Ultra-Sensitive Colorimetric Assay System Based on the Hybridization

Subscriber access provided by University of Otago Library

Article

Ultra-Sensitive Colorimetric Assay System Based on the Hybridization Chain Reaction-Triggered Enzyme Cascade Amplification Shasha Lu, Tao Hu, Shuang Wang, Jian Sun, and Xiurong Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13201 • Publication Date (Web): 07 Dec 2016 Downloaded from http://pubs.acs.org on December 12, 2016

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 free 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 accessible to all readers and 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.

ACS Applied Materials & Interfaces 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 34

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

Ultra-Sensitive Colorimetric Assay System Based on the Hybridization Chain ReactionTriggered Enzyme Cascade Amplification Shasha Lu,†, ‡ Tao Hu,†, ‡ Shuang Wang,†, ‡ Jian Sun,† Xiurong Yang*, †

†

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied

Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China

‡

University of Science and Technology of China, Hefei, Anhui 230026, China

KEYWORDS: Hybridization Chain Reaction, Amplification, Glucose oxidase, Horseradish peroxidase, Cascade.

ABSTRACT: A versatile and ultra-sensitive colorimetric detection platform has been developed based on the hybridization chain reaction (HCR)-triggered enzyme cascade amplification in this work. The proposal involves the preparation of two different hairpin DNA strands consisting of the H1, modified with glucose oxidase (GOx-H1) and H2, modified with horseradish peroxidase (HRP-H2). The H1 and H2 were comprised of complementary sequence of nucleic acid target (T) and interlaced complementary stem-loop sequences. In the nucleic acid detection, the hybridization of T and its complementary sequence induces the autonomous assembly of GOx-H1 and HRP-H2 through the predictable HCR, accompanied by the formation of GOx/HRP enzyme pairs with a multiple enzymatic cascade. In contrast to the crude mixture of free GOx-H1 and HRP-H2, the catalytic performance of enzyme cascade 1

ACS Paragon Plus Environment


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

reaction has been significantly enhanced, which can be determined by monitoring the absorbance change of 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS2-), a typical substrate with hydrogen peroxide for the HRP. Furthermore, this platform can be utilized in the assay of biological substances by the introduction of corresponding aptamer (Apt), complementary strands (Com) and an assistant hairpin DNA strand (HAssist). In view of the signal amplification of HCR and the enhanced catalytic performance of cascaded enzymes, our colorimetric assay system exhibits excellent sensitivity, and the detection limits have been calculated to be 5.2 fM and 0.8 pM for the nucleic acid target (T as a model) and biological substances (ATP as a model), respectively.

INTRODUCTION

The sensitive and selective assay of nucleic acid, biological molecules, and proteins, especially at low physiological levels, is being significant in life sciences due to their pivotal role in mutation identification, gene therapy, and tumor early screen and other life activities.13

In this regard, a variety of amplifying techniques have been developed for the fast and

reliable measurements, such as polymerase chain reaction(PCR),4 hybridization chain reaction (HCR),5, 6 rolling circle amplification (RCA),7-9 strand displacement amplification (SDA),10, 11 and catalyzed hairpin assembly (CHA).12, 13 Recent advances in entropy-driven catalysis,14 in particular, HCR has gained comprehensive attention in terms of an autonomous isothermal replication process triggered by a target DNA,15 without any polymerase or other 2


Page 2 of 34

Page 3 of 34

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


enzymes, and performing experiments under mild conditions.16 For instance, Tang et al. developed a sensitive and real-time detection assay for nucleic acids by combining the HCR with quartz crystal microbalance with dissipation monitoring (QCM-D).17 Huang et al. linked spatially sensitive fluorescence signal pyrene molecules to hairpin DNAs and used the fluorescence method to detect nucleic acids.18 In addition, through the enzyme-free autonomous assembly of DNAzyme subunits, Willner’s group developed a series of amplified detection assays for sensing DNA with high sensitivity.19,

20

Therefore, it is

suggested that HCR shows great potential in the field of amplification detection and has been already employed in several nucleic acids and small biological molecules detection platforms until now.

The enzyme cascade reaction, as another significant signal amplification strategy, is often involved in the high-sensitive nucleic acid detection.21,

22

In a typical case, the reaction

product of one type of enzyme can be used as a substrate for another enzyme. Once the enzymes were organized in appropriate spatial arrangement, the cascade reaction might occur specifically and the catalytic performance would be efficiently improved by avoiding subsidiary reactions.23 In the past decades, inspired by a series of continuous enzymatic reactions in living organisms, DNA nanostructures,24 dendritic charged polymer,25 metal– organic frameworks,26 and hydrogels27 were all used in the construction of scaffolds for enzyme cascade reaction. Another notable advantage of enzyme cascade reactions is that the characteristic absorption peak of their colored end-products could be used as output signal,22 3



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

providing a reliable method for quantitative detection. Willner’s group has made great efforts and already done several outstanding works based on the enzyme cascade reaction. For example, the assembly of the topologically programmed DNA scaffolds would effectively active the bienzyme cascade reaction, during which either enzymes has been attached to the DNA scaffolds.28 Furthermore, by means of the rolling circle amplification (RCA) process, they have handled the ordered assembly of enzymes for the synthesis of metallic nanowires on the DNA scaffold, which was generated by the sequence-specific nucleic acids.29 However, there are almost no reports focusing on the photochemistry biosensor based on the enzyme cascade amplification reaction triggered by the HCR.

