure 2. (A) Investigation of the loading capacity of the hydrogel platform. From 'a' to 'f': concentration of the amylase encapsulated in hydrogel was 0, 0.2, 0.3, 0.4, 0.6 and 1.0 μg/μL, respectively. All the experiments were performed using the same concentration of amylose substrate. (B) PGM signal intensity versus the incubation time in the presence of 0.4 μg/μL amylase and 4 μg/μL amylose. (C) PGM signal intensity versus the different concentrations of amylose at a 2-h incubation time. (D) Effect of EA-21&EB-21 concentration on the PGM signal response to miR-21DNA (10 nM).

Stability and Sensing Performance of the Hydrogel Platform. Good stability is a fundamental requirement for a sensing platform to ensure the reliability of the target detection. Accordingly, the stability of the hydrogel sensing platform under different temperature conditions was first carefully investigated. As indicated by the results shown in Figure S2 and Figure S3, the dark blue color of


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the amylose/I2 mixture solution at the top of the hydrogel underwent no significant change after a 24 h incubation at 37 oC and 1 week storage at 4 oC, suggesting that this hydrogel sensing platform is very stable under the detecting/storage temperature condition. Such a high stability of the hydrogel sensing platform will ensure the accuracy of the miRNA detection in potential practical applications. After verifying the mechanism and optimizing the conditions, the response capacity and specificity of the hydrogel sensing platform were also studied. For the investigation of sensing performance,

different

concentration

of

miR-21DNA

was

added

into

the

sensing

hydrogel-contained tube. After incubation at 37 oC for 4 h, 14 μg/μL of amylose solution was added into the tubes and further incubated at 37 oC for another 2 h. Afterwards, 5 μL of supernatant was used to detect the glucose content with the PGM. As illustrated in Figure 3A, the rise in the concentration of miR-21DNA dramatically increases the PGM signal. A linear relationship was obtained between the PGM signal and log[miR-21DNA] in the range of 0.5−250 fmol (R2 = 0.976). Moreover, a detection limit of ca 0.325 fmol was estimated according to the 3σ rule, which is comparable with that reported for a reported fluorescence-based miRNA detection method.32 This finding suggests that the prepared hydrogel sensing platform is efficient for the sensitive detection of a specific target of miRNA. For the specificity analysis, it is a great challenge to achieve the specific discrimination among the miRNA family members due to their high sequence similarity. The miR-21 DNA target with different number of mismatched bases to the recognition sequence (EA-21 or EB-21) and some other kinds of miRNA-corresponding DNA fragments were used to investigate the sequence-specificity of the hydrogel sensing platform. The results displayed in Figure 3B show that the PGM signal resulting from the addition of miR-21DNA is much higher than that produced by the addition of other DNA target fragments with more than one mismatched base. Moreover, based on these encouraging results, it can be speculated that the identification of



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single-base mismatch can be achieved by reasonably designing the recognition sequence of the two arms of EA&EB. The above results indicate that the specificity of the prepared hydrogel sensing platform is very high, suggesting its potential for application in the specific discrimination of target miRNA among miRNA family members with high sequence similarity.

Figure 3. (A) Correlation of the PGM signal and miR-21DNA concentrations. (B) Investigation of the specificity of the miR-21 hydrogel sensing platform using PGM. (C-E) Validation of the miR-335, miR-155, and miR-122 hydrogel sensors using the amylose/I2 mixture solution: without (a)/with (b) the target sequence. (F) PGM signal response of the miR-335, miR-155 and miR-122 hydrogel sensing platform to miR-155DNA, miR-122DNA, miR-21DNA and miR-335DNA in buffer solution, respectively.

Universal Property of the Hydrogel Platform for Measurement of Multiple Targets. To determine the universal property of the hydrogel platform for the detection of other target miRNA,


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the corresponding hydrogel sensing platform for the detection of miR-335 (breast cancer and prostate cancer),33,34 miR-155 (breast cancer, thyroid carcinoma, colon cancer, and cervical cancer),35 and miR-122 (hepatocellular carcinoma),36 were also prepared by simply adjusting the recognition sequence in the two arms of EA&EB (Scheme S2, Scheme S3, Scheme S4). To verify the successful construction of the hydrogel sensing platforms, the amylose/I2 mixture system was also used. The validation tests shown in Figure 3C, Figure 3D and Figure 3E revealed that the dark blue color of the amylose/I2 solution on the top of the hydrogel clearly became colorless upon addition

of

the

corresponding

target

sequence,

which

resulted

from

the

target-hybridization-activated hydrolysis of the hydrogel and the subsequent release of amylase, which digested amylose to glucose. The sensing performance and specificity of these prepared hydrogel sensing platform were also investigated by using PGM. The results shown in Figure 3F indicate that, except for the addition of the corresponding target sequence, a significant PGM signal change cannot be observed when one hydrogel sensing platform is incubated with other oligonucleotide sequences. Furthermore, a similar PGM signal intensity produced by miR-335DNA, miR-155DNA and miR-122DNA, can be observed compared with that produced by miR-21DNA at the same sequence concentration, suggesting that all three prepared hydrogel sensing platform also have a similar significant sensing performance and high specificity. We speculate that the detection limits of miR-335DNA, miR-155DNA, miR-122DNA are similar to that of miR-21DNA, given that the recognition sequences are identical in length. Together, the above results indicate that the corresponding hydrogel sensing platform for miR-335, miR-155 and miR-122 measurement was formed successfully, and the hydrogel platform has the universal capacity to be used for constructing multitarget miRNA sensing platform simply by adjusting the recognition sequence. Hydrogel Sensing Platform for Target miRNA Assay in Buffer and Serum. Although the ACS Paragon Plus Environment


