Based on DNA Strand Displacement and Functionalized Magnetic

Subscriber access provided by Iowa State University | Library

Article

Based on DNA strand displacement and functionalized magnetic nanoparticles: a promising strategy for enzyme immobilization Jiayi Song, Ping Su, Ruian Ma, Ye Yang, and Yi Yang Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b00595 • Publication Date (Web): 17 Apr 2017 Downloaded from http://pubs.acs.org on April 19, 2017

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.

Industrial & Engineering Chemistry Research 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 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

Industrial & Engineering Chemistry Research

Based on DNA strand displacement and functionalized magnetic nanoparticles: a promising strategy for enzyme immobilization

Jiayi Song, Ping Su*, Ruian Ma, Ye Yang and Yi Yang*

Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, College of Science, Beijing University of Chemical Technology, Beijing 100029, P.R. China.

*Corresponding authors. Email: [email protected]；[email protected] Tel: +86-010-64441521

1

ACS Paragon Plus Environment

Industrial & Engineering Chemistry Research 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

ABSTRACT: The efforts to enhance the stability of enzymes and improve their activity have generated considerable interest because of their wide applications in bioenergy conversion, proteomics research, and bioassays. This study reports a promising strategy for enzyme immobilization based on toehold-mediated DNA strand displacement on functionalized magnetic nanoparticles for the first time. The strategy provided a convenient approach to achieve sequential displacement and immobilization of different enzymes, using alkaline phosphatase (ALP), horseradish peroxidase (HRP), and trypsin as different model enzymes. Taking trypsin as an example, the enzyme immobilization procedure by DNA strand displacement exhibited high reversibility and reproducibility, which could retrieve more than 87% of the enzymatic activity after consecutive hybridization and dehybridization cycles. The thermal stability of the immobilized trypsin was significantly enhanced up to 3.1- and 2.3-fold greater than free enzyme after 45 min incubation at 60°C and 70°C, respectively, and the immobilized enzyme preserved promising enzymatic activity of more than 87% after 10 cycles. Notably, the immobilized enzyme exhibited excellent long-term incubation stability and storage stability as compared with free enzyme, and showed up to 11-fold higher stability than free enzyme towards different solvents. Significantly improved digestion efficiency of myoglobin, glycated hemoglobin, and cytochrome C was achieved with this immobilized enzyme within 10 min, and the obtained sequence coverages were 1.5-, 1.3-, and 1.6-fold higher than conventional in-solution digestion for 12 h. Thus, the developed strategy exhibited a promising alternative platform with high magnetic responsiveness and significantly enhanced properties for the immobilization of important enzymes and their broad applications. KEYWORDS: Immobilized enzyme; magnetic nanoparticles; DNA strand displacement

Introduction Enzymes are powerful biocatalysts, playing important roles in biosensors, industrial processes, and clinical diagnosis.1-3 The key requirements for these applications are good enzyme stability and better enzyme activity.4, 5 However, it is known that the application of native enzymes is usually limited by their denaturation and autodigestion during their application and storage, leading to poor stability, as well as difficulties in their recovery and reusability.6 Alternatively, immobilization of enzymes is a promising method to overcome these problems. In recent years, 2


Page 2 of 25

Page 3 of 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


many structured and morphologically distinct supports such as graphene,7-9 silica particles,10, 11 ordered mesoporous materials,12, 13 metal organic frameworks (MOFs),14, 15 and carbon materials16, 17

have been developed to improve the performance of enzymes. Magnetic nanoparticles as an

ideal immobilization supports have received more attention in recent years because of their large surface-to-volume ratios, the enhancement stability of enzyme, excellent biocompatibility, and easy separation.18-20 Considerable research efforts have been focused on immobilization of enzymes onto magnetic nanoparticles through different immobilized strategies, such as physical adsorption, covalent bonding, and cross-linking, to enhance the performance of immobilized enzymes.21-27 Although satisfactory enzyme activity and enzymatic assays were enhanced by these immobilization procedures, almost all of the configurations reported to date are based on direct immobilization of enzymes to the surface of modified immobilization materials via multiple covalent or noncovalent contacts, leading to the restriction of their conformational freedom or partial denaturation.28, 29 There is thus an urgent need to search for more efficient immobilization procedure to achieve enzyme immobilization under mild conditions and achieve a high level of immobilized enzyme performance. DNA nanotechnology utilizes DNA strands to manipulate the spatial and temporal distribution of matter, the predictability and specificity of Watson-Crick base pairing (A–T, C–G) make DNA a powerful and versatile material for engineering at the nanoscale.30 Because of the high physicochemical stability and pronounced mechanical rigidity, protein immobilization through short double-stranded DNA linkers under mild conditions could remain their active sites fully exposed and retain their biological activity.28, 31, 32 DNA strand displacement reactions, which are initiated through a short single stranded segments of DNA (referred to as toeholds), is the process through which two strands with partial or full complementarity hybridize to each other, displacing one or more pre-hybridized strands in the process under mild conditions, playing a central role in dynamic DNA nanotechnology.30, 33, 34 Due to their superior kinetics and modularity, DNA strand displacement reactions have been widely used to design diverse nanostructures for a variety of applications, such as DNA circuits, DNA biosensor, and DNA catalysis.35-38 Considering the excellent properties of DNA strand displacement and magnetic nanoparticles, it is envisioned that the immobilization enzyme strategy based on toehold-mediated DNA strand displacement on functionalized magnetic nanoparticles could hold great potential for immobilization of enzymes. 3



However, to the best of our knowledge, the study of enzyme immobilization based on this strategy has not been reported. In the present study, for the first time, we have reported the immobilization of ALP, HRP, and trypsin as different model enzymes via an efficient immobilization enzyme procedure based on toehold-mediated DNA strand displacement on modified magnetic nanoparticles. The different model enzymes were achieved sequential displacement and immobilization, and the enzyme immobilization procedure by DNA strand displacement exhibited high reversibility and reproducibility. The results confirmed that enzymes immobilized by this immobilization strategy were quite effective compared with other previous reports. The immobilized trypsin by toehold-mediated DNA strand displacement on modified magnetic nanoparticles was selected for further study. The immobilized trypsin exhibited excellent performance towards reusability, storage stability, thermostable, organic solvents-tolerant, and pH-stable along with potential applications in high throughput enzymatic assays and proteome analysis, as compared with the free enzyme. The outcome suggested that the developed strategy is a promising alternative platform for the immobilization of various kinds of enzymes and their broad applications.