Inspired by the dual amplification ability of HCR and enzyme cascade reaction, we developed a versatile and ultra-sensitive strategy for colorimetric detection by means of a typical enzyme cascade system (glucose oxidase/horseradish peroxidase, GOx/HRP). This platform was first applied in the detection of nucleic acid target (T, as a model) and the overall strategy was illustrated in Scheme 1. (1) The enzymes and two kinds of hairpin DNA strands were crosslinked by bis (sulfosuccinimidyl) suberate (BS3) to form the enzymefunctionalized hairpin DNA strands (GOx-H1/HRP-H2). (2) The presence of T would trigger the HCR of GOx-H1 and HRP-H2, generate the long double stranded DNA frameworks consisting of T-(H1·H2)n, and lead to the multiple cascades of GOx and HRP on the HCR chains. (3) Upon the addition of glucose and 2, 2'–azino–bis (3-ethylbenzothiazoline-6sulphonic acid) (ABTS2-), the glucose will be oxidized by GOx and yield gluconic acid and 4


Page 4 of 34

Page 5 of 34

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


H2O2 as products. And then the as-formed H2O2 acts as the substrate for HRP that oxidizes the ABTS2- to the colored product, ABTS•- (λ= 414 nm).22 In this regard, the produce rate of the ABTS•- is directly related to the catalytic performance of enzyme cascade reaction, likewise is indirectly related to the concentration of T (i.e., the quantity of the generated T(H1·H2)n DNA frameworks). Therefore, the absorbance changes at 414 nm (i.e., the quantity of the generated ABTS•-) could be used to quantify the concentration of T. The integration of the HCR signal amplification performance and enhanced catalytic performance of enzyme cascade reaction will lead to excellent sensitivity. More significantly, the strategy could also be applied to the detection of biological substances (ATP, as a model) with the aid of the corresponding aptamer. In addition to ultrasensitive and universal, our detection platform also had the characteristics of simplicity, fast speed and inexpensiveness.

Scheme 1. Schematic illustration of the detection of nucleic acid target (T). EXPERIMENTAL SECTION

5



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

Chemicals and Apparatus. GOx, HRP, glucose, ABTS2-, BS3, adenosine triphosphate (ATP), adenosine diphosphate (ADP), triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP) were all obtained from Sigma-Aldrich (USA). The ultrapure water (18.2 MΩ) was used throughout all the experiments. All synthetic DNA oligonucleotides were ordered from Sangon Biotechnology Co.Ltd. (Shanghai, China), and the sequences were listed in Table 1. The underlined parts of all kinds of hairpin DNA strands were the sticky ends and loops of them respectively. Table 1 The sequences of DNA strands Name

Sequence

T

5’-GCCCGAAGCCGCAAC-3’

H1

5’-NH2C6-GTTGCGGCTTCGGGCCCAGAAGCCCGAAGC-3’

H2

5’-GCCCGAAGCCGCAACGCTTCGGGCTTCTGG-NH2C6-3’

Apt

5’-ACCTGGGGGAGTATTGCGGAGGAAGGT-3’

Com

5’-ACCTTCCTCCGCAATACTCCCCCAGGT-3’

HAssist

5’-CTCCGCAATACTCCCACCTGGGGGAGTATTGCGGAGGAAGGT-3’

H1 (ATP)

5’-NH2C6-GGGAGTATTGCGGAGTTTTTTCTCCGCAAT-3’

H2 (ATP)

5’-CTCCGCAATACTCCCATTGCGGAGAAAAAA-NH2C6-3’

UV-vis absorption measurements were carried out on a Cary 50 UV-vis spectrophotometer (Varian) and the circular dichroism (CD) spectral measurements were performed on a Jasco J-820 Circular Dichroism Spectra polarimeter (Tokyo, Japan). The atomic force microscopy (AFM) imaging was performed at tapping mode in air using Dimension Icon SPM (Bruker, Germany). The MALDI–TOF mass spectra were carried out by an autoflex III smart beam MALDI-TOF/TOF Mass spectrometer (Bruker, Germany).

Preparation of the Enzyme-Functionalized Hairpin DNA Strands (GOx-H1/HRP-H2). The GOx or HRP-functionalized hairpin DNA strands were prepared by using BS3 as a

6


Page 6 of 34

Page 7 of 34

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


crosslinker based on a modified protocol described previously.30 Briefly, 25 µL of 20 µM enzyme solution was added into the solution of 50-fold excess BS3 and reacted for two hours in phosphate buffer solution (10mM, pH=7.4), allowing one N-hydroxysuccinimide (NHS) ester group of the BS3 to react with the lysine residues on the enzyme surface. And then excess BS3 was removed by a cutoff filter (Amicon, 30 KDa).

In the meantime, the amino-modified hairpin DNA strands were annealed from 95°C to room temperature in two hours to form hairpin structure. Subsequently, the BS3 modified enzyme solution was mixed with 10-fold excess amino-modified hairpin DNA strands which had been annealed and incubated for two hours. In this regard, the other Nhydroxysuccinimide (NHS) ester group of the BS3 will react with the amino of hairpin DNA strands. After that, excess hairpin DNA strands were removed by a cutoff filter (Amicon, 30 KDa). The successful conjunctions (GOx-H1 and HRP-H2) were characterized by 8% native polyacrylamide gel electrophoresis (PAGE) and UV-vis spectroscopy.

Detection of Nucleic Acid Target (T, as a Model). A colorimetric nucleic acid assay was performed using the following procedures. Firstly, the different concentrations of T were added into the mixed solutions of GOx-H1 and HRP-H2, and the solutions were incubated for five hours at room temperature. And then glucose and ABTS2- were added to the formed enzymes’ multiple cascades systems. The final concentration of GOx-H1, HRP-H2, glucose and ABTS2- in mixed solutions were 0.1 µM, 0.1µM, 1mM and 2mM, respectively. The real time increase of absorbance at 414 nm was monitored to measure the catalytic performance 7



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 8 of 34

and quantify the concentration of T.30 As control, the free GOx and HRP, instead of GOx-H1 and HRP-H2, were mixed and incubated.

Detection of Biological Substances (ATP, as a Model). Firstly, equivalent aptamer (Apt) and complementary strands (Com) were mixed and incubated for 30 min at room temperature, following by the addition of different concentrations of ATP and one hour incubation. After that, the assistant hairpin (HAssist) was added and incubated for another 30 min. Then, two kinds of enzyme-functionalized hairpin DNA strands for ATP detection (GOx-H1 (ATP)/HRP-H2 (ATP), final concentration 0.1 µM) were added and incubated for five hours at room temperature. Lastly, glucose (final concentration 1 mM) and ABTS2- (final concentration 2 mM) were added to the formed systems and the real time increase of absorbance at 414 nm was reported.

