New Strategy for Ultrasensitive Aptasensor Fabrication: DâAâD

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New Strategy for Ultrasensitive Aptasensor Fabrication: D-A-D Constitution as Charge Transfer Platform and Recognition Element Zhiqian Xu, Tingting Zhang, Yue Gu, Futong Liu, He Liu, Nannan Lu, Haixin Xu, Xiaoyi Yan, Zhiquan Zhang, and Ping Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05689 • Publication Date (Web): 22 Apr 2019 Downloaded from http://pubs.acs.org on April 22, 2019

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ACS Applied Materials & Interfaces

New Strategy for Ultrasensitive Aptasensor Fabrication: D-A-D Constitution as Charge Transfer Platform and Recognition Element

Zhiqian Xua, Tingting Zhanga, Yue Gua, Futong Liub, He Liua, Nannan Lua, Haixin Xua, Xiaoyi Yana, Zhiquan Zhanga*, Ping Lub*

a College bState

of Chemistry, Jilin University, Changchun 130012, P. R. China

Key Laboratory of Supramolecular Structure and Materials, Jilin University,

Changchun 130012, P. R. China

* Corresponding

author. Tel: +86-431-85168352-7; Fax: +86-431-85168399

E-mail address: [email protected] (Z. Zhang); [email protected] (P. Lu)

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Over the past decade, various sensing systems based on aptamers have attracted a great deal of studies directed to the design of high selective biosensors. In this paper, we described a new-style electrochemical aptamer sensor (aptasensor) via donor-acceptor link substrate, which was characterized by electrochemical methods and other helpful characterization instruments. Molecules with D-A-D configuration always undergo an intrinsic signal amplification due to the elongation of the π electron conjugation. Triphenylamine, a peripheral electron donor, has excellent hole-transport property and is able to assemble on the surface of GCE (glassy carbon electrode) by π-π stacking interaction. To further improve the performance of the ATP sensor, we chose diphenylfumaronitrile containing electron withdrawing group as central core to promote charge transfer which can also combine with aptamers by multi-hydrogen bond function efficiently. Surprisingly, the sensing platform showed a wide liner range from 0.1 pM to 100 nM with a detection limit of 0.018 pM. We examined the ATP in human serum sample, indicating that the novel aptasensor based on D-A-D conjugated polymer holds a great possibility for practical detection of ATP. Moreover, it is foreseeable that the conjugated polymers of D-A structure will have a promising application in the preparation of biosensors.

Keywords: donor-acceptor-donor (D-A-D) structure; charge transfer; adenosine triphosphate; aptamer; EIS


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1. Introduction Adenosine triphosphate (ATP), supplying energy for physiology, has long been playing an important role in organic activities1. The abnormality of ATP concentrations may lead to various diseases, injury of cells, and irregular metabolic activities2. At the same time, ATP can be used as subsidiary medication for the clinical treatment of muscle atrophy, myocardial diseases, hepatitis and so on3. Thus, it is necessary to make a rapid and accurate determination of ATP content in a physiological environment. Up to now, there have been a number of methods to determine this small molecule, such as fluorescence assays, electrophoresis methods, surface-enhanced Raman scattering, high performance liquid chromatography and bioluminescent methods4-8. Nevertheless, lots of regular measurements are time-consuming resulting in unsatisfied sensitivity. Among them, bioluminescent assay based on luciferase is a well-established technique which has been extensively used to detect ATP with the advantages of rapid response and high sensitivity9. In fact, bioluminescence detection relies on the interaction of ATP with luciferin by the catalysis of firefly luciferase10. However, the luciferase cannot be reused and is prone to inactivation, which greatly limits the application of this method. Therefore, it is necessary to establish an ATP measurement system with superior sensitivity and stable reproducibility. In recent years, electrochemical biosensors based on aptamers have caused extensive attention to quantitate biological molecules with high selectivity11-12. Aptamer (Apt) is originated from combinatorial oligonucleotide libraries by SELEX (systematic