Results and discussion Synthesis of the functionalized magnetic nanoparticles and enzyme immobilization Scheme 1 shows the synthetic procedures for the immobilized enzymes by DNA strand displacement reactions. The Fe3O4 magnetic nanoparticles were synthesized using a typical solvothermal reduction method and subsequently coated with tetraethyl orthosilicate (TEOS) to form core-shell structured Fe3O4@SiO2 nanoparticles. The 3-aminopropyltriethoxysilane (APTES) with primary amine groups was then used to fabricate Fe3O4@SiO2@APTES magnetic nanoparticles (FSAMs). The FSAMs were then incubated with 3’-aminated capture DNA (44bases) by glutaraldehyde crosslinking to obtain the capture DNA-functionalized nanoparticles (FSAMs@capDNA). For ALP immobilization, ALP-target DNA conjugates (24bases) were hybridized with partly complementary FSAMs@capDNA by highly specific Watson-Crick hybridization to obtain the FSAMs@DNA-ALP. For trypsin immobilization by toehold-mediated DNA strand displacement reactions, trypsin-target DNA conjugates (44bases) were incubated with FSAMs@DNA-ALP to completely hybridize to obtain the FSAMs@DNA-trypsin. Scheme 2 4


Page 4 of 25

Page 5 of 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


shows the immobilization mechanism in detail by DNA strand displacement reactions. As shown in panel A, directional lines with the hook (at 3’ end) are used to represent DNA, and contiguous DNA bases are abstracted into DNA domains (represented by DNA numbers, Table S2). Domain α* is complementary in sequence to domain α, and the same as β* to β. Panel B shows the trypsin-target

DNA

conjugates

reacted

with

FSAMs@DNA-ALP

to

obtain

the

FSAMs@DNA-trypsin. The DNA strand displacement reaction was facilitated by the ‘toehold’ domains β and β*. Toehold β of the trypsin-target DNA conjugates was hybridized with toehold β* of the FSAMs@DNA-ALP to allow the domain α ‘branch migrate’ until domain α of trypsin-target DNA conjugates displaced domain α of FSAMs@DNA-ALP through a series of reversible single nucleotide dissociation and hybridization steps. After the completion reaction of branch migration, the ALP-target DNA conjugates of FSAMs@DNA-ALP were released, and trypsin-target DNA conjugates were immobilized. In order to evaluate the performance of immobilized enzymes by DNA strand displacement reactions on functionalized magnetic nanoparticles, FSAMs@DNA-trypsin was selected for further study, and the immobilized trypsin on FSAMs (FSAMs@trypsin) by glutaraldehyde without DNA linkers was prepared for comparison. All of the synthesis process and the experimental section of the current study are shown in supplementary information.

5



Scheme 1 Schematic diagram for the synthesis of functionalized magnetic nanoparticles and enzymes immobilization by DNA strand displacement.

Scheme 2 Schematic representation for enzyme immobilization procedure in detail by DNA strand displacement.

Characterization of the functionalized magnetic nanoparticles The morphology of the functional nanoparticles was characterized by TEM. As displayed in Fig. 1A, the as-prepared Fe3O4 was spherical and monodisperse with a mean diameter of 480 nm. The Fe3O4@SiO2 (Fig. 1B) with core-shell architecture after silanization with TEOS showed the good dispersity, and the average size was larger than the pristine Fe3O4. Fig. 1C shows that there was no obvious change in morphology and particle size because of the monolayer modification of APTES. The particle size distributions (inset of Fig.1) of the functional nanoparticles at different steps suggested that Fe3O4, Fe3O4@SiO2, and FSAMs nanoparticles had a single peak centered at 480 nm, 560 nm, and 570 nm, respectively. The EDX elemental mapping was carried out, and the results showed that the elements Fe, O, N and C existed in the FSAMs (Fig.1E-H). The XRD spectra of the as-prepared samples are indicated in Fig. 1D. Characteristic diffraction peaks of all samples were assigned to the (220), (311), (400), (422), (511), and (440) planes, which were indexed to the typical cubic iron oxide Fe3O4 (JCPDS, # 65-3107). The average crystallite sizes of the nanoparticles at crystal indices (311) were calculated to be about 18 nm using the Debye-Scherrer equation.39 The XRD patterns reveal that there is no significant difference in the crystal structure after the surface modification with silica, APTES, and enzyme-target DNA conjugates.

6


Page 6 of 25

Page 7 of 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


Fig. 1 TEM images of (A) Fe3O4, (B) Fe3O4@SiO2, (C) FSAMs, and particle size distribution (inset); (D) XRD patterns of (a) Fe3O4, (b) Fe3O4@SiO2, (c) FSAMs, and (d) FSAMs@DNA-trypsin; (E-H) EDX elemental mapping images of Fe, O, N and C of FSAMs.

The FTIR spectra of the functional nanocomposites are presented in Fig. S1A. The band that appeared at 584 cm−1 was related to the Fe–O vibration. The bands that appeared at 950 and 1095 cm-1 were related to the Si–OH and Si–O–Si stretching vibration, respectively. The absorption at 2926, 2856 cm-1 corresponded to the alkyl chain –CH2 stretching vibrations of APTES, while the bands at 1445 cm-1 should be attributed to the N–H stretching vibration in APTES.39, 40 The zeta-potential measurements (Fig.S1B) with the change of the surface charge from negative to positive values and the TGA analysis (Fig. S1C) with a reduction in the masses of the synthesized magnetic materials indicated the successful modification of silica, APTES, and enzymes. Elemental analysis (Table S3) also indicated that the C, H and N contents significantly increased after modification with silica, APTES, and enzymes. The magnetic properties of the prepared magnetic materials were conducted by VSM at 298 K, and the results are shown in Fig. S1D. The magnetic hysteresis curves of the as-prepared materials exhibited no obvious remanence and coercivity with no hysteresis, indicating a typical superparamagnetic feature at room temperature.39 The magnetization saturation (Ms) values of the bare Fe3O4, FSAMs, and FSAMs@DNA-trypsin were 84.3, 50.5, and 46.2 emu g-1, respectively. This decrease was due to the increased nonmagnetic 7



coatings (silica, APTES, and enzymes). However, the functionalized magnetic materials remained highly magnetically responsive. All samples could easily be separated from solution with a magnet within 10 s, and then readily re-dispersed in water by slightly shaking. The hybridization of double-stranded DNA (dsDNA) was confirmed by Gene Finder (GF), which intercalated in situ into dsDNA to produce strong green fluorescence.20, 41 The more complementary base pairs, the stronger the fluorescence. Confocal laser scanning microscopy (CLSM) analysis of the GF/dsDNA mixtures of FSAMs@DNA-ALP revealed a large number of green fluorescent (Fig. 2A). Furthermore, stronger green fluorescence was observed with the GF/dsDNA mixtures of FSAMs@DNA-trypsin (Fig. 2B), and the FITC-labeled trypsin-target DNA conjugates were present in FSAMs@DNA-trypsin (Fig. 2C). These results indicated that the dsDNA had hybridized, and that trypsin-target DNA conjugates had been immobilized by DNA strand displacement reactions. The ALP and trypsin binding capacity by DNA strand displacement reactions was calculated to be 2.47 mg g-1 and 2.24 mg g -1, respectively, and the trypsin binding capacity on FSAMs without DNA linkers (FSAMs@trypsin) was 15.36 mg g-1.

