A Universal Paper-based Electrochemical Sensor for Zero

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Biological and Medical Applications of Materials and Interfaces

A Universal Paper-based Electrochemical Sensor for Zero-background Assay of Diverse Biomarkers Xiaojuan Liu, Xiuyuan Li, Xin Gao, Lei Ge, Xinzhi Sun, and Feng Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03860 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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 45 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

ACS Applied Materials & Interfaces

A Universal Paper-based Electrochemical Sensor for Zero-background Assay of Diverse Biomarkers

Xiaojuan Liu, Xiuyuan Li, Xin Gao, Lei Ge, Xinzhi Sun, and Feng Li*

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University,

Qingdao 266109, P. R. China.

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 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

Page 2 of 45

ABSTRACT: This paper describes a universal paper-based electrochemical sensing platform that uses a paper modified with signal molecule-labeled DNA and a screenprinted electrode along with target recognition solutions to achieve the detection of multiple types of biomarkers. These assays rely on the target-induced synthesis of Mg2+-dependent DNAzyme for catalyzing the cleavage of substrate DNA from paper, which have been demonstrated by using microRNA recognition probe for miR-21, a phosphorylated hairpin probe for alkaline phosphatase, and a DNA aptamer for carcinoembryonic antigen assays, respectively. Taking advantages of the high specific target-triggered polymerization/nicking and DNAzyme-catalyzed signal amplification, the present assays enable highly sensitive and selective detection of these targets with zero-background. These assays can also be applied to detect target in spiked serum samples, demonstrating the potential for point-of-care detection of clinical samples.

KEYWORDS: Bioassay, DNAzyme, Nucleic acid, Paper, Electrochemical sensor


2

Page 3 of 45 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


1. INTRODUCTION

The development of effective and reliable point-of-care (POC) diagnostic tools to detect disease-related biomarkers is crucial for improving patient care in resourcelimited settings.1-4 Paper, as one of the cheapest and most ubiquitous materials, has drawn increasing interest as a portable, affordable, flexible, and scalable substrate material for the construction of POC tests.5,6 Paper-based POC tests have been fabricated to target glucose as early as the 1950s7,8 and greatly expanded for the detection of diverse biomarkers (e.g. small biomolecules, proteins, active enzymes, and even cancer cells).9-11 Although great progress has been made in the last decades, most of the reported paper-based POC tests are relied on the protein-based measurement by using enzymes and antibodies as recognition elements. However, these POC tests are only suitable for the biomarkers that have recognition enzyme or antibody, which cannot be expanded for the detection of other types of biomarkers, such as nucleic acid biomarkers. In this aspect, nucleic acid-based assay is more attractive due to its amenability of flexible design of recognition strategies for different


3


Page 4 of 45

types of biomarkers. Moreover, it is a new research field for the combination of nucleic acid-based assay onto paper-based POC tests.12,13 Nucleic acid-based assay that employs DNA or RNA as recognition motif has emerged as an effective alternative platform for the determination of genetic or protein biomarkers in biological samples owing to their unique features, such as the strict base sequence controlled hybridization and functional DNA with catalytic activity or recognition ability.14-17 For example, DNAzymes, which are special DNA sequences, can catalyze a wide variety of reactions, such as the cleavage of the ribonucleic acid glycosidic bond.18,19 The unique catalytic activity of DNAzymes makes them fascinating signal amplifiers for highly sensitive detection of various different targets.20-22 Aptamers, another type of functional DNA, which can target distinct molecules with high affinity and specificity that are comparable to that of antigen and antibody.23,24 Furthermore, nucleic acids are more stable and adaptable, which also can be easily obtained by using a commercial DNA synthesizer.25 Therefore, nucleic acids have been served as useful molecular tools in the design of diverse biosensors and nanodevices. Compared with the traditional antigen-antibody diagnostic methods (e.g. ELISA), nucleic acid-based


4

Page 5 of 45 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


assay has the merit of high sensitivity, because its sensitivity can be efficiently improved by using appropriate signal amplification strategies, such as isothermal amplification, catalytic DNA circuit, as well as their combined strategies.26-28 However, the vast majority of the reported nucleic acid-based assays are carried out in solution and it is still a challenge for integrating nucleic acid-based assay onto paper-based POC tests. Additionally, signal readout is considered to be another crucial factor determining the potential application of paper-based POC tests. Until now, a number of signal readout strategies

have

been

developed,

such

as

colorimetry,29

fluorimetry,30

electrochemiluminescence,31 amperometry,32-34 and so on. Compared with the widely used optical methods, electrochemical strategy is a more attractive signal readout scheme for paper-based sensors, because it is more quantitative and resistant to interference from color.35-37 Unfortunately, one of the main limitations of the conventional heterogeneous electrochemical biosensors is the need of specific functionalized electrode for each target, which restricts their potential application for other analytes.38 Another obstacle is the non-specific adsorption by the working electrode, which usually produces high background current, leading to a low sensitivity.