evolution of ligands by exponential enrichment), which are able to build a forceful attachment with specific targets13-14. It is capable of specific recognition because aptamers can produce a conformational fold after binding to the analyte which is similar to antigen-antibody interaction with a high precision15. Due to these specificities, we constructed an electrochemical aptamer measurement system. Reviewing the pioneering literatures, conventional electrode stuff prepared for electrochemical biosensors are nanoparticles, carbon materials, metal organic frameworks and their derivative composites16-19. In general, these materials merely served as a sustaining matrix to bind electroactive substances for further recognition, and this may complicate the fabrication process. In consideration of various electrode materials, conjugated polymers (CPs) have come into our sight due to their unique electrical properties and the ability to carry electroactive substances20-21. Over the past decades, CPs have been maturely used as promising functional materials in light emitting diodes, solar cells, tunable conductive networks and the like22-24. Recently, some photochemically stable CPs have been utilized for biological applications25-26. CPs refer to a class of high molecular polymers having π-π conjugated electronic structures which allows electrons to migrate along the main chain freely, acting like a "molecular wire". And the polymers in the form of molecular wire may increase the response signal by several times compared to the single molecule. For further analysis utilization, it has also performed as an indispensable category for tracing biomolecules in fluorescent platforms27-29. It is reported that comprehensive applications in optoelectronics as well as charge transfer researches relevant to both


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biological and chemical fields made this a vibrant topic30. Among the various kinds of organic conjugated polymers, donor-acceptor-donor (D-A-D) linked CPs with push-pull electronic groups were widely used in the field of photoelectricity due to their certain band gaps, adjustable properties and flexible molecular arrangment31. According to different backbone structures, D-A polymers can be roughly divided into several types, such as thiophene, fluorene, aniline, carbazole, pyrrole and others consisting of alternating saturated and unsaturated bonds (double or triple bonds). D-A-D link plays a key role in charge separation and molecular architecture which ultimately influences the charge transport. Compared with commonly used electrode materials, the sensor based on donor-acceptor constitution can amplify the response signal and get ultra-low detection limits efficiently when connecting with the analytes. The unique delocalized π-electron system provides a channel for electrons and holes to transport. In addition, the energy level structure can be effectively adjusted by changing the number and connection modes of donors or acceptors to obtain more satisfactory electrochemical properties. With the deepening of research, more D-A conjugated polymers have been synthesized. However, D-A-D structure were rarely reported for the manufacture of electrocatalytic techniques32-33. In order to construct the desired sensing device, we chose triphenylamine (TPA) as the donor moiety due to its low ionization potentials and excellent hole transport capability. Notably, the propeller-like TPA involves three benzene rings surrounding a nitrogen atom with the lone pair which has a good electron donating ability to intramolecular charge transfer34. Simultaneously, triphenylamine also keeps an



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amorphous film-forming capacity which can be utilized to self-polymerize on the electrode surface stably35. As a functional material, TPA has found its application in aggregation induced emission (AIE) widely36. However, rarely research reports its application in electrochemical sensors. Diphenylfumaronitrile (DBFN) unit is prone to be an acceptor part owing to its two cyano functionalities substituted on the vinylene bond inserted in the middle of two phenylene groups, which can be incorporated as π-linker37. We combine the two units

to

build

an

organic

π-conjugated

system,

2,3-bis(4′-(diphenylamino)-[1,1′-biphenyl]-4-yl)fumaronitrile (TPATCN), which was been first employed to amplify the impedance signal for electrochemical aptamer sensor. Subsequently, aptamer chains with p-rich electronic structure can be combined by aromatic groups and cyano groups through hydrogen interactions38. Compared with previous literatures, the wonderful performance of TPATCN is attributed to the following features: (1) The connected aromatic rings in the CPs allow a stable attachment to the electrode surface. (2) The D-A substrate can also provide favorable sites for aptamer growth via hydrogen bonds interactions. (3) The substantial intrachain charge transfer in the nontrivial D-A architecture brings active signal transmission. (4) CPs supplied a promising bioaffinity for binding aptamers. Our work can not only build an ultrasensitive aptasensor of ATP but also provide wider application potential of D-A-D constitution for further biosensing construction and medical diagnostics. 2. Experimental