RESULT AND DISCUSSIONS Crosslink of the Hairpin DNA Strands and Free Enzymes. The BS3, a typical water soluble protein crosslinker, was employed to prepare DNA-enzyme conjugates in this study. To verify that we have obtained enzyme-functionalized hairpin DNA strands (i.e. GOx-H1 and HRP-H2 in here) successfully, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (Native-PAGE) were implemented (Figure 1). Enzymes and DNA strands could be stained effectively by silver ions in SDS and native polyacrylamide gels respectively.31,

32

For the SDS-PAGE, the

positions of GOx and GOx-H1 (Figure 1A, lane 2, 3) were obviously higher than the positions 8


Page 9 of 34

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


of HRP and HRP-H2 (Figure 1A, lane 4, 5), which was mainly determined by the molecular weight of GOx and HRP(~72 KDa for GOx, ~43KDa for HRP, Figure S1), and the GOx-H1 and HRP-H2 showed bright stripes at similar locations with free enzymes near their molecular weight, confirming that the enzymes were effectively retained in the processes of modification and purification. However, the relative positions of GOx and GOx-H1 were hard to distinguish, as well as the HRP and HRP-H2, which were because that the molecular weight interval between the stripes of protein marker was too large for the linked small hairpin DNA strands. In order to address this issue, PAGE was employed subsequently. As shown in Figure 1B, the bright strips were observed with regard to pure hairpin DNA strands (H1/H2, Figure 1B, lane 4, 7). However, after the modification procedures, enzymefunctionalized hairpin DNA strands (GOx-H1/HRP-H2) presented bright strips (Figure 1B, lane 3, 6) at the positions nearby the molecular weight of pure enzymes,33 which were greater than the molecular weights of pure hairpin DNA strands, indicating that the hairpin DNA strands were successfully functionalized by enzymes.

Figure 1. Characterization of the forming process of enzyme-functionalized hairpin DNA strands (GOx-H1/HRP-H2) by gel electrophoresis. (A) 8% SDS-PAGE. (1) Protein marker (59



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

245 KDa); (2) GOx; (3) GOx-H1; (4) HRP; (5) HRP-H2. (B) 12% native PAGE. (1) DNA ladder (50 -500 bp); (2) GOx; (3) GOx-H1; (4) H1; (5) HRP; (6) HRP-H2; (7) H2. UV-vis absorption spectroscopy was used to further confirm our prepared enzymefunctionalized hairpin DNA strands (Figure S2). Both the characteristic absorption peaks of enzymes and hairpin DNA strands were shown in enzyme-functionalized hairpin DNA strands, 450 nm for GOx, 403 nm for HRP and 260 nm for hairpin DNA strands, consistent with the previous report.32 The strength of peaks of enzymes were similar before and after modification, which indicated that few of the enzyme-functionalized hairpin DNA strands were removed in the ultrafiltration processes. More important is that the number of hairpin DNA strands on each enzyme could be roughly calculated by the strength of absorption peaks.32 About 3.38 H1 loading on each GOx and 7.20 H2 loading on each HRP, the non-oneto-one relationship between hairpin DNA strands and enzymes would be likely to have some impacts on the following two aspects: (1) the secondary structure of enzymes and hairpin DNA strands (2) the efficiency of HCR. Studies on the Secondary Structure of Enzymes and Hairpin DNA Strands. With above consideration in mind, the CD spectra of free enzymes, hairpin DNA stands and enzymefunctionalized hairpin DNA strands were carried out (Figure 2) to study their secondary structures changes in the process of modification and purification. Free enzymes possess two negative peaks at 208 nm and 222 nm, and hairpin DNA strands possess a positive peak at approximately 280 nm and a negative peak at 250 nm, which were consistent with the

10


Page 10 of 34

Page 11 of 34

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


literature reported.34,

35, 36

However, it is difficult to judge the changes of their secondary

structures due to the disturbance of each other. Herein, the difference CD spectra of enzymes were worked out (black dotted curves) by subtracting the normalized CD spectra of free hairpin DNA strands, both peak positions and intensities were similar to that of enzyme, which indicated that the secondary structures as well as the contents of enzymes were well maintained in the process of modification and the following purification.37 In like manner, the difference CD spectra of hairpin DNA strands were also computed by subtracting the CD spectra of enzymes (blue dotted curves), the positions of peaks kept unchanged38 but the peak intensities obviously decreased, which were because the excess hairpin DNA strands could be separated from enzyme-functionalized hairpin DNA strands in the ultrafiltration purification process.

In other words, both the CD spectral features of hairpin DNA strands and enzymes were unchanged after modification (Figure 2). The stable secondary structures of them were very favorable for maintaining the HCR efficiency of the hairpin DNA strands and the catalytic performance of enzymes.

Figure 2. (A) CD spectra of GOx (black curve), H1 (blue curve) and GOx-H1 (red curve); (B) CD spectra of HRP (black curve), H2 (blue curve) and the HRP-functionalized H2 (red curve). 11



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 black dotted curves of A and B are difference CD spectra of GOx and HRP respectively. And the blue dotted curves of A and B are difference CD spectra of H1 and H2 respectively. .

Analysis of HCR Efficiency. We studied the HCR efficiencies of hairpin DNA strands and enzyme-functionalized hairpin DNA strands. For the pure hairpin DNA strands (H1/H2), different concentrations of T were used to trigger the HCR and the result was illustrated in Figure 3A.32 In the control groups (Figure 3A, lane 2-4), either alone or in mixture, the band positions were very low because both H1 and H2 existed in the form of monomer, indicating that these two hairpin DNA strands could stably coexisted in solution. With the addition of T, a series of new bright stripes which were higher than the positions of hairpin DNA strands could clearly be seen on the PAGE gel (Figure 3A, lane 5-10), indicating that the H1 and H1 had been assembled to long double strands by T-triggered HCR. More interestingly, the positions of these stripes changed with the concentration of T when the concentrations of hairpin DNA strands were constant. When the concentration of T, namely, the ratio of concentrations of T and hairpin DNA strands was high (1:10 to 1:1), superfluous T would compete hairpin DNA strands with each other and limit the assembly degree of hairpin DNA strands, so the molecular weight of assembly products were relatively small (Figure 3A, lane 5-8). However, when the concentration ratio of T and hairpin DNA strands decreases to 1:50 or lower, the HCR of hairpin DNA strands could reach the maximum degree and the molecular mass of assembly products could reach a summit, so the stripe would be very high and the strips of hairpin DNA strands appeared again (Figure 3A, lane 9, 10). These results 12


Page 12 of 34

Page 13 of 34

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


indicated that the HCR efficiencies of pure hairpin DNA strands (H1/H2) triggered by T was very high. Furthermore, the AFM image and cross-section analysis of the product of HCR were shown in Figure S3A. Long chains of micrometer-scale could be clearly seen, and the height of the chain was about 2 nm, consistent with the reported height of double-stranded DNA39. The results were in good agreement with the PAGE, further proved the high HCR efficiencies of pure hairpin DNA strands.

