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An amplified electrochemical aptasensor based on hybridization chain reactions and catalysis of silver nanoclusters Ling Chena,b, Liang Shaa,b, Yuwei Qiua,b, Guangfeng Wanga,b*, Hong Jianga,b and Xiaojun Zhanga,b* 5

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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x In the present study, based on the mimic oxidase catalytic character of nucleic-acid-stabilized silver nanoclusters (DNA /AgNCs) and hybridization chain reactions for signal amplification, a label-free sensitive “turn-on” electrochemical aptasensor for amplified determination of lysozyme was demonstrated. First, the designed DNA duplex was modified on the electrode. With the specific binding of the target, lysozyme and its aptamer, lysozyme-binding DNA sequence was liberated exposing the induced DNA sequence, which in turn triggered the formation of the supersandwich DNA structure. Because cytosine-rich sequence was designed ingeniously on the DNA sequence, DNA/AgNCs were formed on the supersandwich DNA structure. The peroxidase-like character of DNA/AgNCs produced detectable electrochemical signal for lysozyme aptasensor which showed a satisfying sensitive detection of lysozyme with a low detection limit of 42 pM and wide linear range of 10−10 M to 10−5 M.

1. Introduction Recently, nucleic-acid-stabilized silver nanoclusters (DNA /AgNCs) have received special attention due to their high luminescence quantum yields, tunable emissions, excellent photostability, subnanometer size, controllability by the size and the protecting layer of the nanoclusters, water solubility and biocompatibility1 which makes them become attractive materials for the development of optical sensor and biolabels.2 Up to now, numerous effective fluorescent assays have been reported based on the interesting photophysical properties of DNA/AgNCs3 including the fluorescent labels for sensing of metal ions such as Hg2+ or Cu2+,4 bioactive thiols such as cysteine, homocysteine or glutathione,5 probing enzyme activities such as tyrosinase or glucose oxidase,6 detection of aptamer−substrate recognition complexes7 such as detection of thrombin or ATP, and analysis of single nucleotide mutation in DNA8 or microRNAs (miRNAs).9 DNA/AgNCs conjugates were also used for cell imaging.10 However, to the best of our knowledge, almost all of the DNA/AgNCs based sensors are originated from the fluorescence character of DNA/AgNCs at present, which may limit their further applications due to the disadvantages of the fluorimetry such as the requirement of expensive instruments and sensitivity to the interferences.11 Thus, novel character of DNA/AgNCs and its further application are well worth expecting. As we know, although natural enzymes play various important roles in organism, their wide applications still encounter some limitations due to some disadvantages of the natural enzymes such as high-cost and difficult purification processes, timeconsuming, being sensitive to environmental conditions, and easy loss of catalytic activity restricts.12 Therefore, more and more attention has been paid to constructing artificial enzyme mimics in recent years. Nanostructure materials that possess mimic enzymes activity have specially received considerable interest,13 because enzyme mimics based on nanostructures have advantages This journal is © The Royal Society of Chemistry [year]

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over natural enzymes in low-cost, high stability under harsh conditions and high catalytic activity.14 Therefore nanomaterials are promising oxidase mimics. This thought inspired us to study the oxidase mimic character of the special nanomaterials, DNA/AgNCs. According to the literature most reported peroxidase-like nanomaterials showed their mimic oxidase character in detecting H2O2 or substances related with the H2O2 reaction such as bilirubin,15 immunoprotein,16 melamine,17 l-lactate,18 choline and acetylcholine,19 etc. In these sensors, the signals are from the catalytic reaction and the signals almost have no amplification or only were amplified by the surface effect of nanomaterials. As we know, amplification of recognition events is a central element in analytical or bioanalytical science.20 In an attempt to improve the sensitivity of sensors, various signal amplification strategy have been proposed including the use of indicators,21 nanomaterials22 and enzymes23 as labels. In spite of the attractive sensitivity of these methods, extra complicated modification or conjugation steps are commonly required in these schemes. If properly designed, DNA can assemble into various structures by DNA nanoassembly, which is a simple and effective approach for signal enhancement via probe hybridization.24 Hybridization chain reaction (HCR) has evolved widely used technique as a fascinating strategy for sensitive assay since Dirks and Pierce first introduced the concept in 2004.25 HCR is an initiatortriggered reaction and an enzyme-free process in which the polymerization of oligonucleotides can be led into a long nicked dsDNA molecule with a hybridization event be triggered by an initiator. It possesses the inherent advantages, for instance mild conditions required, a low background and great potential in signal amplification.26 Therefore, it has the capacity to be a fascinating strategy for signal enhancement. It is well known that the high specificity and affinity to the corresponding targets and ease of their easy preparation, great stability make aptamers-based sensors increasingly attractive.27 In [journal], [year], [vol], 00–00 | 1