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2.1 Reagents and Apparatus This section can be found in the Supporting Information. 2.2 Synthesis of the TPATCN The synthetic route of TPATCN was depicted in Scheme S1 (Supporting Information) according to Lu's group39. The 2,3-bis(4-bromophenyl)fumaronitrile (M2) was attained with an efficient productivity according to previous reports40-41. Then, M2 was coupled with (4-(diphenylamino)phenyl)boronic acid (M3) via Suzuki reaction to afford the target compound TPATCN with a high yield. The purity and chemical structure of TPATCN were well characterized by NMR, FTIR, UV and XPS analysis. 2.3 Fabrication of the ATP aptasensor The attempt to exploit D-A-D polymers for creating aptasensor was summarized in Scheme 1. Firstly, the bare GCE was polished with the 0.3 and 0.05 µm alumina slurry and then ultra-sonicated in nitric acid (1/1, ν/ν), 100 % ethanol and ultrapure water for 3 min successively. Afterwards, the polished GCE was dried by nitrogen steam and 10 µL of the TPATCN solution (tetrahydrofuran, 0.5 mg mL-1) was dropped on the surface. After the conjugated polymer assembled in the air, 10 µL of 2 µM aptamer (10 mM Tris buffer, 7.4) was coated on the TPATCN/GCE to incubate for 12 h at room temperature in a humid environment. Subsequently, the electrode was washed gently by ultra water to eliminate the unreacted aptamer. Finally, the acquired Apt/TPATCN/GCE was immersed in the 0.5 mM BSA (10 mM PBS, 7.4) for 10 min to block the residual sites. Rinse the electrode surface with distilled water before using.



Scheme 1. Construction process of the ATP aptasenor.

2.4 Electrochemical Impedance Spectroscopy (EIS) Measurements Compared with other conventional electrochemical methods, EIS tests can obtain more information about kinetics and the attachments of transducer surface in the field of biosensors42. In this work we used it to detect the process of aptasensor fabrication and the future target assays. EIS measurements were carried out in PBS (10 mM, 7.4) containing 5 mM K3[Fe(CN)6]/ K4[Fe(CN)6] and 0.1 M KCl. First of all, the modified electrode was immersed in the PBS (10 mM, 7.4) for 60 minutes to get the initial blank response. Next, the electrode was immersed in PBS (10 mM, 7.4) with different concentrations of ATP for testing. The most common representation of impedance data is the Nyquist spectrum which is fitted to equivalent Randles circuit43. When both the electrochemical polarization and the concentration polarization exist, the Randles electronic circuit is consisted of a semicircle part at a high frequencies amounts to the charge transfer resistance and a linear part at a low frequencies corresponding to the diffusion limited process. The direct current potential was set at 0.20 V and the frequency range was from 0.01 to 105 Hz with the amplitude wave of 5


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mV. 3. Results and Discussions 3.1 Design of the aptasensor The probe aptamer labeled with 5'-NH was attached to the functionalized TPATCN by multi-hydrogen bonds. During the preparation of the sensor, we use cyclic voltammetry (CV) and EIS to confirm the different state of electrode surface by recording the electrochemical characteristic of potassium ferricyanide, the benchmark redox system. As revealed in Fig. 1a, the TPATCN-modified electrode exhibited higher electron-transfer resistance (Rct) than the bare GCE, which may be because the certain band gap hinders the migration of internal electrons. When the aptamer is attached to the film of the D-A linked polymer, the Rct increased more than doubled. It was because the negatively charged phosphate backbone in aptamer chain kept the [Fe(CN)6]3-/4- from approaching the electrode surface because of the effect of charge repulsion. BSA was used for the last step of the whole process, and the magnify in the impedance value proved that residual active sites were blocked successfully. Cheeringly, the signal of ATP attached sensor system showed a significant enhancement and we can use the device to carry out the following detection. The result of CV curves (Fig. 1b) was consistent with the EIS characterization, illustrating the preparation of the aptasensor as our expected.