Encouraged by the above results, we further explored the efficiency of the HCR of enzyme-functionalized hairpin DNA strands (GOx-H1/HRP-H2) by 5% native PAGE (Figure 3B). In the present of T, a series of bright stripes appeared (Figure 3B, lane 2-5) and the position of these stripes became higher with the decrease of the concentration ratio of T and hairpin DNA strands (Figure 3B, lane 3, 4, 5), consistent with the result of HCR of pure hairpin DNA strands. However, when we continued to reduce the ratio, the stripes’ position of lane 2 was similar to that in lane 3, which demonstrated that the HCR had reached the maximum degree when the ratio was as high as 1:10 (Figure 3B, lane 3). Figure 3C shows the AFM image and cross-section analyses of the HCR-assembled GOx-H1 and HRP-H2. We could see that the lengths of the HCR products were ranges in the nanometer to micrometerscale, which indicated that the GOx-H1 and HRP-H2 would be assembled into long double DNA strands of different lengths. This result was coincided with that our PAGE bands were distributed in the 500-1500 bp.

13



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 3. (A) Characterization of the HCR of pure hairpin DNA strands by 8% native PAGE. (1) DNA ladder (20-500 bp); (2) H1; (3) H2; (4) H1+H2; (5-10) H1+H2+T. The concentration of H1 and H2 in lane 2, 3 was 1µM, while in lane 4-10 was 500nM. The concentrations of T were 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 1 nM from lane 5 to 10, respectively. (B) Characterization of the HCR of enzyme-functionalized hairpin DNA strands by 5% native PAGE. (1) DNA ladder (100-5000 bp); (2) GOx-H1 + HRP-H2 + 10 nM T; (3) GOx-H1 + HRP-H2 + 100 nM T; (4) GOx-H1 + HRP-H2 + 200 nM T; (5) GOx-H1 + HRP-H2 + 500 nM T. All of the concentrations of enzyme-functionalized hairpin DNA strands in lane 2-5 were 1µM. (C) The AFM image of HCR-assembled GOx-H1 and HRP-H2 and cross-section analyses of GOx (green line), HRP (red line) and double DNA strands (blue line).

Another important inspiration was that the concentration ratio of T and enzymefunctionalized hairpin DNA strands (GOx-H1/HRP-H2) should be strictly controlled in the 14


Page 14 of 34

Page 15 of 34

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


construction of the detection platform. We defined the lowest ratio which could trigger the HCR reaction to the maximum degree, 1:10 for our system, as the critical value. The number of enzyme-functionalized hairpin DNA strands assembled by the HCR was positively correlated with the concentration of T when the ratio was below the critical value. However, when the ratio was higher than the critical value, the mechanism of signal amplification will change because the inhibitory effect of excess T on HCR. Therefore, our following studies were carried out in the range of near the critical ratio (1:10).

Feasibility Study. In order to demonstrate the effect of the multiple enzyme cascades on the catalytic performance, the produce rate of the ABTS•- was used to measure catalytic performance of different catalytic systems by observing its characteristic absorption peak at 414 nm.30, 32 The blank buffer showed no catalytic performance (Figure 4A, line 1) and the system of free enzymes showed obvious catalytic performance (Figure 4A, line 2). In addition, the catalytic performance of systems which containing one or both of the enzymefunctionalized hairpin DNA strands were only a little reduced (Figure 4A, line 3, 4, 5) compared to free enzymes (Figure 4A, line 2), suggesting that the hairpin DNA strands on the surface of enzymes could not obviously influence the catalytic performance of enzymes, which was accorded with the unchanged secondary structures in CD spectra (Figure 2). Furthermore, the simple mixture of free enzymes and hairpin DNA strands exhibited similar catalytic performance with only free enzymes no matter whether T existed (Figure 4A, line 2, 6, 7). The enzymes were dispersed in the solution irregularly in all of the above systems. 15



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

Under these circumstances, the distance between GOx (~8 nm of height) 40 and HRP (~6 nm of height) 41 was micrometer-scale (Figure S3B), with relatively low catalytic performance, which was agree with previous study.32

However, compared with free enzymes, the system consisting of both enzymefunctionalized hairpin DNA strands demonstrated an approximately 107% higher catalytic performance in the present of T, which was calculated based on the changes of absorbance in 1500 s at 414 nm (Figure 4A, line 2, 8).32 The corresponding UV-vis absorption spectroscopy and photograph (inset) at 1500 s was shown in Figure S4. The color of system 8 was much deeper than other systems. The enhancement was caused by the multiple cascades of enzymes, in which both enzymes were spatially organized on the double stranded DNA formed by T triggered HCR of enzyme-functionalized hairpin DNA strands. The distance between two kinds of enzymes was reduced to nanometer-scale (Figure 3C),32 which enabled the hydrogen peroxide (H2O2) generated by GOx to form a high local concentration contiguous to the HRP and facilitated the oxidation of ABTS2- accordingly.22, 28 So these results demonstrated that the catalytic performance of enzyme cascade reaction could be greatly enhanced by the addition of T as it could trigger the HCR of GOx-H1 and HRP-H2, proving the feasibility of our strategy powerfully.