5


Page 6 of 45

Although our group recently reported homogeneous electrochemical methods can solve the problem of electrode functionalization, they still suffer from the problem of high background currents owing to the non-completely inhibited diffusion of signal molecules towards the electrode surface.39,40 Therefore, it is one of the biggest challenges for analytical methods to eliminate the background signals. To address these issues, we attempt to separate the signal molecules from the working electrode by immobilizing them on an isolated paper, which cannot diffuse or non-specific adsorb to the surface of the electrode without the target-induced cleavage. Besides the background signal, the development of universal sensing platform for multiple bioassays is a continuous analytical challenge as there are many different types of disease-related biomarkers, such as some specific nucleic acids, enzymes, and proteins. For example, microRNAs (miRNAs) are a kind of nucleic acid biomarker, which have the functions of regulating gene expression. The abnormal repression of certain miRNA has been demonstrated to be related to many diseases, such as cardiovascular disease, cancers, and tissue injury.41-47 Alkaline phosphatase (ALP) is an enzyme with the capability of catalyzing the hydrolysis of a phosphomonoester into


6

Page 7 of 45 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


an inorganic phosphate.48 In routine clinical assays, ALP is commonly utilized as an important biomarker for diagnosis of many diseases, including diabetes, liver dysfunction, and cancers.49-51 Carcinoembryonic antigen (CEA), one of serum tumor biomarkers, is a useful screening tool in annual medical checkups. High concentration of serum CEA has been proved to be assisted with many cancers, such as breast cancer, gastric cancer, lung cancer, and ovarian carcinoma.52-54 Herein, a specially designed universal paper-based electrochemical sensor (PES) was prepared for highly sensitive analysis of different types of biomarkers. Three biomarkers, namely miRNA, ALP, and CEA, were employed as the model biomarkers of nucleic acid, enzyme, and protein, respectively. The presence of the target biomarkers initiated the isothermal autonomous polymerization of the sequence-specific Mg2+dependent DNAzymes by using its complementary DNA sequence as a template. These DNAzymes were then released to catalyze the cleavage of the ferrocene (Fc)labeled DNA (Fc-DNA) strand, leading to the release of signal molecules from the paper surface and generating an increased electrochemical signal on a screen-printed electrode (SPE).


7


Page 8 of 45

2. EXPERIMENTAL SECTION

2.1. Materials and Apparatus. The materials and apparatus used in the experiment were described in Section S1.1 and S1.2 (supporting information). 2.2. Immobilization of Fc-DNA on Paper. The paper was designed to be circular pieces with 5 mm diameter using CorelDraw X4 and cut by LASER mini. The laser has a speed engraving speed of 40%, a speed engraving strength of 30%. The circular paper was treated by NaIO4 for forming aldehyde groups to immobilize Fc-DNA through Schiff alkali reaction. In brief, 50 pieces of the circular paper were immersed into 50 mL solution (containing 50 mM NaIO4 and 700 mM LiCl) and incubated at 55 C in dark chamber for 3 h, washed with Milli-Q water for three times and then dried in room temperature. Subsequently, 30 μL of 25 μM Fc-DNA were dropped onto the circular paper at 37 C for 2 h. Then, the paper was washed three times with Tris-HCl buffer and ultrapure water, respectively, and finally dried in room temperature. 2.3. Preparation of PES. The obtained Fc-DNA modified paper was combined with a commercial SPE to prepare a prototype of PES, by sticking them onto a soft plastic


8

Page 9 of 45 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


slide using double sided adhesive tape. To improve the performance of the electrochemical response, the working electrode of SPE was modified with carbon nanotubes (CNTs). Briefly, 3μg CNTs dispersed in water was spotted on the working electrode of the SPE and dried in room temperature. 2.4. Electrochemical Detection of MiRNA. Different concentrations of miR-21 was added into the miRNA recognition solution containing 1 µM P1, 0.1 U µL-1 KF polymerase, 0.1 U µL-1 Nt.BbvCI, and 100 µM dNTPs and then incubated in a centrifuge tube at 37 °C for 2 h. Subsequently, the reaction solution was transferred from the centrifuge tube to the circular paper on PES at room temperature for 1.5 h. After this, the circular paper on PES was folded onto the surface of SPE for electrochemical detection. For selectivity investigation, 10-5 M interference miRNA strands were used instead of 10-6 M miR-21, respectively. The fluorescent detection of miRNA was detailed in Section S1.3 (supporting information). The procedures for electrochemical detection of ALP and CEA were similar to that of miRNA, which were detailed in Section S1.4 and S1.5 (supporting information).


9


Page 10 of 45

2.5. Detection of MiRNA in Serum Samples. For human serum samples detection, different concentrations of miR-21 were dissolved in 10% human serum according to the standard addition method. Then 5 µL of spiked miR-21 serum solution was added into 45 µL miRNA recognition solution and incubated in a centrifuge tube at 37 °C for 2 h. The final miR-21 concentration in the recognition solution are 10, 100, and 1000 pM, respectively. Subsequently, the solution was dropped onto the circular paper on PES and then detected as described above. 2.6. Non-denaturing PAGE Analysis. The solution with different contents were added in 6 × loading buffer and loaded on the 8% non-denaturing polyacrylamide gel. The gel electrophoresis was carried out in 1× TBE buffer (89 mM Tris-Borate, 2.0 mM EDTA, pH 8.3) at 110 V for 45 min at room temperature. After being strained by GelRed dye solution for 30 min, the gel was imagined using the gel imaging system.

3. RESULTS AND DISCUSSION

3.1. Preparation and Characterization of PES. The foldable PES was prepared by assembling Fc-DNA functionalized paper and CNTs modified SPE. The paper was first


10

Page 11 of 45 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


cut to the desired dimensions and then functionalized with aldehyde groups (CHO-) through oxidating cellulose with periodate (IO4-) (Figure S1, supporting information).55 Next, the Fc-DNA with amino groups (NH2-) were immobilized on the surface of paper via Schiff base reaction, as shown in Figure 1. The SEM images revealed that the morphology of treated paper cellulose had no obvious change when compared to unmodified paper (Figure S2, supporting information). The functionalization of paper cellulose with aldehyde groups and Fc-DNA were demonstrated by X-ray photoelectron spectroscopy (XPS). For all of the three survey scans, sharp peaks of C1s and O1s at 285 and 532 eV were observed owing to the fact that cellulose was made up of C and O elements (Figure S3A, supporting information). In addition to C and O elements, the survey scan of Fc-DNA modified paper revealed the presence of N and P elements (curve iii), indicating the successful immobilization of Fc-DNA onto the paper surface. Compared with the high-resolution C1s XPS spectrum of untreated paper (Figure S3B, supporting information), the functionalization of paper with aldehyde groups was demonstrated by the appearance of new component peak at 288.4 eV belonging to the C=O in the spectrum of oxidated paper (Figure S3C, supporting information).