Fig. 1. Nyquist plots of the EIS (a) and CV curves (b) obtained at different modified electrodes: (black curve) GCE; (red curve) TPATCN/GCE; (blue curve) Apt/TPATCN/GCE; (pink curve) BSA/Apt/TPATCN/GCE; (green curve) ATP/BSA/Apt/TPATCN/GCE. Measurements were performed in PBS (10 mM, pH 7.4) containing 5.0 mM [Fe(CN)6]3-/4- and 0.1 M KCl.

XPS spectrum was used to characterize the surface chemistries of electrode during different modification steps. As exhibited in Fig. 2a, two predominant peaks of TPATCN appeared at 285 eV (C1s) and 400 eV (N1s) respectively owing to its unique structure. After the immobilization of aptamer, the photoelectronic signals of 134 eV (P2p) and 533 eV (O1s) showed up, representing the phosphate skeleton from the oligonucleotide chains, which can prove that the aptasensor was successfully built. In order to validate the idea, we characterized the functional groups and some bonding modes by Fourier transform infrared (FTIR) absorption spectroscopy (as pellet in potassium bromide) in Fig. 2b. The characteristic absorption peak at 2352 cm-1 was attributed to the cyano group and the peak at 3135 cm-1 was attributed to the C-H stretching mode in the aromatic nucleus44-45. The stretching of C=C and C-C bonds of conjugated aromatic rings led to two obvious absorption peaks at 1592 and 1492 cm-1 46.

The band at 1278 cm-1 has been confirmed to C-N stretching of tertiary amines47.


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The peaks that appeared in the 700-900 cm-1 range were assigned to aromatic ring substituents and C-H out-of-plane vibration from 1,4-disubstituted benzene rings47. With the existence of aptamers, we found that the peak of cyano group (2340 cm-1) produced a redshift. The same variety also occurred in the C-H vibration of the benzene ring. The locational change can be explained by the presence of hydrogen bonds which can average electron cloud density. Moreover, a FTIR band at about 1000-1200 cm-1 was caused by asymmetric stretching mode of phosphate skeleton vibration, while the band between 500 and 650 cm-1 was due to symmetric P-O stretching vibrations48. All the results of XPS and FTIR spectrum indicated that the ATP aptamer had been already attached to the electrode substrate. Static water contact angle measurements were performed to investigate the hydrophilic properties of every step of the functionalization process with a silicon pellet. The droplet image was achieved through a digital camera as shown in Fig. 2c, Fig. 2d and Fig. 2e. The concrete values were listed in Table S1. We found that the contact angle increased markedly after coated with the conjugated polymer due to the π-surface of the aromatic rings. When aptamers covered, the entire interface got an improvement of the hydrophilicity. This change may be contributed by the additionally peripheral hydroxyl groups from phosphate backbone and numerous base rings in the aptamer chain49. Tests for contact angles corroborated the complete introduction of aptamer and further indicated the successful fabrication of the biosensor.



Fig. 2. XPS spectra (a) and FTIR spectra (b) of TPATCN (black line) and Apt/TPATCN (red line). Contact angle characterization of bare (c), TPATCN modified (d) and Apt/TPATCN modified (e) silicon wafer.