Figure 4B showed the change trend of the catalytic performance of the system 7 and 8 in the presence of different concentrations of T, and the corresponding UV-vis absorption spectroscopy and photograph (inset) at 1500 s were shown in Figure S5A and B, respectively. 16


Page 16 of 34

Page 17 of 34

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 changes of absorbance of the ABTS•- in 1500 s at 414 nm were used to measure the catalytic performance.32 As shown in Figure S5A, the absorbance at 1500 s does not change with the increases of concentration of T, indicating that the addition of T could hardly affect the catalytic performance of enzymes for system 7 (Figure 4B, black samples). However, the catalytic performance of system 8 would enhance along with the increased concentration of T (Figure S5B and red samples in Figure 4B). For the system of simple mixture of free enzymes and hairpin DNA strands (system 7), the addition of different concentrations of T would trigger the HCR of different number of hairpin DNA strands but wouldn’t cause any change in the distance between the two kinds of enzymes. Therefore, the concentration of T would not affect the catalytic performance of the enzymes (black samples). However, for the system of enzyme-functionalized hairpin DNA strands (system 8), the number of enzymefunctionalized hairpin DNA strands assembled by the HCR, as well as the number of enzymes which were spatial organized on the double stranded DNA and the catalytic performance were positively correlated with the concentration of T when the concentration ratio of T and enzyme-functionalized hairpin DNA strands was below the critical value (1× 107 fM in this circumstance). Increasing the concentration of T in succession didn’t induce higher catalytic performance, which was attributed to the lower assembly degree of hairpin DNA strands caused by the excess T. The above results fully demonstrated that our platform had the potential to detect the concentration of T in a certain concentration range and the range could be regulated by the concentration of enzyme-functionalized hairpin DNA strands.

17



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. (A) Time-dependent changes of absorbance in the presence of different enzyme catalytic systems. All of the concentration of free enzymes and enzyme-functionalized hairpin DNA strands were 0.1 µM while the concentration was 1µM for H1/H2 and 10 pM for T. (B) The catalytic performance of system 7 (black samples) and 8 (red samples) triggered by different concentrations of T.

Detection of T. In order to test and verify the detection performance of our platform in a relatively low concentration range, we fixed the concentrations of enzyme-functionalized hairpin DNA strands as 0.1 µM and studied the effect of different concentrations of T below the critical value (1×107 fM in this circumstance) on the catalytic performance. In the absence of T, the GOx-H1 and HRP-H2 stably coexisted in solution, with lower catalytic performance. However, upon the addition of T, the GOx-H1 and HRP-H2 would carry out the HCR and assembled into long double-stranded DNA structure. Meanwhile, the GOx and HRP would be organized on the long double-stranded DNA spatially, with the enhancement of catalytic performance of the enzyme cascade reaction. What’s more, the concentration of T below the critical value would affect the cascade degree, as well as the catalytic performance. The catalytic performance of a series of HCR systems triggered by different concentrations of T (0-1×107 fM) were tested and the real time absorbance kinetic curves at 414 nm were 18


Page 18 of 34

Page 19 of 34

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


shown in Figure 5A. A relatively low catalytic performance was observed in the absence of T (Figure 5A, line 1) and the catalytic performance could be enhanced gradually with the increase of the concentration of T (Figure 5A, line 2-9). Nevertheless, when the concentration of T increased to 1 nM or above, the absorbance tended to form a platform at 1500 s (Figure 5A, line 10, 11). This should be attributed to the inhibitory effect of excess T on the assembly of the enzyme-functionalized hairpin DNA strands, which was studied in Figure 3 and Figure 4B. We then used the changes of absorbance of the ABTS•- in 1500 s as a measurement standard to quantify the T.22, 30, 32 As can be seen in Figure 5B, a semilogarithmic dependence was obtained between the changes of absorbance in 1500 s and the concentration of T in the range of 1×101–1 ×104 fM (the linear correlation coefficient is 0.9813). The limit of detection was 5.2 fM based on 3S/N, which is comparable or a little better than most of reported amplification nucleic acid detection methods which were only depends on the amplification performance of HCR (Table S1).14, 17, 19, 20, 42-44 For example, the sensitivity of them could even reach 1×10-14 M19 or 1×10-13 M20, which were comparable to that of our dual amplification platform. So we carefully analyzed the reason why the sensitivity of our dual amplification platform did not significantly improved compared with these single amplification platforms. We attributed this to the effect of enzymatic modification on the HCR. The modification of enzymes greatly reduced the lengths of HCR products, with the corresponding reduction of the HCR amplification performance. So the sensitivity may be 19



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

much lower than other HCR methods. However, we introduced enzyme cascade reaction which also has the amplification performance into our detection platform. Integrated above factors, our dual amplification platform had comparable amplification performance with aforementioned single amplification platforms. Importantly, the main purpose of this work was to establish a new detection platform and we have applied it into the ultrasensitive detection of target successfully. In future research, we may put more focus on how to further improve the sensitivity.

Figure 5. (A) The real time absorbance kinetic curves of HCR systems triggered by different concentrations of T. The blank 0 was shown as a control and the concentration of T was increased from line 1 to line 11 (0, 101, 5×101, 1×102, 5×102, 1×103, 5×103, 1×104, 1× 105, 1 × 106, 1 × 107 fM). (B) Plot of the changes of absorbance in 1500 s against T concentration. The concentrations of enzyme-functionalized hairpin DNA strands (GOxH1/HRP-H2) were 0.1 µM in the system.

Detection of ATP. Based on similar principle with the detection of T, we designed an ATP detection strategy. The design process was shown in Figure 6A. When ATP exists, the ATP would compete the aptamer (Apt) with the complementary strands (Com) and leave the Com alone. The concentration of alone Com was proportional to the concentration of ATP. 20


Page 20 of 34

Page 21 of 34

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 alone Com would open the loop of assistant hairpin strand (HAssist) and the HAssist could trigger the self-assembly of two kinds of enzyme-functionalized hairpin DNA strands for ATP (GOx-H1 (ATP)/HRP-H2 (ATP)). The assembly of them would enhance the catalytic performance of enzyme cascade reactions and reflect the concentration of ATP. The effect of different concentrations of ATP was displayed in Figure 6B, C. The catalytic performance enhanced gradually with the increased concentration of ATP and tended to form a platform when the concentration reached 1×106 pM. We use the changes of absorbance of the ABTS•in 1500 s as a measurement standard to quantify the concentration of ATP. As shown in Figure 6C, the semilogarithmic dependence was obtained from 1 pM to 1×103 pM (the linear correlation coefficient is 0.9824) and the detection limit could reach 0.8 pM based on 3S/N, which could be comparable with most of the detection methods of ATP based on aptamer (Table S2).45-51