Fig. 2 CLSM images of (A) GF/FSAMs@DNA-ALP hybrids, (B) GF/FSAMs@DNA-trypsin hybrids, and (C) FITC-labeled FSAMs@DNA-trypsin nanoparticles. The GF intercalated into the dsDNA of the FSAMs@DNA-enzyme nanoparticles indicating the successful hybridization of DNA and the immobilization of enzyme on FSAMs@capDNA.

Displacement and immobilization of different enzymes To test the performance of displacement and immobilization of different enzymes of the immobilization enzyme procedure by DNA strand displacement reactions, we employed three types of common enzymes as guest molecules, including ALP, HRP, and trypsin. These enzymes were respectively incubated with target DNA to prepare ALP-target DNA (24bases) conjugates, 8


Page 8 of 25

Page 9 of 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


HRP-target DNA (34bases) conjugates, and trypsin-target DNA (44bases) conjugates. A scheme illustrating the displacement and immobilization of different enzymes is shown in scheme S1. Based on the DNA strand displacement reactions, for HRP immobilization, the above prepared FSAMs@DNA-ALP particles were incubated with HRP-target DNA conjugates to obtain the FSAMs@DNA-HRP. Furthermore, the FSAMs@DNA-trypsin was prepared by incubation of the trypsin-target DNA conjugates with FSAMs@DNA-HRP. In order to characterize the enzymes immobilization by DNA strand displacement reactions, the HRP-target DNA conjugates and trypsin-target DNA conjugates were labeled by fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RhB), respectively. As shown in Fig. 3, the stronger green fluorescence (Fig. 3A) of FSAMs@DNA-HRP and the red fluorescence (Fig. 3C) of FSAMs@DNA-trypsin were observed by confocal laser scanning microscopy (CLSM) compared with the relative dark green fluorescence (Fig. 3B) of FSAMs@DNA-trypsin after trypsin immobilization by DNA strand displacement reactions. The intensity change of the green fluorescence and the appearance of red fluorescence demonstrated that the enzymes have been successfully immobilized by DNA strand displacement reactions and the efficiency of the displacement reaction was ca. 70%. As well, it could be speculated that the immobilized enzyme, which lost its activity after multiple use or was not suitable for the enzymatic analysis, could be easily displaced with any target enzyme for immobilization by rationally designing the DNA sequence to trigger a toehold-mediated DNA strand displacement reaction. Based on this strategy, sequential displacement and immobilization of different enzymes on single FSAMs@capDNA could be achieved easily under mild conditions. Thus, it was revealed that the attractive immobilization strategy by DNA strand displacement proved to be significantly effective for displacement and immobilization of different enzymes for different purpose, throwing light on the promising application in highly efficient enzyme immobilization.

9



Fig. 3 CLSM images of (A) FITC-labeled HRP-target DNA conjugates on FSAMs@DNA-HRP nanoparticles; (B) FITC-labeled HRP-target DNA conjugates on the FSAMs@DNA-trypsin nanoparticles after trypsin immobilization by DNA strand displacement reactions; (C) RhB-labeled trypsin-target DNA conjugates on the FSAMs@DNA-trypsin nanoparticles after trypsin immobilization by DNA strand displacement reactions.

Reversible enzyme immobilization To investigate the surface regeneration and reversible enzyme immobilization of the immobilization procedure (the procedure is shown in scheme S2), the DNA duplex was dehybridized by 0.05 M NaOH to remove the enzyme-target DNA conjugates. As shown in Fig. 4, it was possible to remove the enzyme from the surface of FSAMs@DNA-ALP by dehybridizing the DNA duplex, as indicated by the dramatic decrease observed in the enzymatic activity (less than 1.8%) (cycle 1B, ALP). Similar phenomenon could be observed for trypsin immobilized by DNA strand displacement reactions (less than 2.0%, cycle 1B, Trypsin). Moreover, the reversibility of the enzyme immobilization process and the ability to retrieve the enzymatic activity were demonstrated by consecutive hybridization and dehybridization cycles. The results of these experiments clearly showed that the relative activity of the immobilized enzyme remained greater than 96% (cycle 2A, ALP), 87% (cycle 2A, Trypsin) and less than 2.6% (cycle 2B, ALP), 2.9% (cycle 2B, Trypsin) before and after the dehybridization step, respectively. In order to further confirm the surface regeneration and reversible enzyme immobilization of the immobilization procedure, the ALP-target DNA conjugates and trypsin-target DNA conjugates were labeled by FITC and RhB, respectively. As shown in Fig. S2, the intensity and amount change of the fluorescence demonstrated that the DNA duplex has been dehybridized after treatment with 0.05 M NaOH and the enzyme-target DNA conjugates have been immobilized on the surface of FSAMs@capDNA by DNA strand displacement. These results therefore confirmed that the immobilization enzyme procedure by DNA strand displacement exhibited high reversibility and reproducibility. Furthermore, the reversibility of the enzyme immobilization process, which 10


Page 10 of 25

Page 11 of 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


resulted in the reusability of FSAMs@capDNA for enzyme immobilization by DNA strand displacement, reduced the cost of the synthetic process.

Fig. 4 Reversible enzyme immobilization and surface regeneration of FSAMs@DNA-trypsin by DNA strand displacement. (A) Relative activity of FSAMs@DNA-ALP and FSAMs@DNA-trypsin, cycles 1 and 2 represent the first and second hybridization steps of ALP-target DNA conjugates with FSAMs@capDNA and trypsin-target DNA conjugates with FSAMs@DNA-ALP, respectively; (B) Relative activity after removal of the enzyme-target DNA conjugates from the surface of FSAMs@DNA-enzyme by 0.05 M NaOH, cycles 1 and 2 represent the first and second dehybridization steps, respectively. The high reversibility and reproducibility of the enzyme immobilization strategy were confirmed by two consecutive cycles of enzyme-target DNA conjugate hybridization and dehybridization.