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consequence, a variety of aptamer-based sensing programs have been suggested for the detection of proteins based on luminescence,28 quartz crystal microbalance,29 colorimetry,37 fluorescence31 and electrochemistry.32 In contrast, simple electrochemical aptamer-based sensing methods have drawn considerable attention recently due to their advantages of portability, simple instrumentation, low cost, fast response, high sensitivity. 33,34 In consideration of many previous electrochemical aptasensors labeled with electroactive materials such as methylene blue, ferrocene and enzyme,35 which comes about complex, laborious, and time consuming labeling process and even has an effect on the binding affinity between the targets and their aptamers 36, it is necessary to develop a label-free electrochemical aptasensor. Herein, inspired by the thirst for the exploration of novel character of DNA/AgNCs, we studied the oxidase mimic character of DNA template Ag NCs. Based on the peroxidase-like character of DNA/AgNCs, the advantages of signal amplified HCR and electrochemical aptamer-based sensing methods, we proposed a “turn-on”, label-free and sensitive electrochemical strategy based on initiator-triggered HCR signal amplification with DNA/AgNCs as oxidase mimics for aptamer monitoring. In this aptamer-based sensing, lysozyme was studied as a model. Lysozyme is an ubiquitous enzyme widely distributed in diverse organisms, such as virus, bacteria, plants, insects, mammalian tissues and secretions.37 It has been broadly used as an antiinflammatory drug in the pharmaceutical and as a natural antibacterial agent in food industries.38 Besides its so useful properties and wide application, lysozyme can induce allergic reactions even in minute amounts. Thus, it is important to develop highly sensitive biosensors for detection of lysozyme.

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2.1. Materials and reagents All HPLC-purified oligonucleotides were purchased from Sangon Biotechnology Co. Ltd (Shanghai, China) with the following sequences: S1: 5’-CTA AGT AAC TCT GCA CTC TTA TAT ATC ATA GAA TTG GTA GAT-(CH2)6-SH-3’ (partially complementary DNA sequence with the lysozyme binding aptamer). The lysozyme binding aptamer (S2): 5’-ATC TAC GAA TTC ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG-3’. Hairpin DNA probe 1 (HP1): 5’-AGA GTG CAG AGT TAC TTA- GAA ACA TCT AAG TAA CTC TG-3’, which have a loop of 6 nucleotides and a stem of 13 base pairs with an extra tail of 6 nucleotides. Hairpin DNA probe 2 (HP2): 5’-CTA AGT AAC TCT GTG AAT ACA GAG TTA CTT AGC CCC CCC CCC CC-3’, which have a loop of 6 nucleotides and a stem of 13 base pairs with an extra tail of 6 nucleotides and 12 cytosine nucleotides. Both HP1 and HP2 have two sections complementary sequences with each other. Hairpin DNA probe 3 (HP3): 5’-CTA AGT AAC TCT GTG AAT ACA GAG TTA CTT AG-3’. S3: 5’-CAG AGT TAC TTA GCC CCC CCC CCC C-3’. Tris (hydroxymethyl) aminomethane (Tris), Lysozyme, Thrombin, Bovine serum hemoglobin (BHb), Bovine serum albumin (BSA) and 6-mercaptohexanol (MCH) (in order to block possible remaining active sites and avoid non-specific adsorption) 2 | Journal Name, [year], [vol], 00–00