AFM (3D) images revealed the heights of different surfaces for further analysis. Obviously, the topographies varied after each step of modification. As shown in Fig. S1b, the TPATCN film presented a different scene from the bare silicon wafer with an average height of about 5 nm. As shown in Fig. S1c, in the presence of aptamers, the surface changed not only in height, but also morphology. The aptasensor exhibited a wave-like 3D shape whereas the height (9.6 nm) doubled due to the immobilization of aptamers. The result indicated that the idea of fabricating this ATP sensor can be realized. 3.2 Characterization of TPATCN Fig. S2 proved the 1H NMR spectrum of TPATCN that can clearly explain the position of the proton. The multiplicity of peaks was expressed in the following: s,


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singlet; d, doublet; t, triplet; m, multiplet. 1H NMR(500M Hz, CDCl3): δ = 7.95 (d, J = 8.5 Hz, 4H), 7.75 (d, J = 8.5 Hz, 4H), 7.55 (d, J = 8.7 Hz, 4H), 7.35-7.3 (m, 8H), 7.18 (d, J = 7.6 Hz, 12H), 7.09 (dd, J = 13.8, 6.4 Hz, 4H). All peaks were produced consistent with the structure of the given polymer. For further confirming, scanning electron microscope (SEM) was applied to identify the morphological properties of TPATCN. As observed in Fig. 3a, the original powder presented like granules with monomers stacking together apart from aptamers binding. Actually, the steric hindrance causes intramolecular motion to be suppressed. The difference was that the monomers in dilute solution assembled into a chain-like structure with a size of 5-10 µm (Fig. 3b), which have enough spaces and active sites exposed to aptamers. When we reduce the magnification of the microscope (Fig. 3c), we can see the uniform film on the electrode surface which is available for the preparation of aptasensor. From the given structure, the SEM image was in accordance with the theory of molecular wires mentioned before. In order to further characterize the organic molecule we synthesized, we performed element mapping and energy-dispersive X-ray spectroscopy (EDS) analysis by transmission electron microscopy. Fig. 3d shows a portion of TPATCN whose morphology matches the SEM image. As Fig. 3e-f exhibited, the element mapping analysis also displayed a homogeneous distribution of the conjugated polymer. EDS analysis can be found in Fig. S3.



Fig. 3. SEM images of the original powder (a), the specific structure at high magnification (b) and the self-polymerized film at low magnification (c) dissloved in dilute solutions. TEM image of TPATCN (d) in dilute solution. Element mapping (e-f) of TPATCN.

UV absorption spectrum was carried out to illustrate the structure of donor and acceptor sections in THF solution. From the Fig. S4, we can see that the spectral line has two prominent peaks at 309 nm and 491 nm in accord with two components and no other impurity peaks exist50.


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3.3 Optimization of the incubation time of ATP Incubation time of ATP is a meaningful parameter in this experiment. Accordingly, we checked the Rct changes [ΔRct/Rct0 (%)] by immersing the modified electrode into PBS (10 mM, 7.4) with 1 pM ATP for diverse time. In this statement, ΔRct= Rct- Rct0, Rct refers to the value of electron transfer resistance with the existence of ATP and Rct0 refers to the value of electron transfer resistance without ATP. As revealed in Fig. 4, the Rct changes had an upward trend with time transforming from 20 min to 60 min. When ATP incubated for more than an hour, the curve began to be flat because that binding sites of aptamers had been filled with ATP molecules. The inserted picture is a direct representation of the impedance during the measurement. Therefore, 60 min was chosen as the optical incubation time for ATP test.

Fig. 4. Optimization of ATP incubation time for the aptasensor in PBS (10 mM, 7.4). Inset: Nyquist plots of the EIS for the aptasensor after different incubation time with 1.0 pM ATP in PBS (10 mM, 7.4).