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significant sensing performance of the hydrogel sensing platform has been well demonstrated by using a DNA-sequenced target and under the ideal buffer conditions, for practical application, it is necessary to further demonstrate the reliability of the prepared hydrogel sensing platform in RNA-sequenced target assay and under complex detection conditions, for example in serum. The miR-21, miR-122, miR-155 and miR-335 in buffer solution were first measured by using four different prepared hydrogel sensing platform. The results shown in Figure 4A indicate that, except for the presence of the corresponding target miRNA, a significant increase of the PGM signal, compared with that resulting from the blank solution, cannot be observed when one hydrogel sensing platform was incubated with other miRNA sequences. Additionally, a slightly lower but similar PGM signal produced by these miRNA sequences can be observed compared with that produced by the corresponding DNA-sequenced target (shown in Figure 3) at the same sequence concentration, suggesting that the prepared hydrogel sensing platform also has a significant sensing performance and high specificity for the RNA-sequenced target. Then, the miR-21, miR-122, miR-155 or miR-335 spiked into the 10% fetal bovine serum were also measured by using the four corresponding prepared hydrogel sensing platform. As shown in Figure 4B, except for the presence of the corresponding target miRNA, a significant PGM signal also cannot be observed when one hydrogel sensing platform was incubated with other miRNA sequence spiked into the 10% fetal bovine serum, which is similar with that obtained under the ideal buffer condition (shown in Figure 4A). All these results further indicate that the hydrogel sensing platform still has a high specificity and capacity to respond to the miRNA target under the complex conditions and has great potential for application in target miRNA detection in complex biological sample.


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Figure 4. (A) PGM signal response of the miR-21, miR-335, miR-155 and miR-122 hydrogel sensing platform to miR-335, miR-155, miR-122 and miR-21 in buffer solution, respectively. (B) PGM signal response of the miR-21, miR-335, miR-155 and miR-122 hydrogel sensing platform to miR-335, miR-155, miR-122 and miR-21 in 10% fetal bovine serum-contained buffer solution, respectively.

Cellular Extracts Assay of Target miRNA Using the Hydrogel Sensing Platform. After carefully investigating the various characteristics of the hydrogel sensing platform, we then used the corresponding hydrogel sensing platform to test the target miRNAs, including miR-21, miR-122 and miR-155, in the RNA extracted from the HeLa cells, HepG2 cells, MCF-7 cells, and L02 cells, respectively. The obtained results are shown in Figure 5. The PGM signal for miR-21 in HeLa cells and MCF-7 cells was much higher, while only a slight increase was observed in HepG2 cells and a normal signal was observed in L02 cells. These findings indicate that there was a relatively higher expression level of miR-21 in HeLa cells and MCF-7 cells than that in HepG2 cells and L02 cells, which is also consistent with the reported related literature.37-39 We also found that miRNA-155 is over expressed in MCF-7 cells,40 but a lower expression level was detected in HeLa cells, HepG2



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cells and L02 cells. Additionally, miR-122, downregulated in liver cancer,41 showed slightly higher expression in L02 cells than in HeLa, HepG2, and MCF-7 cells. These results indicate that the expression of certain endogenous miRNAs can be significantly different between normal and cancerous cells, which is not unexpected, as some miRNAs are thought to function as oncogenes or tumor suppressors, which are correlated with various human cancers and shown different expression level in various cancerous cells and normal cells.35-41 The detection results are consistent with the expression levels of these miRNAs previously reported, further indicating the effectiveness of the hydrogel sensing platform for miRNAs detection in real biological-samples.

Figure 5. PGM signal response of the miR-21, miR-155 and miR-122 hydrogel sensing platform to RNA extracts obtained from HeLa cells, HepG2 cells, MCF-7 cells, and L02 cells, respectively.

CONCLUSION In summary, we have successfully developed an innovative amylase-trapping DNA hydrogel sensing platform for detection of multiple miRNAs in buffer and in real samples from cellular extracts. Such a simple, sensitive and portable amylase-trapping DNA hydrogel-based method may circumvent the drawbacks of the current detection techniques in testing target miRNAs. It can be


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tailored as a simple and efficient detection method for low-level miRNAs in blood or cancer cells for the diagnosis of cancer and the warning or monitoring of cancer metastasis in clinical applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Oligonucleotide sequences used in this work (Table S1), working principle of the hydrogel sensing platform for target miRNA (Scheme S1-S4), and other additional information as noted in the text, including Figures S1-S3. AUTHOR INFORMATION Corresponding Author * [email protected].

Fax: +86-731-8882 1848

ORCID Yanmei Si: 0000-0001-8424-8238 Jishan Li: 0000-0001-8144-361X Notes The authors declare no competing financial interest. ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China (21475036,



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