21



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 6. (A) Schematic illustration of the detection of ATP by enzyme cascade reaction. (B) The real time absorbance kinetic curves of HCR systems of conjugations for ATP (GOx-H1 (ATP)/HRP-H2 (ATP)) triggered by different concentrations of ATP. The blank 0 was shown as a control and the concentration of ATP was increased from line 1 to line 10 (0, 1×100, 5×100, 1×101, 5×101, 1×102, 1×103, 1×104, 1×105, 1×106 pM). (C) Plot of the changes of absorbance at 1500 s against ATP concentration. The concentrations of conjugations (GOx-H1 (ATP)/HRP-H2 (ATP)) were 0.1 µM in the system.

Specificity Study. The specificity of our system for nucleic acid target (T) or biological substances (ATP) with suitable aptamer were evaluated. Various sequences which were one base pair mismatched, deleted or inserted at different positions (middle or end) of T were used to study the specificity of our detection platform for T (Figure 7A) and the analogues of ATP (ADP, CTP, UTP, GTP) were used to study the specificity for ATP (Figure 7B). Only T and ATP exhibited a significant enhancement for the catalytic performance, whereas all of others displayed nearly the same performance with the blank control group under the same experimental conditions. These results clearly suggested that our system possesses a prominent specificity for the detection of T and ATP.

22


Page 22 of 34

Page 23 of 34

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 7. (A) Specificity studies of T. The concentrations of all sequences were 10 pM and the concentrations of enzyme-functionalized hairpin DNA strands (GOx-H1/HRP-H2) were 0.1 µM. (B) Specificity studies of ATP. The concentrations of ATP and analogues were 1 nM and the concentrations of enzyme-functionalized hairpin DNA strands (GOx-H1 (ATP)/HRPH2 (ATP)) were 0.1 µM.

CONCLUSIONS

CONCLUSIONS

In conclusion, we have successfully designed a versatile ultra-sensitive colorimetric assay system based on the hybridization chain reaction-triggered enzyme cascade amplification. The signal amplification relies on the inherent signal amplification of HCR and the enhanced catalytic performance of multiple cascaded enzymes. In our work, we firstly studied the construction process and the influencing factors of the detection platform systematically by gel electrophoresis, AFM, CD spectrum and UV-vis spectrum. And then, we confirmed the feasibility of the platform for detection and applied it to nucleic acid (T, as a model) detection. We also expanded this system to the detection of biological substances (ATP, as a model) with suitable aptamer. To our best knowledge, this is the first enzyme cascade amplification photochemistry biosensor triggered by HCR. We believe that our method would be an ultrasensitive, highly specific and universal strategy for the detection of various nucleic acids and biological substances with suitable aptamer.

23



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

ASSOCIATED CONTENT Supporting Information Available: The MALDI–TOF mass spectra of GOx and HRP, UVvis spectra of free enzymes and enzyme-functionalized hairpin DNA strands and AFM images and cross-section analysis of HCR-assembled H1 and H2, GOx and HRP. The visual color and absorbance corresponding to the Figure 4. The comparison of our work with other nucleic acid or ATP detection methods. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Prof. Xiurong Yang, State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. E-mail: [email protected]. Tel.: +86 431 85262056; Fax: +86 431 85689278.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (Grant Nos. 21435005, 21627808 and 21605139).

REFERENCES

24


Page 24 of 34

Page 25 of 34

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) Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A. Gold Nanoparticles for Biology and Medicine. Angew. Chem. Int. Ed. 2010, 49 (19), 3280-3294. (2) Zhu, X.; Zhao, T.; Nie, Z.; Liu, Y.; Yao, S., Non-Redox Modulated Fluorescence Strategy for Sensitive and Selective Ascorbic Acid Detection with Highly Photoluminescent NitrogenDoped Carbon Nanoparticles via Solid-State Synthesis. Anal. Chem. 2015, 87 (16), 85248530.

(3) Wang, Z.; Sun, N.; He, Y.; Liu, Y.; Li, J., DNA Assembled Gold Nanoparticles Polymeric Network Blocks Modular Highly Sensitive Electrochemical Biosensors for Protein Kinase Activity Analysis and Inhibition. Anal. Chem. 2014, 86 (12), 6153-6159.

(4) Henihan, G.; Schulze, H.; Corrigan, D. K.; Giraud, G.; Terry, J. G.; Hardie, A.; Campbell, C. J.; Walton, A. J.; Crain, J.; Pethig, R.; Templeton, K. E.; Mount, A. R.; Bachmann, T. T. Label- and Amplification-Free Electrochemical Detection of Bacterial Ribosomal RNA. Biosens. Bioelectron. 2016, 81, 487-494. (5) Guo, J.; Wang, J.; Zhao, J.; Guo, Z.; Zhang, Y., Ultrasensitive Multiplexed Immunoassay for Tumor Biomarkers Based on DNA Hybridization Chain Reaction Amplifying Signal. ACS Appl. Mater. Interface 2016, 8 (11), 6898-6904. (6) Yang, C.; Shi, K.; Dou, B.; Xiang, Y.; Chai, Y.; Yuan, R., In Situ DNA-Templated Synthesis of Silver Nanoclusters for Ultrasensitive and Label-Free Electrochemical Detection of MicroRNA. ACS Appl. Mater. Interface 2015, 7 (2), 1188-1193. 25