11


Page 12 of 45

A commercial SPE was used as a signal readout part of the foldable PES. To improve the affinity of working electrode to single strand DNA (ssDNA), the working electrode of SPE was modified with CNTs because of their excellent electrical conductivity and their ability of non-covalently adsorption of ssDNA through π-π stacking interactions.56 It is obviously that higher different pulse voltammogram (DPV) current was obtained after modification

(Figure

S4,

supporting

information),

indicating

the

improved

electrochemical response of SPE electrode toward the released Fc-DNA. Figure S5 (supporting information) revealed the surface morphology of the working electrode before and after modification. After modification, the surface was covered with cylindrical CNTs, which not only remarkably increased the surface area of the electrode, but also increased the affinity of the electrode toward ssDNA. Then, the Fc-DNA functionalized paper and a commercial SPE were stuck onto a soft plastic to assemble into a foldable PES device (Figure S6, supporting information). The prepared PES was used as a universal platform for diverse biomarkers assay. The whole assay includes two steps. The first step is the recognition of the target biomarker by rational designed DNA probe to output Mg2+-dependent DNAzyme strands through


12

Page 13 of 45 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


the polymerization/nicking reaction. The second step is the electrochemical response of PES to the released DNAzymes. It is noteworthy that no washing step was contained in the assays based on PES. 3.2. Principle of MiRNA Assay. Figure 2 illustrates the principle for miRNA assay by using lung cancer-specific miR-21 as a target biomarker. In the first step, miR-21 was incubated with its recognition solution that mainly consists of ingeniously designed ssDNA probe (P1), KF polymerase, and nicking endonuclease Nt.BbvCI. P1 contains a recognition sequence (domain I), which is complementary to the sequence of miR-21. Hybridization between miR-21 and P1 initiated the polymerization via the activity of KF to extend the 3’-end and form the double-stranded DNA (dsDNA) with recognition site for the Nt.BbvCI (domain II). By using the activity of Nt.BbvCI to cut one strand of the dsDNA, new replication site could be generated for KF, and the downstream Mg2+dependent DNAzyme strand (complementary to domain III) would be displaced and released. The cycle of polymerization, nicking, and displacement led to an isothermal exponential amplification and the generation of a large amount of DNAzyme strands. Then, the DNAzyme strands folded into the catalytically active loop structure and bound


13


Page 14 of 45

to Fc-DNA immobilized on the paper. With the assistant of Mg2+, they could catalyze the cleavage of Fc-DNA at the 3’-phosphoester bond of the ribonucleotide A (rA), resulting in the release of DNAzyme strands and cleaved Fc-labeled shorter ssDNA (Fc-SDNA). Then, the released DNAzyme could bind to another substrate to trigger a recycle cleavage process for the release of multiple Fc-SDNA from the paper, which could subsequently diffuse and adsorb to the surface of CNTs modified working electrode (CNTs-WE), generating a significantly increased DPV signal. However, in the absence of miR-21, the polymerization and release of DNAzyme could not be happen in the first step. As a result, the substrate Fc-DNA could not be cleaved for releasing Fc-SDNA from paper. Thus, no background signal of Fc was observed. Therefore, highly sensitive miRNA assay was achieved by monitoring the change of the electrochemical signal. 3.3. Feasibility of MiRNA Assay. The recognition of miR-21 in the first step was validated by non-denaturating polyacrylamide gel electrophoresis (PAGE) (Figure S7A, supporting information). The appearance of new electrophoresis bands in lines iv and iii indicated the formation of P1-miR-21 complex and the extension of the complex in the presence of KF. After the addition of Nt.BbvCI, a new dim band, which was separated


14

Page 15 of 45 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


from the bright band of the extended P1-miR-21 complex (line ii), appeared at the same electrophoresis distance as that of DNAzyme strands (line i), demonstrating the converting of miR-21 recognition to the generation of the DNAzyme strands. Furthermore, DPV response of PES toward recognition solutions with different contents was also recorded and presented in Figure S7B (supporting information). Obviously, a strong electrochemical signal was observed when target miR-21 was incubated with the recognition solution containing KF, and Nt.BbvCI (curve iv). In the absence of KF, Nt.BbvCI, or miR-21, the DNAzyme strand could not be generated or released. As a result, no current was obtained without any one of them (curves i, ii, iii), suggesting that PES can achieve zero-background detection of miRNA. 3.4. Reasons for Zero-background Detection. Two reasons are considered to be responsible for achieving zero-background detection. One reason is that Fc-DNA can not be diffused or adsorbed to the surface of working electrode without the cleavage by DNAzyme, because Fc-DNA is immobilized on a piece of paper and isolated to the electrode surface. This isolation avoids not only the non-specific adsorption-induced background signals existed in most of the heterogeneous electrochemical methods, but