Analysis of enzyme activity It is known that enzyme activity is dependent on the ionization state of the amino acids in the active site, so pH plays a significant role in maintaining the proper conformation of an enzyme.42 The influence of pH on the activity of free trypsin, FSAMs@DNA-trypsin, and FSAMs@trypsin was determined in the range of 5.0 to 10.5, maximum activities of free trypsin, FSAMs@DNA-trypsin, and FSAMs@trypsin were observed at pH values of 7.5, 8.0, and 8.0, respectively (Fig. 5A). The FSAMs@DNA-trypsin sustained higher enzymatic activity over a broad pH range of 8.0-10.5 in comparison with both free trypsin and FSAMs@trypsin. The enzymatic activity of FSAMs@DNA-trypsin was 99.3% at pH 8.5, 76.7% at pH 9.5, and 4.7% at pH 10.5 in comparison with the values of 92.8%, 52.9%, and 1.5% for FSAMs@trypsin at the respective pH. Under similar conditions, it showed 2.6-, 9.6-, and 12.2-fold higher enzymatic activities than the free enzyme, respectively. The difference in pH tolerance for free trypsin, FSAMs@DNA-trypsin, and FSAMs@trypsin was caused by the change of the pendent groups and the enzyme microenvironment. The aldehyde group of glutaraldehyde and the residual amino of enzymes could result in the Schiff base and further form the stable secondary amine formation, 11



Page 12 of 25

which would affect the enzyme active center and give the higher stability of FSAMs@trypsin in pH than that of the free enzyme.42 In addition, the strong covalent bond may also lead to the spatial rigid structure that could affect the intra-molecular forces and the conformation of the enzymes. Furthermore, enzyme immobilized by DNA linkers could expose more active sites of the enzyme and might preserve more stable conformation of the enzyme, giving higher pH tolerance than FSAMs@trypsin and free enzyme. Similar phenomenon that the shift in optimum pH values after immobilization could be observed in the temperature tolerance assay, in which the enzymatic activity of FSAMs@DNA-trypsin also significantly improved at higher temperature compared with free trypsin and FSAMs@trypsin. The optimum temperature for both FSAMs@DNA-trypsin and FSAMs@trypsin was 45°C, which was 10°C higher than the free enzyme (Fig. 5B). The FSAMs@DNA-trypsin

retained

much

higher

enzymatic

activity

compared

to

both

FSAMs@trypsin and free enzyme at a temperature range of 45°C to 70°C, which suggested that the enzyme immobilized on magnetic nanoparticles by DNA linkers possessed more stable three-dimensional structure. Therefore, it can be deduced that the enzyme immobilization procedure based on DNA strand displacement and functionalized magnetic nanoparticles could serve as an ideal platform and remarkably increase the pH and temperature tolerance of the enzyme.

Fig. 5 Relative activity of free trypsin, FSAMs@DNA-trypsin, and FSAMs@trypsin: (A) at different pH values and (B) at different temperature; (C) effects of high temperature, strong alkali, and DNA strand displacement on the enzymatic activity of FSAMs@DNA-ALP.

To further evaluate the immobilized enzymes, the Michaelis−Menten constant (Km) and maximum catalytic rate (Vmax) were applied to determine kinetic parameters.43 Higher Km indicates lower affinity of the substrate toward the enzyme, and Vmax value implies the enzymatic reaction rate. Compared with the free enzymes, the Km values of FSAMs@trypsin and FSAMs@DNA-trypsin were a little higher (see Table 1). The cross-linking by glutaraldehyde led 12


Page 13 of 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


to an increase in Km of FSAMs@trypsin, which might be attributed to the additional steric hindrance and partial loss of enzyme conformation flexibility.44 The decrease in Km of FSAMs@DNA-trypsin compared with FSAMs@trypsin might be attributed to the better mass transfer or change into the more active conformation of the enzyme.24, 45 In contrast, the Vmax value for FSAMs@DNA-trypsin and FSAMs@trypsin were about 5.6 and 4.0 times higher than that of the free trypsin, respectively. The main reason is that the high concentration of trypsin after immobilization was in a very limited space of functionalized magnetic nanoparticles with high surface area-to-volume, and the interactive frequency between trypsin and substrates was also increased by the decreased diffusion within the matrix,45 especially for trypsin immobilization by DNA strand displacement because of the more flexibility of the DNA linkers. Previous report have demonstrated that inward-facing active sites can limit substrate access to the enzyme.46 Enzyme immobilized by DNA strand displacement on the surface of functionalized magnetic nanoparticles exposed more active sites compared with cross-linking by glutaraldehyde; therefore, FSAMs@DNA-trypsin preserved higher Vmax than FSAMs@trypsin.

Table 1 Kinetic parameters of the free trypsin, FSAMs@trypsin, and FSAMs@DNA-trypsin.

Enzymes

Km (mM)

Vmax(mmol min−1 mg protein−1)

Free trypsin FSAMs@trypsin FSAMs@DNA-trypsin

1.14 1.83 1.68

43.61 175.45 244.07

In order to further evaluate the superior performance of the immobilization enzyme strategy designed in this study by DNA strand displacement reactions, effects of strong alkali, high temperature, and DNA strand displacement on enzymatic activity were evaluated through the pre-incubation of FSAMs@DNA-ALP in 0.05 M NaOH or 90°C for 10 min to dehybridize the DNA duplex to release the enzyme-target DNA conjugates, respectively. After the dehybridization, the supernatant and the nanoparticles were collected to evaluate the enzymatic activity, and the initial activity of FSAMs@DNA-ALP was considered as 100%. As shown in Fig.5C, the almost complete loss of enzyme activity of the supernatant (0.7% for strong alkali and 0.4% for high temperature) and the nanoparticles (2.7% for strong alkali and 3.3% for high temperature) was observed by either incubating with strong alkali or high temperature. However, the remaining 13



enzyme activity after immobilization by DNA strand displacement was 50.6% for supernatant and 30.2% for nanoparticles, and the enzyme activity of the supernatant were about 72.3- and 126.5-fold higher than that of the supernatant of strong alkali and high temperature, respectively. The results revealed that although the DNA duplex could be dehybridized by high temperature or alkali, the three-dimensional structure of the enzyme could be destroyed, resulting in the irreversible inactivation of the enzyme. However, the immobilized enzyme procedure by DNA strand displacement reactions carried out under mild conditions had little effect on enzyme activity. The released ALP-target DNA conjugates after immobilizing trypsin by DNA strand displacement could be further reused, which was beneficial for saving the production cost, thereby confirming the superiority of this system. Stability test High enzyme stability is important for applications. Deactivation of enzymes by high temperature is the major reason for enzyme inactivation.47 The tolerance of high temperature of FSAMs@DNA-trypsin nanoparticles was investigated at temperatures of 50, 60, and 70°C for 90 min with 15 min intervals and compared with that of the free enzyme and FSAMs@trypsin. As shown in Figure 6A and S3, FSAMs@DNA-trypsin could retain a high degree of activity (more than 94%) at 50°C after 60 min incubation compared to the 50% and 25% enzymatic activities for FSAMs@trypsin and free trypsin, respectively, and could still exhibited promising enzymatic activities of about 83% and 52% after 45 min incubation at 60°C and 70°C, which were about 3.1and 2.3-fold greater than free trypsin (2.9- and 17.3-fold higher than FSAMs@trypsin) at the respective temperatures. The thermal deactivation constants (kd) and half-lives (t1/2) of free trypsin, FSAMs@DNA-trypsin, and FSAMs@trypsin are presented in Table S4. The kd value of FSAMs@DNA-trypsin was significantly lower than that of both free trypsin and FSAMs@trypsin. The half-lives (t1/2) of FSAMs@DNA-trypsin were 216.6 min, 85.6 min, and 33.6 min at 50 °C, 60 °C, and 70 °C, respectively, which were about 5.4-, 2.3-, and 1.3-fold greater than that of the free trypsin at the respective temperatures. It has been demonstrated that immobilized enzymes always have more stable structural configurations than the free ones, especially under harsh conditions.48 Furthermore, magnetic nanoparticles are good absorbers of the radiation, contributing to higher temperature at their surface, which inevitably influences the enzyme 14