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

(7) Ali, M. M.; Li, Y. Colorimetric Sensing by Using Allosteric-DNAzyme-Coupled Rolling Circle Amplification and a Peptide Nucleic Acid–Organic Dye Probe. Angew. Chem. Int. Ed. 2009, 48 (19), 3512-3515. (8) Ye, T.; Chen, J.; Liu, Y.; Ji, X.; Zhou, G.; He, Z., Periodic Fluorescent Silver Clusters Assembled by Rolling Circle Amplification and Their Sensor Application. ACS Appl. Mater. Interface 2014, 6 (18), 16091-16096. (9) Cheglakov, Z.; Weizmann, Y.; Basnar, B.; Willner, I., Diagnosing viruses by the rolling circle amplified synthesis of DNAzymes. Org. Biomol. Chem. 2007, 5 (2), 223-225. (10) Qian, L.; Winfree, E. Scaling Up Digital Circuit Computation with DNA Strand Displacement Cascades. Science 2011, 332 (6034), 1196-1201. (11) He, J.-L.; Wu, Z.-S.; Zhou, H.; Wang, H.-Q.; Jiang, J.-H.; Shen, G.-L.; Yu, R.-Q. Fluorescence Aptameric Sensor for Strand Displacement Amplification Detection of Cocaine. Anal. Chem. 2010, 82 (4), 1358-1364. (12) Zheng, A.-X.; Wang, J.-R.; Li, J.; Song, X.-R.; Chen, G.-N.; Yang, H.-H. Enzyme-Free Fluorescence Aptasensor for Amplification Detection of Human Thrombin via TargetCatalyzed Hairpin Assembly. Biosens. Bioelectron. 2012, 36 (1), 217-221. (13) 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 (34), 13918-13921.

26


Page 26 of 34

Page 27 of 34

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


(14) Ma, C.; Wang, W.; Mulchandani, A.; Shi, C. A Simple Colorimetric DNA Detection by Target-Induced Hybridization Chain Reaction for Isothermal Signal Amplification. Anal. Biochem. 2014, 457, 19-23. (15) Li, W.; Yang, X.; He, L.; Wang, K.; Wang, Q.; Huang, J.; Liu, J.; Wu, B.; Xu, C., SelfAssembled DNA Nanocentipede as Multivalent Drug Carrier for Targeted Delivery. ACS Appl. Mater. Interface 2016, 8 (39), 25733-25740. (16) Wang, F.; Lu, C. H.; Willner, I. From Cascaded Catalytic Nucleic Acids to EnzymeDNA Nanostructures: Controlling Reactivity, Sensing, Logic Operations, and Assembly of Complex Structures. Chem. Rev. 2014, 114 (5), 2881-2941. (17) Tang, W.; Wang, D.; Xu, Y.; Li, N.; Liu, F. A Self-Assembled DNA NanostructureAmplified Quartz Crystal Microbalance with Dissipation Biosensing Platform for Nucleic Acids. Chem. Commun. 2012, 48 (53), 6678-6680. (18) Huang, J.; Wu, Y.; Chen, Y.; Zhu, Z.; Yang, X.; Yang, C. J.; Wang, K.; Tan, W. PyreneExcimer Probes Based on the Hybridization Chain Reaction for the Detection of Nucleic Acids in Complex Biological Fluids. Angew. Chem. Int. Ed. 2011, 50 (2), 401-404. (19) Wang, F.; Elbaz, J.; Orbach, R.; Magen, N.; Willner, I., Amplified Analysis of DNA by the Autonomous Assembly of Polymers Consisting of DNAzyme Wires. J. Am. Chem. Soc. 2011, 133 (43), 17149-17151.

27



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

(20) Shimron, S.; Wang, F.; Orbach, R.; Willner, I., Amplified Detection of DNA through the Enzyme-Free Autonomous Assembly of Hemin/G-Quadruplex DNAzyme Nanowires. Anal. Chem. 2012, 84 (2), 1042-1048. (21) Vriezema, D. M.; Garcia, P. M. L.; Sancho Oltra, N.; Hatzakis, N. S.; Kuiper, S. M.; Nolte, R. J. M.; Rowan, A. E.; van Hest, J. C. M. Positional Assembly of Enzymes in Polymersome Nanoreactors for Cascade Reactions. Angew. Chem. Int. Ed. 2007, 46 (39), 7378-7382. (22) Freeman, R.; Sharon, E.; Tel-Vered, R.; Willner, I. Supramolecular Cocaine−Aptamer Complexes Activate Biocatalytic Cascades. J. Am. Chem. Soc. 2009, 131 (14), 5028-5029. (23) Shaw, A. S.; Filbert, E. L. Scaffold Proteins and Immune-Cell Signalling. Nat. Rev. Immunol. 2009, 9 (1), 47-56. (24) Zhao, M.; Zhuo, Y.; Chai, Y.; Xiang, Y.; Liao, N.; Gui, G.; Yuan, R. Dual Signal Amplification Strategy for the Fabrication of an Ultrasensitive Electrochemiluminescenct Aptasensor. Analyst 2013, 138 (21), 6639-6644. (25) Zhang, L.; Jiang, L.; Liu, Y.; Yin, Q. Ionic Strength-Modulated Catalytic Efficiency of a Multienzyme Cascade Nanoconfined on Charged Hierarchical Scaffolds. RSC Adv. 2015, 5 (63), 50807-50812. (26) Linko, V.; Eerikainen, M.; Kostiainen, M. A. A Modular DNA Origami-Based Enzyme Cascade Nanoreactor. Chem. Commun. 2015, 51 (25), 5351-5354.

28


Page 28 of 34

Page 29 of 34

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


(27) Lilienthal, S.; Shpilt, Z.; Wang, F.; Orbach, R.; Willner, I. Programmed DNAzymeTriggered Dissolution of DNA-Based. Hydrogels: Means for Controlled Release of Biocatalysts and for the Activation of Enzyme Cascades. ACS Appl. Mater. Interface 2015, 7 (16), 8923-8931.

(28) Wilner, O. I.; Weizmann, Y.; Gill, R.; Lioubashevski, O.; Freeman, R.; Willner, I., Enzyme Cascades Activated on Topologically Programmed DNA Scaffolds. Nat. Nano. 2009, 4 (4), 249-254. (29) Wilner, O. I.; Shimron, S.; Weizmann, Y.; Wang, Z.-G.; Willner, I., Self-Assembly of Enzymes on DNA Scaffolds: En Route to Biocatalytic Cascades and the Synthesis of Metallic Nanowires. Nano Lett. 2009, 9 (5), 2040-2043.