15


Page 16 of 45

also the non-completely inhibited diffusion-induced background signals presented in homogeneous electrochemical methods.38-40,42 Another reason is the high specificity of the recognition solution to output DNAzyme strands by the target-triggered polymerization/nicking reaction. The high specificity is likely due to the fact that the recognition solution does not contain DNAzyme strand and this strand cannot be synthesized in the absence of target. In our previous work, we found that the addition of DNAzyme strands to the sensing system produced slightly increased background signals, even though the DNAzyme strands were blocked to an inactive structure.[57] This probably attributed to the presence of thermodynamic competition balance between the active and inactive DNAzyme conformation. The small amount of active DNAzyme strands generated slightly increased background signals. Therefore, we anticipated that the absence of DNAzyme strand in the recognition solution could solve the problem of background signals generated by the DNAzyme conformational change. To demonstrate this speculation, a fluorescent strategy was designed, in which a fluorophore (Cy5) and a quencher (BHQ2) labeled substrate FQ-DNA was used instead of Fc-DNA (Figure S8, supporting information). In this strategy, weak fluorescence was


16

Page 17 of 45 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


observed from FQ-DNA solution (Figure S9A, curve i, supporting information), owing to the fact that the fluorescence of Cy5 cannot be completely quenched by BHQ2. If any one of miR-21, KF, or Nt.BbvCI was absent, the fluorescence was as weak as that of FQ-DNA solution (Figure S9B, supporting information), suggesting the absence of DNAzyme strand for increasing the background signals. In contrast, strong fluorescence was observed from the recognition solution with addition of target miR-21 (Figure S9A, supporting information), indicating the high specificity of miRNA-triggered generation and releasing of DNAzyme strands. 3.5. Analytical Performance for MiRNA Detection. We also optimized some experimental conditions that can affect the detection performance (detailed in section S2,

supporting

information).

Under

the

optimal

experimental

conditions,

the

electrochemical response of PES toward miR-21 was investigated by monitoring the DPV signals in the presence of miR-21 with different concentrations. As displayed in Figure 3A, the DPV currents of PES gradually increased with elevated miR-21 concentration, suggesting that the DNAzyme-catalyzed cleavage and release of FcSDNA from paper is highly dependent on miR-21 concentration. Figure 3B showed the


17


Page 18 of 45

relationship between the peak currents and the concentrations of miR-21. Evidently, miR-21 could be directly assayed with a range from 1 fM to 1 μM. For quantitative assay, the DPV peak currents exhibited a good linear relationship with the logarithm of miR-21 concentration. The correlation equation is ip = -2.42 + 5.33 log CmiRNA (R2 = 0.987), where ip is the peak current value and CmiRNA is the concentration of miR-21 (Figure 3C). The selectivity of this strategy for miR-21 assay was further tested by detecting different miRNA sequences, including miR-141, miR-155, miR-199a, miR-143, miR-21 with five-, three-, and one-base mismatched, as well as miR-21 without mismatch. As shown in Figure 3D, high DPV current was only detected when miR-21 was present (curve viii), whereas very weak DPV currents were obtained from the one-, three-, and five-base mismatched miRNA (curves v-vii), and even no current was observed when mismatched miRNA (curves i-iv) were present. These results demonstrate the high selectivity of the present recognition strategy for miRNA assay, which is even able to distinguish one base mismatch. Moreover, the stability of PES was also investigated by recording the electrochemical response of the sensor toward 1 μM, 10 nM, and 100 pM


18

Page 19 of 45 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


miR-21 after different days. For all of the three miRNA concentrations, the DPV signals were only slightly decreased after stored PES at room temperature for 35 days (Figure S13, supporting information), indicating the high stability of the prepared PES. 3.6. Detection of MiRNA in Serum Sample. The applicability for testing miRNA in complex biological matrixes was investigated by detecting the recoveries of miR-21 spiked in 10% diluted human serum samples. The results were analyzed and summarized in Table S2 (Section S3 in supporting information). The measured miR-21 concentrations in the spiked serum samples were statistically close to the added miR-21 concentrations with recoveries from 93 % to 102%, suggesting the potential applicability of PES for clinical miRNA assay in human serum samples. 3.7. ALP Assay Based on PES. Since the response of PES to DNAzyme is only involved in the second step, this sensor can be easily adopted to detect different biomarkers by simply changing the recognition probe. To demonstrate this, a new recognition probe HP1 was designed and applied for the detection of ALP. As illustrated in Figure 4A, HP1 has a 3’-phosphorylated hairpin structure with a protruding configuration at 5’-terminus. The protruding strand consists of two domains, which are


19


Page 20 of 45

the same sequence to domains II and III in P1. When ALP was present, the phosphoryl group at the 3’ terminus of HP1 was removed, resulting in the generation of active substrate for KF. This initiated the following polymerization/nicking reaction to release DNAzyme strands, which then produced readout signal on PES (Figure 4B, curve iv). However, in the absence of ALP, HP1 was an inactive substrate for KF, which could not trigger the following reactions. As a result, no signal was observed on PES (curve iii). Moreover, KF and Nt.BbvCI also played important roles in this assay and no current was observed when either of them was absent (curve i, ii). These results indicated the feasibility of the proposed PES for ALP assay based on PES. Figure 4C revealed that DPV currents of PES gradually increased as the concentration of ALP increased. The relationship between the peak currents and ALP concentrations was shown in Figure 4D and Figure S14 (supporting information). This assay exhibited a good linear relationship between the peak currents and the logarithm of ALP concentrations ranging from 1 to 105 mU L-1 with the correlation equation of ip = 1.30 + 7.03 log CALP (R2 = 0.981) (the inset of Figure 4D). The selectivity of this assay for ALP activity was further investigated by using lysozyme as an interfering enzyme