Page 14 of 25

Page 15 of 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


structure to some extent.24 However, because of the high stability and mechanical rigidity of the DNA linkers,28 enzyme immobilized by DNA strand displacement gives the possibility that enzymes were not directly immobilized on the surface of modified magnetic nanoparticles. These understandings can explain why FSAMs@DNA-trypsin became heat-resistance in comparison with

FSAMs@trypsin

and

the

native

ones.

The

results

therefore

revealed

that

FSAMs@DNA-trypsin possessed remarkably higher stability towards temperature, and the enzymatic reactions could be achieved at high temperatures, which was beneficial for improving the reaction efficiency. The long-term incubation stability of free trypsin, FSAMs@DNA-trypsin, and FSAMs@trypsin was investigated through the incubation of enzymes at 37°C for different time. As shown in Figure 6B, the free trypsin lost more than 98% of its enzymatic activity only after 6 h, however, the FSAMs@DNA-trypsin and FSAMs@trypsin still preserved fabulous enzymatic activities of about 80% and 70%, respectively. After 12 h of incubation, the FSAMs@DNA-trypsin maintained more than 75% activity compared to the complete loss of activity for free trypsin, and could still preserve 22% of original activity after incubation for 96 h. The results demonstrated that FSAMs@DNA-trypsin possessed much stronger tolerance towards the long-term incubation, superior to the free enzyme. In addition, the long-term storage stability of enzymes was carried out under storage conditions at 4°C and room temperature for different days. It was worthy noted that the FSAMs@DNA-trypsin nanoparticles preserved more than 73% of the initial activity at 4°C for 88 days and 60% at room temperature for 29 days, respectively, superior to FSAMs@trypsin (Fig. 6C and S4). The loss of enzymatic activity in this period might be attributed to protein denaturation and degradation during long-term storage. However, the free trypsin lost more than 64% and 99% of the overall activity under the same conditions after 9 days at 4°C and 7 days at room temperature, respectively. Previous studies have reported that enzyme immobilization on magnetic nanoparticles could help the enzyme molecules to be well dispersed on the material substrates to improve their biological activity, and the immobilized enzymes contributed to the decreased auto-digestion during the storage period and the increased stability of the enzyme compared with native ones.19, 24, 48 Enzymes directly immobilized on the surface of solid substrates for a long time generally denature to give unfolded proteins, which are much more susceptible by successive incubation or storage, and the reduction of enzyme activity may be caused by the 15



irreversible conformation change of the enzyme during cross-linking by glutaraldehyde, as observed by other researchers.49, 50 However, the high physicochemical stability and pronounced mechanical rigidity of the DNA linkers between the enzymes and magnetic nanoparticles could protect the enzyme being away from the magnetic nanoparticles and preserve more stable conformation flexibility, giving higher stability for long-term reaction. Therefore, the long-term incubation stability and the long-term storage stability of the FSAMs@DNA-trypsin were enhanced significantly by the immobilized enzyme on FSAMs via DNA strand displacement reactions. It was therefore revealed that the FSAMs@DNA-trypsin was an attractive platform for long-term reactions with little loss of performance. The tolerance of organic solvents of free trypsin and FSAMs@DNA-trypsin nanoparticles was investigated through the incubation of enzymes with different commonly available solvents (25% v v-1). The FSAMs@DNA-trypsin nanoparticles possessed excellent tolerance towards all tested solvents as compared with the free enzyme (Fig. 7). The free trypsin almost completely lost of its activity after incubation for 4 h; however, the FSAMs@DNA-trypsin could still showed a high degree of activity (more than 81%) for almost all tested solvents. The FSAMs@DNA-trypsin possessed excellent tolerance towards DMF among these solvents, retaining approximately 11-fold higher enzymatic activity than the free enzyme. Furthermore, a significant increase in stability of the tolerance of organic solvents (more than 7-fold for free enzyme) was retained by the FSAMs@DNA-trypsin through either increasing the incubation time or altering the concentration of DMF (Fig. S5). Organic solvents have been known to be helpful for the unfolding of proteins,51 so the deactivation of free trypsin was assisted by incubating with the organic solvents. However, the immobilization procedure by DNA strand displacement on functionalized magnetic nanoparticles could protect the enzyme from deactivation in organic solvents by decreasing the probability of the immobilized proteins to undergo denaturing unfolding–refolding motions. These results reported in this study demonstrated that the immobilized trypsin exhibited excellent solvents stability properties than free trypsin, and that this immobilized trypsin system by DNA strand displacement on functionalized magnetic nanoparticles therefore represented an attractive platform for prospective applications and a significant reduction in the cost of the overall process.

16


Page 16 of 25

Page 17 of 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


Fig. 6 Stability of the FSAMs@DNA-trypsin nanoparticles compared with the equivalent free enzymes and FSAMs@trypsin: (A) tolerance of high temperature at 50°C, (B) long-term incubation at 37°C, and (C) long-term storage at 4°C; (D) reusability of FSAMs@DNA-trypsin and FSAMs@trypsin.

Fig. 7 Tolerance of the FSAMs@DNA-trypsin and free trypsin towards different organic solvents.

Reusability test The reusability of immobilized enzyme is a crucial aspect in bio-applications and also plays a key role in its economic significance. The nanoparticles prepared in this study could be easily collected by a magnet after the reaction within 10 s, and could be re-dispersed to the nanoscale size by slightly shaking. As shown in Fig. 6D, the FSAMs@DNA-trypsin nanoparticles 17