(30) Fu, J.; Liu, M.; Liu, Y.; Woodbury, N. W.; Yan, H. Interenzyme Substrate Diffusion for an Enzyme Cascade Organized on Spatially Addressable DNA Nanostructures. J. Am. Chem. Soc. 2012, 134 (12), 5516-5519. (31) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels. Anal. Chem. 1996, 68 (5), 850-858. (32) Xin, L.; Zhou, C.; Yang, Z.; Liu, D. Regulation of an Enzyme Cascade Reaction by a DNA Machine. Small 2013, 9 (18), 3088-3091. (33) Bassam, B. J.; Caetano-Anollés, G.; Gresshoff, P. M. Fast and Sensitive Silver Staining of DNA in Polyacrylamide Gels. Anal. Biochem. 1991, 196 (1), 80-83.

29



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

(34) Witt, S.; Singh, M.; Kalisz, H. M. Structural and Kinetic Properties of Nonglycosylated Recombinant Penicillium amagasakiense Glucose Oxidase Expressed in Escherichia coli. Appl. Environ. Microb. 1998, 64 (4), 1405-1411. (35) Chen, X.; Ruan, C.; Kong, J.; Deng, J. Fresen. J. Spectroelectrochemical Investigation of Direct Electron Transfer Between Resting Horseradish Peroxidase and its Oxidation States Promoted by DNA. Anal. Chem. 2000, 367 (2), 172-177. (36) Lafer, E. M.; Möller, A.; Nordheim, A.; Stollar, B. D.; Rich, A. P. Antibodies Specific for Left-Handed Z-DNA. Natl Acad. Sci. 1981, 78 (6), 3546-3550. (37) Cao, L.; Ye, J.; Tong, L.; Tang, B. A New Route to the Considerable Enhancement of Glucose Oxidase (GOx) Activity: The Simple Assembly of a Complex from CdTe Quantum Dots and GOx, and Its Glucose Sensing. Chem.– Eur. J. 2008, 14 (31), 9633-9640. (38) Kypr, J.; Kejnovská, I.; Renčiuk, D.; Vorlíčková, M. Circular Dichroism and Conformational Polymorphism of DNA. Nucleic Acids Res. 2009, 37 (6), 1713-1725. (39) Lyubchenko, Y. L., Preparation of DNA and Nucleoprotein Samples for AFM Imaging. Micron 2011, 42 (2), 196-206. (40) Wilson, R.; Turner, A. P. F., Glucose Oxidase: An Ideal Enzyme. Biosens. Bioelectron. 1992, 7 (3), 165-185. (41) Zhang, J.; Chi, Q.; Dong, S.; Wang, E., In Situ Electrochemical Scanning Tunnelling Microscopy Investigation of Structure for Horseradish Peroxidase and its Electricatalytic Property. Bioelectrochemistry 1996, 39 (2), 267-274.

30


Page 30 of 34

Page 31 of 34

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) Chen, Y.; Chen, L.; Ou, Y.; Guo, L.; Fu, F. Enzyme-Free Detection of DNA Based on Hybridization Chain Reaction Amplification and Fluorescence Resonance Energy Transfer. Sensor. Actuat. B: Chem. 2016, 233, 691-696. (43) Chao, J.; Li, Z.; Li, J.; Peng, H.; Su, S.; Li, Q.; Zhu, C.; Zuo, X.; Song, S.; Wang, L.; Wang, L. Hybridization Chain Reaction Amplification for Highly Sensitive Fluorescence Detection of DNA with Dextran Coated Microarrays. Biosens. Bioelectron. 2016, 81, 92-96. (44) Ravan, H. Isothermal RNA Detection Through the Formation of DNA Concatemers Containing HRP-Mimicking DNAzymes on the Surface of Gold Nanoparticles. Biosens. Bioelectron. 2016, 80, 67-73. (45) Liu, Z.; Chen, S.; Liu, B.; Wu, J.; Zhou, Y.; He, L.; Ding, J.; Liu, J. Intracellular Detection of ATP Using an Aptamer Beacon Covalently Linked to Graphene Oxide Resisting Nonspecific Probe Displacement. Anal. Chem. 2014, 86 (24), 12229-12235. (46) Li, L. J.; Tian, X.; Kong, X. J.; Chu, X. A G-quadruplex-based Label-Free Fluorometric Aptasensor for Adenosine Triphosphate Detection. Anal. Sci. 2015, 31 (6), 469-473. (47) Fang, B.-Y.; Yao, M.-H.; Wang, C.-Y.; Wang, C.-Y.; Zhao, Y.-D.; Chen, F. Detection of Adenosine Triphosphate in HeLa Cell using Capillary Electrophoresis-Laser Induced FluorescenceDetection Based on Aptamer and Graphene Oxide. Colloid. Surface. B. 2016, 140, 233-238.

31



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

(48) Ma, K.; Wang, H.; Li, H.; Wang, S.; Li, X.; Xu, B.; Tian, W. A Label-Free Aptasensor for Turn-On Fluorescent Detection of ATP Based on AIE-Active Probe and Water-Soluble Carbon Nanotubes. Sensor. Actuat. B: Chem. 2016, 230, 556-558. (49) Wen, C.; Huang, Y.; Tian, J.; Hu, K.; Pan, L.; Zhao, S. A Novel Exonuclease III-Aided Amplification Assay Based on a Graphene Platform for Sensitive Detection of Adenosine Triphosphate. Anal. Methods 2015, 7 (9), 3708-3713. (50) Guo, Y.; liu, J.; Yang, G.; Sun, X.; Chen, H.-Y.; Xu, J.-J. Multiple Turnovers of DNAzyme for Amplified Detection of ATP and Reduced Thiol in Cell Homogenates. Chem. Commun. 2015, 51 (5), 862-865. (51) Yang, Y.; Huang, J.; Yang, X.; Quan, K.; Wang, H.; Ying, L.; Xie, N.; Ou, M.; Wang, K. Aptazyme–Gold Nanoparticle Sensor for Amplified Molecular Probing in Living Cells. Anal. Chem. 2016, 88 (11), 5981-5987.

32


Page 32 of 34

Page 33 of 34

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


Table of Contents Graphic and Synopsis

33



Page 34 of 34

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 Paragon Plus Environment

34

Ultra-Sensitive Colorimetric Assay System Based on the Hybridization

Recommend Documents