20

Page 21 of 45 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


and bovine serum albumin (BSA), streptavidin, hemoglobin as interfering proteins. As shown in Figure 4E, no DPV current was observed in the presence of interfering enzyme and proteins, whereas a high DPV current was observed when ALP was present. Therefore, this assay exhibited high selectivity for distinguishing ALP from other interfering enzyme and proteins. 3.8. CEA Assay Based on PES. To further demonstrate the versatility of PES, we designed another recognition strategy that undergone conformational change to trigger the polymerization/nicking reaction and the following electrochemical response. In this case, CEA was employed as a target biomarker, which could be recognized by an ingeniously designed self-blocked hairpin probe (HP2, containing the aptamer sequence marked as domain IV). As illustrated in Figure 5A, the binding between CEA and its aptamer sequence triggered a conformational change of HP2, exposing its occluded stem region (domain V). The exposed domain V of HP2 could hybridize with domain IV’ of another rational designed ssDNA probe (P2) to form dsDNA, which initiated the subsequent polymerization/nicking reaction, leading to the generation of DNAzyme strands. Then, the released DNAzyme strands produced an amplified signal response


21


Page 22 of 45

on PES. In the absence of CEA, HP2 and P2 were designed to kinetically hinder any spontaneous interaction between them. As a result, no obvious DPV current was observed in the absence of CEA, which is similar with the case when KF or Nt.BbvCI was absent (Figure 5B, curve i-iii). Comparatively, strong DPV current was obtained when CEA was present (curve iv). The electrochemical response of PES toward different CEA concentrations was shown in Figure 5C. As expected, remarkable increases in the DPV currents were clearly observed with the increase of CEA concentrations. The resulting calibration curve showed that CEA concentrations ranging from 1 to 1500 fg mL-1 could be directly detected by this strategy (Figure 5D and Figure S15, supporting information). Furthermore, the inset of Figure 5D revealed a good linear correlation between the DPV peak currents and the logarithm of CEA concentrations with the correlation equation of

ip = 4.14 + 4.65 log CCEA (R2 = 0.984). The selectivity of the strategy for CEA assay was evaluated by using interfering proteins to replace CEA, which indicated that only CEA induced a significant DPV current, while the interfering proteins had negligible effect on DPV current (Figure 5E).


22

Page 23 of 45 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. CONCLUSIONS

In summary, we designed a universal PES for sensitive and selective detection of diverse

biomarkers

based

on

the

isothermal

signal

amplification

via

the

polymerization/nicking reaction and DNAzyme-catalyzed cleavage of Fc-DNA from paper. The assays based on PES possess several unique features. Firstly, PES has been demonstrated to be a universal sensing platform for a variety of biomarkers due to the flexible design of the recognition probe. Secondly, the sensing strategies based on PES have the merit of zero-background current, leading to highly sensitive bioassays. Thirdly, the prepared PES exhibits desired stability. Additionally, the bioassays based on PES exhibit high selectivity for target biomarkers. Thus, the proposed PES holds great potential for POC diagnosis in resource-limited settings.

ASSOCIATED CONTENT Supporting Information


23


Page 24 of 45

Experimental Section, Optimization of Experimental Conditions, Supporting Tables, Supporting Figures, and References.

AUTHOR INFORMATION *Corresponding Author Tel/Fax: (86) 53286080855 E-mail address: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 21775083, 21575074, and 31501570), the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201710), the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (No. 663-1114304), the Special Foundation for Distinguished Taishan Scholar of Shandong Province (No.


24

Page 25 of 45 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


ts201511052), and Major Program of Shandong Province Natural Science Foundation (No. ZR2018ZC0127).

REFERENCES (1) Daar, A. S.; Thorsteinsdottir, H.; Martin, D. K.; Smith, A. C.; Nast, S.; Singer, P. A. Top Ten Biotechnologies for Improving Health in Developing Countries. Nat. Genet. 2002, 32, 229–232. (2) Turner, A. P. F. Biosensors: Sense and Sensibility. Chem. Soc. Rev. 2013, 42, 3184–3196. (3) Ji, H. W.; Wu, L.; Pu, F.; Ren, J. S.; Qu, X. G. Point-of-Care Identification of Bacteria Using Protein-Encapsulated Gold Nanoclusters. Adv. Healthc. Mater. 2018, 7, 1701370. (4) Wu, L.; Qu, X. G. Cancer Biomarker Detection: Recent Achievements and Challenges. Chem. Soc. Rev. 2015, 44, 2963–2997. (5) Gong, M. M.; Sinton, D. Turning the Page: Advancing Paper-Based Microfluidics for Broad Diagnostic Application. Chem. Rev. 2017, 117, 8447–8480.


25


Page 26 of 45

(6) Zhang, J. J.; Lu, Y. Biocomputing for Portable, Resettable, and Quantitative Pointof-Care Diagnostics: Making the Glucose Meter a Logic-Gate Responsive Device for Measuring Many Clinically Relevant Targets. Angew. Chem. Int. Ed. 2018, 57, 9702– 9706. (7) Comer, J. P. Semiquantitative Specific Test Paper for Glucose in Urine. Anal.

Chem. 1956, 28, 1748–1750. (8) Free, A. H.; Adams, E. C.; Kercher, M. L.; Free, H. M.; Cook, M. H. Simple Specific Test for Urine Glucose. Clin. Chem. 1957, 3, 163–168. (9) Wang, C. C.; Hennek, J. W.; Ainla, A.; Kumar, A. A.; Lan, W. J.; Im, J.; Smith, B. S.; Zhao, M. X.; Whitesides, G. M. A Paper-Based “Pop-up” Electrochemical Device for Analysis of Beta-Hydroxybutyrate. Anal. Chem. 2016, 88, 6326–6333. (10) Yang, X. S.; Shi, D. M.; Zhu, S. M.; Wang, B. J.; Zhang, X. J.; Wang, G. F. Portable Aptasensor of Aflatoxin B1 in Bread Based on a Personal Glucose Meter and DNA Walking Machine. ACS Sens. 2018, 3, 1368–1375.