Page 18 of 25

maintained more than 93% of their original enzymatic activity after 5 cycles and still showed a high degree of activity (more than 87%) after 10 cycles in comparison with 83% and 70% for the FSAMs@trypsin, respectively. The decrease about the enzyme activity was often observed for the immobilized biocatalyst after repeated usage, which might be responsible for the low leaching, weak

autodigestion,

and

gradual

denaturation

of

the

enzyme.24

However,

the

FSAMs@DNA-trypsin system prepared in this study still showed excellent reusability especially in comparison with previous reports that the loss of enzymatic activity of the immobilized enzymes was 23% after the 7 cycle,52 39% after 7 cycles,24 and up to 60% after 6 cycles.53 The excellent reusability of FSAMs@DNA-trypsin indicated that the immobilized enzyme strategy on functionalized magnetic nanoparticles could retain the enzyme activity well and decrease the loss or inactivation of the enzyme after multiple reuses. Protein analysis In order to investigate the digestion efficiency of the immobilized trypsin by DNA strand displacement reactions, a protein solution of myoglobin (Myo) was chosen as a model protein for FSAMs@DNA-trypsin digestion compared with the conventional solution-based digestion using free trypsin. The digestion procedure is illustrated in scheme S3. The FSAMs@DNA-trypsin nanoparticles could be easily retrieved from solutions using an external magnet after digestion, and the supernatant solution was directly analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). As shown in Fig. 8, Table 2, and Table S5, sequence coverage turned out to be 95% corresponding to 21 matched peptides within 5 min, superior to those obtained with free trypsin after 12 h (62% and 14, respectively). These results therefore highlighted the higher digestion efficiency of FSAMs@DNA-trypsin compared with many other immobilized trypsin nanomaterials.54-57 The great improvement with better matched peptides and faster digestion time was achieved by FSAMs@DNA-trypsin compared with native ones because of the high enzyme concentration in the reaction area and improved enzymatic activity after immobilization. Furthermore, recent works have demonstrated the importance of active site orientation for the enzymatic activity in single-enzyme systems immobilized on a surface,58 and proteins immobilization through the DNA linkers can expose more their active sites to give better performance.28 Therefore, the significantly improved digestion efficiency of 18


Page 19 of 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


FSAMs@DNA-trypsin toward Myo was achieved by the immobilization procedure on magnetic nanoparticles based on toehold-mediated DNA strand displacement. The glycated hemoglobin (HbA1c) and cytochrome C (Cyt-C) as potential substrates were further investigated to evaluate the digestion performance of the FSAMs@DNA-trypsin nanoparticles. As shown in Fig. S6 for the digestion of HbA1c, we still observed a higher sequence coverage and the better matched peptides compared with free trypsin after 12 h (Table 2 and Table S6). As shown in Fig. S7, Table 2, and Table S7, similar results were also observed using Cyt-C as a model protein substrate. The FSAMs@DNA-trypsin could offer higher enzymatic activity and faster digestion compared with other immobilized trypsin reactors,21, 41, 54, 55

thereby confirming the superiority of this system. These results indicated that

FSAMs@DNA-trypsin could be potentially used for high throughput protein digestion.

Fig. 8 MALDI-TOF mass spectra of the supernatant from (A) FSAMs@DNA-trypsin and (B) free trypsin digestion of Myo, respectively. The peaks marked with M represent matched peptides originating Myo.

Table 2 MALDI-TOF-MS results obtained by FSAMs@DNA-trypsin and free enzyme (n = 3) Protein Digestion method

Myo

HbA1c

Cyt-c

Present

Free

Present

Free

Present

Free

study

enzyme

study

enzyme

study

enzyme

19



Page 20 of 25

Digestion time

5 min

12 h

10 min

12 h

10 min

12 h

Sequence coverage (%)

95

62

85

63

86

54

Peptides matched

21

14

16

11

21

10

Conclusions In summary, we presented a novel mild highly efficient enzyme immobilization strategy based on toehold-mediated DNA strand displacement on functionalized Fe3O4 magnetic nanoparticles. The strategy carried out under mild conditions had little effect on enzyme activity compared with high temperature or alkali and achieved dynamic immobilization of enzymes with high reversibility and reproducibility. The results confirmed that immobilized enzymes by the developed immobilization strategy were quite effective compared with other previous reports. The immobilized trypsin in this study exhibited excellent long-term storage and incubation stability, thermal stability, pH-stable, organic solvents-tolerant along with potential applications in high throughput enzymatic assays and proteome analysis, as compared with the free enzyme. Based on this strategy, sequential displacement and immobilization of different enzymes were achieved, and it could be speculated that the immobilized enzyme, which lost its activity after multiple use or was not suitable for the enzymatic analysis, could be easily displaced with any target enzyme by rationally designing the DNA sequence to trigger a toehold-mediated DNA strand displacement reaction. The developed strategy in the current study exhibited a promising alternative platform with high magnetic responsiveness and significantly enhanced properties for the immobilization of industrially important enzymes and their broad applications.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant No. 21675008), the Beijing Natural Science Foundation (Grant No. 2132048) and the Fundamental Research Funds for the Central Universities (Grant No. JD1516).

AUTHOR INFORMATION *Corresponding

author:

Yi

Yang,

Ping

Su.

Email:

[email protected]; Tel: +86-010-64441521. 20


[email protected];

Page 21 of 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


ASSOCIATED CONTENT Supporting Information: Experimental section and supporting figures.

REFERENCES 1.

Wang, Z.; Huang, P.; Bhirde, A.; Jin, A.; Ma, Y.; Niu, G.; Neamati, N.; Chen, X., A nanoscale

graphene oxide–peptide biosensor for real-time specific biomarker detection on the cell surface. Chem. Commun. 2012, 48, 9768-9770. 2.

DiCosimo, R.; McAuliffe, J.; Poulose, A. J.; Bohlmann, G., Industrial use of immobilized

enzymes. Chem. Soc. Rev. 2013, 42, 6437-6474. 3.

Zhang, Y.; Arugula, M. A.; Wales, M.; Wild, J.; Simonian, A. L., A novel layer-by-layer assembled

multi-enzyme/CNT

biosensor

for

discriminative

detection

between

organophosphorus

and

non-organophosphrus pesticides. Biosens. Bioelectron. 2015, 67, 287-295. 4.

Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.; Moore, J. C.; Robins, K.,

Engineering the third wave of biocatalysis. Nature 2012, 485, 185-194. 5.

Zhanga, Y.; Gea, J.; Zheng, L., Enhanced activity of immobilized or chemically modified

enzymes. ACS Catal. 2015, 5, 4503-4513. 6.

Kuwahara, Y.; Yamanishi, T.; Kamegawa, T.; Mori, K.; Che, M.; Yamashita, H., Lipase-embedded

silica nanoparticles with oil-filled core–shell structure: stable and recyclable platforms for biocatalysts. Chem. Commun. 2012, 48, 2882-2884. 7.

Hermanová, S.; Zarevúcká, M.; Bouša, D.; Pumera, M.; Sofer, Z., Graphene oxide immobilized

enzymes show high thermal and solvent stability. Nanoscale 2015, 7, 5852-5858. 8.

Rezaei, A.; Akhavan, O.; Hashemi, E.; Shamsara, M., Ugi four-component assembly process: an

efficient approach for one-Pot multifunctionalization of nanographene oxide in water and Its application in lipase immobilization. Chem. Mater. 2016, 28, 3004-3016. 9.