3.4 Sensitivity of the ATP Sensor



In order to monitor the analytical performance of the established sensor, EIS was directed to detect different concentrations of ATP. As shown in Fig. 5, the [ΔRct/ Rct0(%)] value has a good liner correlation with the logarithm of the ATP concentration from 0.1 pM to 100 nM. The regression equation is ΔRct/Rct0(%) = 23.86 Log C (pM) + 59.04 (R2 = 0.992), and the low detection limit is 0.018 pM (S/N = 3), which is close to that reported by Chen’s group51 and is lower than that of relevant reports based on aptamers52-54. Such a sensitive platform may be attributed to its unique structure which can provide efficient electron delocalization throughout the D-A conjugated backbone and an advantageous environment for aptamers immobilization. Table S2 summarized the relevant studies of ATP measurement, which are used to identify the sensor we proposed.

Fig. 5. Typical impedance spectra (a) corresponding to the aptasensor incubated in different ATP concentrations with PBS (10 mM, 7.4): curves from inner to outer represent 0 pM, 0.1 pM, 0.5 pM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM and 100 nM ATP, respectively. Rct changes with ATP concentrations (b). Inset in (b): corresponding calibration curve of the aptasensor.

3.5 Reproducibility, stability and selectivity To assess the reproducibility of the developed sensor, five sensing systems were


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fabricated to detect 1.0 pM ATP respectively. The consequence was presented in Fig. S5 and the relative standard deviation (RSD) was 2.78%. We can learn that the reproducibility of the sensor is feasible. In addition, we assessed the stability by reserving a modified electrode under 4℃ for 15 days and tested the performance every other day. As revealed in Fig. S6, the system retained 90.2% of the initial signal after a storage period of 15 days, suggesting a good stability of the developed aptasensor. Furthermore, to discuss the effect of analogous molecules and evaluate the selectivity of the ATP sensor, we investigated the value of [ΔRct/Rct0 (%)] in different solutions containing AMP, UTP, CTP, ADP and GTP. As shown in Fig. S7, the sensitive sensor we set up exhibited a remarkable EIS response to ATP and ignorable responses to other components, indicating a glorious selectivity for ATP detection. It was proved that the aptamer has sufficient specificity to recognize ATP. 3.6 Application in real samples In order to prove the value of the sensor in practical applications, we used the blood of healthy volunteer to detect the ATP content. After the blood sample was centrifuged, we mixed PBS buffer solution with the supernatant to make the result within the liner range. Table S3 provided the calculated final results, which were consistent with the values in the literature55-56. Thus we can conclude that this aptasensor exhibited certain prospect to monitor ATP in actual samples. Spike recovery test was used to evaluate the accuracy of analytical method as Table S4 shown. According to the result, it can be proved that the ATP aptasensor has feasibility of practical application in the biological environment.



4. Conclusion In general, we have manufactured an original aptamer sensing unit for ultrasensitive detection of ATP based on a D-A-D type charge transfer platform for the first time. As anticipated, the dipolar push-pull electronic system in D-A-D structure ensured superior charge transfer when ATP was attached. Satisfactorily, we can get a wide liner range from 0.1 pM to 100 nM and a low detection limit of 0.018 pM through a simple operation. Compared with other ATP aptamer sensors, we can affirm that the strategy in this study offers the following advantages: (1) Simple electrode preparation without the optimization of electropolymerization, needless of any enzyme labels. (2) Better sensing capabilities were gained over a high carrier mobility of D-A-D constitution. It is worth to say that our present work yields a new idea for electrochemical aptamer sensor, and can be extended to other D-π-A architecture to explore more electrochemical functions. The advantageous fabrication of conjugated polymer holds a great prospect for the subsequent research in electronic sensing platform and our research will continue along this way.

Supporting Information Reagents and apparatus. Synthesis route of TPATCN. Characterization of the TPATCN. Reproducibility, stability and selectivity of the aptasensor. Characterization of the bare, TPATCN and Apt-TPATCN surface. Comparison with previous reports. Application in real samples.


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Acknowledgement This work acknowledges support from the National Natural Science Foundation of China (No. 21375045) and Natural Science Foundation of Jilin Province (No. 20180101195JC).



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