26

Page 27 of 45 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


(11) Yamada, K.; Shibata, H.; Suzuki, K.; Citterio, D. Toward Practical Application of Paper-Based Microfluidics for Medical Diagnostics: State-of-the-Art and Challenges.

Lab Chip 2017, 17, 1206–1249. (12) Xu, G. L.; Nolder, D.; Reboud, J.; Oguike, M. C.; van Schalkwyk, D. A.; Sutherland, C. J.; Cooper, J. M. Paper-Origami-Based Multiplexed Malaria Diagnostics from Whole Blood. Angew. Chem. Int. Ed. 2016, 55, 15250–15253. (13) Liu, M.; Hui, C. Y.; Zhang, Q.; Gu, J.; Kannan, B.; Jahanshahi-Anbuhi, S.; Filipe, C. D. M.; Brennan, J. D.; Li, Y. F. Target-Induced and Equipment-Free DNA Amplification with a Simple Paper Device. Angew. Chem. Int. Ed. 2016, 55, 2709–2713. (14) Wang, F. A.; Liu, X. Q.; Willner, I. DNA Switches: From Principles to Applications Molecular Engineering of DNA: Molecular Beacons. Angew. Chem. Int. Ed. 2014, 53, 1098–1129. (15) Zhang, H. Q.; Li, F.; Dever, B.; Li, X. F.; Le, X. C. DNA-Mediated Homogeneous Binding Assays for Nucleic Acids and Proteins. Chem. Rev. 2013, 113, 2812–2841.


27


Page 28 of 45

(16) Wang, K. M.; Tang, Z. W.; Yang, C. J.; Kim, Y.; Fang, X. H.; Li, W.; Wu, Y. R.; Medley, C. D.; Cao, Z. H.; Li, J.; Colon, P.; Lin, H.; Tan, W. H. Molecular Engineering of DNA: Molecular Beacons. Angew. Chem. Int. Ed. 2009, 48, 856–870. (17) Pu, F.; Ren, J. S.; Qu, X. G. Nucleobases, Nucleosides, and Nucleotides: Versatile Biomolecules for Generating Functional Nanomaterials. Chem. Soc. Rev. 2018, 47, 1285–1306. (18) Tram, K.; Kanda, P.; Salena, B. J.; Huan, S. Y.; Li, Y. F. Translating Bacterial Detection by DNAzymes into a Litmus Test. Angew. Chem. Int. Ed. 2014, 53, 12799– 12802. (19) Freage, L.; Wang, F.; Orbach, R.; Willner, I. Multiplexed Analysis of Genes and of Metal Ions Using Enzyme/DNAzyme Amplification Machineries. Anal. Chem. 2014, 86, 11326−11333. (20) Cha, T. G.; Pan, J.; Chen, H. R.; Robinson, H. N.; Li, X.; Mao, C. D.; Choi, J. H. Design Principles of DNA Enzyme based Walkers: Translocation Kinetics and PhotoRegulation. J. Am. Chem. Soc. 2015, 137, 9429–9437.


28

Page 29 of 45 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


(21) Liu, X. J.; Xu, N. N.; Gai, P. P.; Li, F. Triplex DNA Formation-Mediated Strand Displacement Reaction for Highly Sensitive Fluorescent Detection of Melamine. Talanta 2018, 185, 352–358. (22) Hwang, K.; Wu, P. W.; Kim, T.; Lei, L.; Tian, S. L.; Wang, Y. X.; Lu, Y. Photocaged DNAzymes as a General Method for Sensing Metal Ions in Living Cells.

Angew. Chem. Int. Ed. 2014, 53, 13798–13802. (23) Mascini, M.; Palchetti, I.; Tombelli, S. Nucleic Acid and Peptide Aptamers: Fundamentals and Bioanalytical Aspects. Angew. Chem. Int. Ed. 2012, 51, 1316–1332. (24) Meng, H. M.; Liu, H.; Kuai, H. L.; Peng, R. Z.; Mo, L. T.; Zhang, X. B. Aptamerintegrated DNA Nanostructures for Biosensing, Bioimaging and Cancer Therapy. Chem.

Soc. Rev. 2016, 45, 2583–2602. (25) Hu, R.; Zhang, X.; Xu, Q.; Lu, D. Q.; Yang, Y. H.; Xu, Q. Q.; Ruan, Q.; Mo, L. T.; Zhang, X. B. A Universal Aptameric Biosensor: Multiplexed Detection of Small Analytes via Aggregated Perylene-Based Broad-Spectrum Quencher. Biosens. Bioelectron. 2017, 92 ,40– 46.