Zhang, G.; Ma, J.; Wang, J.; Li, Y.; Zhang, G.; Zhang, F.; Fan, X., Lipase immobilized on

graphene oxide as reusable biocatalyst. Ind. Eng. Chem. Res. 2014, 53, 19878-19883. 10. Lu, C.; Liu, X.; Li, Y.; Yu, F.; Tang, L.; Hu, Y.; Ying, Y., Multifunctional janus hematite-silica nanoparticles: mimicking peroxidase-like activity and sensitive colorimetric detection of glucose. ACS Appl. Mater. Interfaces 2015, 7, 15395-15402. 11. Santos-Moriano, P.; Monsalve-Ledesma, L.; Ortega-Munoz, M.; Fernandez-Arrojo, L.; Ballesteros, A. O.; Santoyo-Gonzalez, F.; Plou, F. J., Vinyl sulfone-activated silica for efficient covalent immobilization of alkaline unstable enzymes: application to levansucrase for fructooligosaccharide synthesis. RSC Adv. 2016, 6, 64175-64181. 12. Zhou, Z.; Hartmann, M., Progress in enzyme immobilization in ordered mesoporous materials and related applications. Chem. Soc. Rev. 2013, 42, 3894-3912. 13. Jiang, Y.; Shi, L.; Huang, Y.; Gao, J.; Zhang, X.; Zhou, L., Preparation of robust biocatalyst based on cross-linked enzyme aggregates entrapped in three-dimensionally ordered macroporous silica. ACS Appl. Mater. Interfaces 2014, 6, 2622-2628. 14. Shieh, F.-K.; Wang, S.-C.; Yen, C.-I.; Wu, C.-C.; Dutta, S.; Chou, L.-Y.; Morabito, J. V.; Hu, P.; Hsu, M.-H.; Wu, K. C. W.; Tsung, C.-K., Imparting functionality to biocatalysts via embedding

21



enzymes into nanoporous materials by a de novo approach: size-selective sheltering of catalase in metal-organic framework microcrystals. J. Am. Chem. Soc. 2015, 137, 4276-4279.. 15. Li, P.; Moon, S.-Y.; Guelta, M. A.; Harvey, S. P.; Hupp, J. T.; Farha, O. K., Encapsulation of a nerve agent detoxifying enzyme by a mesoporous zirconium metal-organic framework engenders thermal and long-term stability. J. Am. Chem. Soc. 2016, 138, 8052-8055. 16. Kim, J. H.; Hong, S.-G.; Wee, Y.; Hu, S.; Kwon, Y.; Ha, S.; Kim, J., Enzyme precipitate coating of pyranose oxidase on carbon nanotubes and their electrochemical applications. Biosens. Bioelectron. 2017, 87, 365-372. 17. Kowalewska, B.; Jakubow, K., The impact of immobilization process on the electrochemical performance, bioactivity and conformation of glucose oxidase enzyme. Sens. Actuators, B 2017, 238, 852-861. 18. Wu, W.; Jiang, C. Z.; Roy, V. A. L., Designed synthesis and surface engineering strategies of magnetic iron oxide nanoparticles for biomedical applications. Nanoscale 2016, 8, 19421-19474. 19. Yang, Y.; Zhu, G.; Wang, G.; Li, Y.; Tang, R., Robust glucose oxidase with a Fe3O4@C-silica nanohybrid structure. J. Mater. Chem.B 2016, 4, 4726-4731. 20. Yang, Y.; Su, P.; Zheng, K.; Wang, T.; Song, J.; Yang, Y., A self-directed and reconstructible immobilization strategy: DNA directed immobilization of alkaline phosphatase for enzyme inhibition assays. RSC Adv. 2016, 6, 36849-36856. 21. Zhao, M.; Zhang, X.; Deng, C., Rational synthesis of novel recyclable Fe3O4@MOF nanocomposites for enzymatic digestion. Chem. Commun. 2015, 51, 8116-8119. 22. Wang, S.; Su, P.; Huang, J.; Wu, J.; Yang, Y., Magnetic nanoparticles coated with immobilized alkaline phosphatase for enzymolysis and enzyme inhibition assays. J. Mater. Chem.B 2013, 1, 1749-1754. 23. Atacan, K.; Cakiroglu, B.; Ozacar, M., Improvement of the stability and activity of immobilized trypsin on modified Fe3O4 magnetic nanoparticles for hydrolysis of bovine serum albumin and its application in the bovine milk. Food Chem. 2016, 212, 460-468. 24. Shen, Y.; Guo, W.; Qi, L.; Qiao, J.; Wang, F.; Mao, L., Immobilization of trypsin via reactive polymer grafting from magnetic nanoparticles for microwave-assisted digestion. J. Mater. Chem.B 2013, 1, 2260-2267. 25. Shao, Y.; Jing, T.; Tian, J.; Zheng, Y., Graphene oxide-based Fe3O4 nanoparticles as a novel scaffold for the immobilization of porcine pancreatic lipase. RSC Adv. 2015, 5, 103943-103955. 26. Hu, T.-G.; Cheng, J.-H.; Zhang, B.-B.; Lou, W.-Y.; Zong, M.-H., Immobilization of alkaline protease on amino-functionalized magnetic nanoparticles and its efficient use for preparation of oat polypeptides. Ind. Eng. Chem. Res. 2015, 54, 4689-4698. 27. Wang, H.; Zhang, W.; Zhao, J.; Xu, L.; Zhou, C.; Chang, L.; Wang, L., Rapid decolorization of phenolic azo dyes by immobilized laccase with Fe3O4/SiO2 nanoparticles as support. Ind. Eng. Chem. Res. 2013, 52, 4401-4407. 28. Niemeyer, C. M., Semisynthetic DNA–protein conjugates for biosensing and nanofabrication. Angew. Chem., Int. Ed. 2010, 49, 1200-1216. 29. Fruk, L.; Mueller, J.; Weber, G.; Narvaez, A.; Dominguez, E.; Niemeyer, C. M., DNA-directed immobilization of horseradish peroxidase-DNA conjugates on microelectrode arrays: towards electrochemical screening of enzyme libraries. Chem. - Eur. J. 2007, 13, 5223-5231. 30. Zhang, D. Y.; Seelig, G., Dynamic DNA nanotechnology using strand-displacement reactions. Nat. Chem. 2011, 3, 103-113. 22