29


Page 30 of 45

(26) Zhao, Y. X.; Chen, F.; Li, Q.; Wang, L. H.; Fan, C. H. Isothermal Amplification of Nucleic Acids. Chem. Rev. 2015, 115, 12491–12545. (27) Reid, M. S.; Le, X. C.; Zhang, H. Q. Exponential Isothermal Amplification of Nucleic Acids and Assays for Proteins, Cells, Small Molecules, and Enzyme Activities– An EXPAR Example. Angew. Chem. Int. Ed. 2018, 57, 11856–11866. (28) Brown, C. W.; Lakin, M. R.; Horwitz, E. K.; Fanning, M. L.; West, H. E.; Stefanovic, D.; Graves, S. W. Signal Propagation in Multi-Layer DNAzyme Cascades Using Structured Chimeric Substrates. Angew. Chem. Int. Ed. 2014, 53, 7183–7187. (29) Chen, J. H.; Zhou, S. G.; Wen, J. L. Disposable Strip Biosensor for Visual Detection of Hg2+ Based on Hg2+-Triggered Toehold Binding and Exonuclease IIIAssisted Signal Amplification. Anal. Chem. 2014, 86, 3108–3114. (30) Hui, C. Y.; Liu, M.; Li, Y. F.; Brennan, J. D. A Paper Sensor Printed with Multifunctional Bio/Nano Materials. Angew. Chem. Int. Ed. 2018, 57, 4549–4553. (31) Liu, H. X.; Zhou, X. M.; Liu, W. P.; Yang, X. K.; Xing, D. Paper-Based Bipolar Electrode Electrochemiluminescence Switch for Label-Free and Sensitive Genetic Detection of Pathogenic Bacteria. Anal. Chem. 2016, 88, 10191–10197.


30

Page 31 of 45 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


(32) Rackus, D. G.; Shamsi, M. H.; Wheeler, A. R. Electrochemistry, Biosensors and Microfluidics: A Convergence of Fields. Chem. Soc. Rev. 2015, 44, 5320–5340. (33) Wang, Y.; Luo, J. P.; Liu, J. T.; Li, X. R.; Kong, Z.; Jin, H. Y.; Cai, X. X. Electrochemical Integrated Paper-Based Immunosensor Modified with Multiwalled Carbon Nanotubes Nanocomposites for Point-of-Care Testing of 17β-Estradiol.

Biosens. Bioelectron. 2018, 107, 47–53. (34) Glavan, A. C.; Christodouleas, D. C.; Mosadegh, B.; Yu, H. D.; Smith, B. S.; Lessing, J.; Fernández-Abedul, M. T.; Whitesides, G. M. Folding Analytical Devices for Electrochemical ELISA in Hydrophobic RH Paper. Anal. Chem. 2014, 86, 11999–12007. (35) Labib, M.; Sargent, E. H.; Kelley, S. O. Electrochemical Methods for the Analysis of Clinically Relevant Biomolecules. Chem. Rev. 2016, 116, 9001–9090. (36) Ding, J. W.; Li, B. W.; Chen, L. X.; Qin, W. A Three-Dimensional Origami PaperBased Device for Potentiometric Biosensing. Angew. Chem. Int. Ed. 2016, 55, 13033– 13037. (37) Hu, J. B.; Stein, A.; Bühlmann, P. A Disposable Planar Paper-Based Potentiometric Ion-Sensing Platform. Angew. Chem. Int. Ed. 2016, 55, 7544–7547.


31


Page 32 of 45

(38) Yang, Y. M.; Liu, X. J.; Wu, M.; Wang, X. Z.; Hou, T.; Li, F. Electrochemical Biosensing Strategy for Highly Sensitive Pesticide Assay Based on Mercury IonMediated

DNA

Conformational

Switch

Coupled

with

Signal

Amplification

by

Hybridization Chain Reaction. Sens. Actuators B: Chem. 2016, 236, 597–604. (39) Liu, X. J.; Song, M. M.; Hou, T.; Li, F. Label-Free Homogeneous Electroanalytical Platform for Pesticide Detection Based on Acetylcholinesterase-Mediated DNA Conformational Switch Integrated with Rolling Circle Amplification. ACS Sens. 2017, 2, 562–568. (40) Liu, X. J.; Li, W.; Hou, T.; Dong, S. S.; Yu, G. H.; Li, F. Homogeneous Electrochemical Strategy for Human Telomerase Activity Assay at Single-Cell Level Based on T7 Exonuclease-Aided Target Recycling Amplification. Anal. Chem. 2015, 87, 4030–4036. (41) Bartel, D. P. MicroRNAs: Target Recognition and Regulatory Functions. Cell 2009, 136, 215–233.


32

Page 33 of 45 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


(42) Hou, T.; Li, W.; Liu, X. J.; Li, F. Label-Free and Enzyme-Free Homogeneous Electrochemical Biosensing Strategy Based on Hybridization Chain Reaction: A Facile, Sensitive and Highly Specific MicroRNA Assay. Anal. Chem. 2015, 87, 11368–11374. (43) Rodriguez, N. M.; Linnes, J. C.; Fan, A.; Ellenson, C. K.; Pollock, N. R.; Klapperich, C. M. Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens. Anal. Chem. 2015, 87, 7872–7879. (44) Dong, H. F.; Lei, J. P.; Ding, L.; Wen, Y. Q.; Ju, H. X.; Zhang, X. J. MicroRNA: Function, Detection, and Bioanalysis. Chem. Rev. 2013, 113, 6207–6233. (45) Sawyers, C. L. The Cancer Biomarker Problem. Nature 2008, 452, 548–552. (46) Li, J. B.; Tan, S.; Kooger, R.; Zhang, C. Y.; Zhang, Y. MicroRNAs as Novel Biological Targets for Detection and Regulation. Chem. Soc. Rev. 2014, 43, 506–517. (47) Deng, R. J.; Tang, L. H.; Tian, Q. Q.; Wang, Y.; Lin, L.; Li, J. H. Toehold-initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells.

Angew. Chem. Int. Ed. 2014, 53, 2389–2394.


33


Page 34 of 45

(48) Coleman, J. E. Structure and Mechanism of Alkaline Phosphatase. Annu. Rev.

Biophys. Biomol. Struct. 1992, 21, 441–483. (49) Christenson, R. H. Biochemical Markers of Bone Metabolism: An Overview. Clin.

Biochem. 1997, 30, 573–593. (50) Zhang, L. F.; Hou, T.; Li, H. Y.; Li, F. A Highly Sensitive Homogeneous Electrochemical Assay for Alkaline Phosphatase Activity Based on Single Molecular Beacon-Initiated T7 Exonuclease Mediated Signal Amplification. Analyst 2015, 140, 4030–4036. (51) Qian, Z. S.; Chai, L. J.; Tang, C.; Huang, Y. Y.; Chen, J. R.; Feng, H. Carbon Quantum Dots-Based Recyclable Real-Time Fluorescence Assay for Alkaline Phosphatase with Adenosine Triphosphate as Substrate. Anal. Chem. 2015, 87, 2966– 2973. (52) Han, J.; Li, Y.; Feng, J.; Li, M.; Wang, P.; Chen, Z.; Dong, Y. J. A Novel Sandwich-Type Immunosensor for Detection of Carcino-Embryonic Antigen using Silver Hybrid Multiwalled Carbon Nanotubes/Manganese Dioxide. Electroanal. Chem. 2017,

786, 112–119.


34

Page 35 of 45 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


(53) Ge, L.; Wang, W. X.; Hou, T.; Li, F. A Versatile Immobilization-Free Photoelectrochemical Biosensor for Ultrasensitive Detection of Cancer Biomarker Based on Enzyme-Free Cascaded Quadratic Amplification Strategy. Biosens.

Bioelectron. 2016, 77, 220–226. (54) Wang, W. X.; Ge, L.; Sun, X. M.; Hou, T.; Li, F. Graphene-Assisted Label-Free Homogeneous Electrochemical Biosensing Strategy Based on Aptamer-Switched Bidirectional DNA Polymerization. ACS Appl. Mater. Interfaces 2015, 7, 28566–28575. (55) Zhang, H.; Zhao, Z.; Lei, Z.; Wang, Z. X. Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance.

Anal. Chem. 2016, 88, 11358–11363. (56) Wang, Y.; Luo, J. P.; Liu, J. T.; Li, X. R.; Jin, H. Y.; Cai, X. X. Electrochemical Integrated Paper-Based Immunosensor Modified with Multi-Walled Carbon Nanotubes Nanocomposites for Point-of-Care Testing of 17β-estradiol. Biosens. Bioelectron. 2018,

107, 47–53.


35


Page 36 of 45

(57) Liu, X. J.; Xu, N. N.; Gai, P. P.; Li, F. Triplex DNA Formation-Mediated Strand Displacement Reaction for Highly Sensitive Fluorescent Detection of Melamine. Talanta 2018, 185, 352–358.


36

Page 37 of 45 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


FIGURES CAPTIONS Figure 1. Schematic illustration of the progress for immobilizing Fc-DNA on paper: functionalizing paper with CHO- groups and then immobilizing NH2-labeled Fc-DNA on paper through Schiff base reaction. Figure 2. Schematic illustration of the principle for miRNA assay based on PES: the recognition of miR-21 in the first step (left); electrochemical response of PES to the released DNAzymes in the second step (lower right); CNTs-WE before and after adsorption of Fc-SDNA (upper right). Figure 3. (A) DPV currents of PES response to increased miR-21 concentrations: (i-xii) 0, 10-15, 10-14, 10-13, 10-12, 10-11, 10-10, 10-9, 10-8, 10-7, 0.5×10-7, and 10-6 M, respectively. (B) Calibration curve corresponding to the peak current versus miR-21 concentration. Inset: calibration curve of low miR-21 concentrations in dot square frame. (C) The linear relationship between the peak current and lgCmiRNA. (D) Selectivity investigation by detecting a few interference miRNA strands: (i-vii) miR-141, miR-155, miR-199a, miR143, miR-21 with five-, three-, one-mismatch, and (viii) target miR-21.


37


Page 38 of 45

Figure 4. (A) Schematic illustration of the principle for ALP assay. (B) DPV currents of PES response to HP1 solution mixed with (i) Nt.BbvCI and ALP; (ii) KF and ALP; (iii) Nt.BbvCI and KF; (iv) Nt.BbvCI, KF, and ALP. (C) DPV currents of PES response to increased ALP concentrations: (i–xii) 0, 1, 5, 10, 50, 100, 500, 1000, 5000, 104, 5×104, 105 mU L-1, respectively. (D) Calibration curve corresponding to the peak current versus ALP concentration. Inset: the linear relationship between the peak current and lgCALP. (E) Selectivity investigation by detecting a few enzyme and proteins: (i-iv) lysozyme, BSA, streptavidin, hemoglobin, and (v) target ALP. Figure 5. (A) Schematic illustration of the principle for CEA assay. (B) DPV currents of PES response to HP2 and P2 solution mixed with (i) Nt.BbvCI and ALP; (ii) KF and ALP; (iii) Nt.BbvCI and KF; (iv) Nt.BbvCI, KF, and ALP. (C) DPV currents of PES response to increased CEA concentrations: (i–xii) 0, 1, 1.5, 3, 5, 15, 30, 50, 150, 300, 500, 1500 fg mL-1, respectively. (D) Calibration curve corresponding to the peak current versus CEA concentration. Inset: the linear relationship between the peak current and lgCCEA. (E) Selectivity investigation by detecting a few proteins: (i-iv) BSA, insulin, prostate specific antigen, fibronectin, and (v) target CEA.


38

Page 39 of 45 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



39


Page 40 of 45

Figure 1.


40

Page 41 of 45 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


Figure 2.


41


Page 42 of 45

Figure 3.


42

Page 43 of 45 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


Figure 4.


43


Page 44 of 45

Figure 5.


44

Page 45 of 45 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


For TOC only


45

A Universal Paper-based Electrochemical Sensor for Zero

Recommend Documents