Page 22 of 25

Page 23 of 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


31. Schröder, H.; Hoffmann, L.; Müller, J.; Alhorn, P.; Fleger, M.; Neyer, A.; Niemeyer, C. M., Addressable microfluidic polymer chip for DNA‐directed immobilization of oligonucleotide‐tagged compounds. Small 2009, 5, 1547-1552. 32. Fruk, L.; Kuhlmann, J.; Niemeyer, C. M., Analysis of heme-reconstitution of apoenzymes by means of surface plasmon resonance. Chem. Commun. 2009, 230-232. 33. Zhang, D. Y.; Winfree, E., Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 2009, 131, 17303-17314. 34. Monserud, J. H.; Macri, K. M.; Schwartz, D. K., Toehold-mediated displacement of an adenosine-binding aptamer from a DNA duplex by its ligand. Angew. Chem., Int. Ed. 2016, 55, 13710-13713. 35. Song, T.; Garg, S.; Mokhtar, R.; Bui, H.; Reif, J., Analog computation by DNA strand displacement circuits. ACS Synth. Biol. 2016, 5, 898-912. 36. Liao, R.; He, K.; Chen, C.; Chen, X.; Cai, C., Double-strand displacement biosensor and quencher-free fluorescence strategy for rapid detection of microRNA. Anal. Chem. 2016, 88, 4254-4258. 37. Bi, S.; Yue, S.; Wu, Q.; Ye, J., Initiator-catalyzed self-assembly of duplex-looped DNA hairpin motif based on strand displacement reaction for logic operations and amplified biosensing. Biosens. Bioelectron. 2016, 83, 281-286. 38. Yang, X.; Tang, Y.; Traynor, S. M.; Li, F., Regulation of DNA strand displacement using an allosteric DNA toehold. J. Am. Chem. Soc. 2016, 138, 14076-14082. 39. Wu, J.; Su, P.; Yang, Y.; Huang, J.; Wang, Y.; Yang, Y., Immobilization of HSA on polyamidoamine-dendronized magnetic microspheres for application in direct chiral separation of racemates. J. Mater. Chem.B 2014, 2, 775-782. 40. Lei, C.; Xu, C.; Nouwens, A.; Yu, C., Ultrasensitive ELISA+ enhanced by dendritic mesoporous silica nanoparticles. J. Mater. Chem.B 2016, 4, 4975-4979. 41. Song, J.; Su, P.; Yang, Y.; Wang, T.; Yang, Y., DNA directed immobilization enzyme on polyamidoamine tethered magnetic composites with high reusability and stability. J. Mater. Chem.B 2016, 4, 5873-5882. 42. Wang, S.; Su, P.; Ding, F.; Yang, Y., Immobilization of cellulase on polyamidoamine dendrimer-grafted silica. J. Mol. Catal. B Enzym. 2013, 89, 35-40. 43. Pla-Tolos, J.; Moliner-Martinez, Y.; Molins-Legua, C.; Campins-Falco, P., Colorimetic biosensing dispositive based on reagentless hybrid biocomposite: Application to hydrogen peroxide determination. Sens. Actuators, B 2016, 231, 837-846. 44. Zhang, Y.; Yong, Y.; Ge, J.; Liu, Z., Lectin agglutinated multienzyme catalyst with enhanced substrate affinity and activity. ACS Catal. 2016, 6, 3789-3795. 45. Huang, Y.; Shan, W.; Liu, B.; Liu, Y.; Zhang, Y.; Zhao, Y.; Lu, H.; Tang, Y.; Yang, P., Zeolite nanoparticle modified microchip reactor for efficient protein digestion. Lab. Chip. 2006, 6, 534-539. 46. Lin, J.-L.; Palomec, L.; Wheeldon, I., Design and analysis of enhanced catalysis in scaffolded multienzyme cascade reactions. ACS Catal. 2014, 4, 505-511. 47. Liang, H.; Jiang, S.; Yuan, Q.; Li, G.; Wang, F.; Zhang, Z.; Liu, J., Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection. Nanoscale 2016, 8, 6071-6078. 48. Rodrigues, R. C.; Ortiz, C.; Berenguer-Murcia, A.; Torres, R.; Fernandez-Lafuente, R., Modifying enzyme activity and selectivity by immobilization. Chem. Soc. Rev. 2013, 42, 6290-6307. 23



49. Lopez-Gallego, F.; Betancor, L.; Mateo, C.; Hidalgo, A.; Alonso-Morales, N.; Dellamora-Ortiz, G.; Guisan, J. M.; Fernandez-Lafuente, R., Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports. J. Biotechnol. 2005, 119, 70-75. 50. Migneault, I.; Dartiguenave, C.; Bertrand, M. J.; Waldron, K. C., Glutaraldehyde: Behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. Biotechniques. 2004, 37, 790-796,798-802. 51. Chen, W.-Y.; Chen, Y.-C., Acceleration of microwave-assisted enzymatic digestion reactions by magnetite beads. Anal. Chem. 2007, 79, 2394-2401. 52. Bayramoğlu, G.; Yılmaz, M.; Şenel, A. Ü.; Arıca, M. Y., Preparation of nanofibrous polymer grafted magnetic poly(GMA-MMA)-g-MAA beads for immobilization of trypsin via adsorption. Biochem. Eng. J. 2008, 40, 262-274. 53. Goradia, D.; Cooney, J.; Hodnett, B. K.; Magner, E., The adsorption characteristics, activity and stability of trypsin onto mesoporous silicates. J. Mol. Catal. B: Enzym. 2005, 32, 231-239. 54. Jiang, H.; Yuan, H.; Liang, Y.; Xia, S.; Zhao, Q.; Wu, Q.; Zhang, L.; Liang, Z.; Zhang, Y., A hydrophilic immobilized trypsin reactor with N-vinyl-2-pyrrolidinone modified polymer microparticles as matrix for highly efficient protein digestion with low peptide residue. J. Chromatogr. A 2012, 1246, 111-116. 55. Jiang, B.; Yang, K.; Zhang, L.; Liang, Z.; Peng, X.; Zhang, Y., Dendrimer-grafted graphene oxide nanosheets as novel support for trypsin immobilization to achieve fast on-plate digestion of proteins. Talanta 2014, 122, 278-284. 56. Wei, L.; Zhang, W.; Lu, H.; Yang, P., Immobilization of enzyme on detonation nanodiamond for highly efficient proteolysis. Talanta 2010, 80, 1298-1304. 57. Li, Y.; Yan, B.; Deng, C.; Tang, J.; Liu, J.; Zhang, X., On-plate digestion of proteins using novel trypsin-immobilized magnetic nanospheres for MALDI-TOF-MS analysis. Proteomics 2007, 7, 3661-3671. 58. Liu, Y.; Ogorzalek, T. L.; Yang, P.; Schroeder, M. M.; Marsh, E. N. G.; Chen, Z., Molecular orientation of enzymes attached to surfaces through defined chemical linkages at the solid-liquid interface. J. Am. Chem. Soc. 2013, 135, 12660-12669.

24


Page 24 of 25

Page 25 of 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


Table of Contents Graphic

A promising strategy for enzyme immobilization based on toehold-mediated DNA strand displacement on functionalized magnetic nanoparticles was developed for the first time in this study.

25


Based on DNA Strand Displacement and Functionalized Magnetic

Recommend Documents