were purchased from Sangon Biotechnology Co. Ltd. (Shanghai, China). Sodium borohydride (NaBH4), 3, 3’, 5, 5’-tetramethylbenzidine (TMB) and other reagents were obtained from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). These reagents were of analytical grade and used without further purification. Ultrapure water (18 MΩ·cm) was used in all experiments. The experiments were conducted at room temperature. To evaluate the practicality of the proposed sensor, human urine samples were collected as an example. human urine samples were kindly obtained from the Yijishan Hospital (Wuhu, China). These samples were diluted with 0.05 M PBS (pH 7.4) for further use. Apparatus: All electrochemical measurements were recorded on a CHI 660B electrochemistry work station (CH Instruments Inc., Shanghai, China). A conventional three-electrode configuration was used, with a modified gold working electrode (2 mm in diameter), a saturated calomel electrode (SCE) as reference electrode, and a platinum wire as auxiliary electrode. Highresolution transmission electron micrographs (HRTEM) observations for the morphological measurements of DNA/AgNCs were obtained with a Tecnai G220S-TWIN transmission electron microscope (FEI). Centrifugation was performed using a HERMLEZ 36 HK apparatus (Wehingen, Germany). Ultrapure water was obtained through PSDK2-10-C (Beijing, China). All pH measurements were measured with a Model pHs-3c meter (Shanghai, China). The high performance liquid chromatography (HPLC) analyses were carried out using a Shimazu chromatographic system (Tokyo, Japan). Chromatographic separation was carried out using a C18 column (250× 4.6 mm, 5 µm) purchased from Angilent Co., Ltd. (Beijing, China). The mobile phase consisted of a mixture of methanol/formic (50:50 (v/v)). The flow rate was 1 mL min-1.

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Before preparation of the aptasensor, the gold working electrode was first immersed in piranha solution at 90 ◦C for 5 min for chemical pretreatment,39 followed by adequately washing with ultrapure water. Subsequently, the electrode was polished sequentially with 0.3 and 0.05 µm alumina powder and sonicated in ethanol and ultrapure water for 3 min each. Afterwards, the electrode was voltammetrically cycled in 0.1 M H2SO4 with the potential between -0.2 and 1.5 V at 0.1 V/s until a representative steady-state cyclic voltammogram was obtained.

2.3. Preparation of the aptasensor based on supersandwich DNA structure and reduction of Ag nanoclusters Prior to use, all the oligonucleotides were dissolved in 0.05 M PBS buffer (pH 7.4) and stored at 4 oC for one night. The DNA solution was heated to 90 oC for 5 min to remove aggregates, and then cooled slowly to room temperature. Preparation of the aptasensor includes these following steps: At first, the immobilization of DNA duplex onto the pre-cleaned electrode was performed as follows: the mixture of the two partially complementary sequences (S1 and S2, 1 µM each) in This journal is © The Royal Society of Chemistry [year]

Nanoscale Accepted Manuscript

DOI: 10.1039/C4NR06664B

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Scheme 1. Fabrication process of the electrochemical amplified determination of lysozyme based on HCR and catalysis of DNA/AgNCs.

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Scheme 1 illustrated the fabrication of the HCR signal amplified electrochemical aptasensor based on the peroxidase-like character of DNA/AgNCs. First, S1 and S2 were designed (the details were shown as section 2.1.). S1 was 3’-terminal thiolated S1, and S2 was a designed lysozyme binding aptamer and partially complementary with S1. With partial base pairs, the DNA duplex was assembled on the gold electrode. In the presence of lysozyme, special binding of lysozyme to S2 with high affinity denatured the duplex and liberated lysozyme-binding S2 into the solution. Then, S1 was left on the surface of electrode for the further HCR process. With the addition of hairpin DNA, HP1 that contains 19 bases completely complementary with S1 at 5’terminal, and HP2 that has two sections complementary sequences with HP1, it would open the hairpin structure of HP1 and expose the remainder of the sequence of HP1, which would hybridize with parts sequence of HP2 and then open HP2. Alternately, the exposed sequence of HP2 would then match with the complementary sequences in HP1. Thus HCR would be triggered and it would generate a supersandwich DNA structure of HP2-HP1-S1 complex on the gold electrode. Because cytosine-rich sequence was designed on the 3’-end of HP2, in the presence of Ag+ and NaBH4, DNA/AgNCs were formed on the supersandwich DNA structure. Based on the peroxidase-like character of DNA/AgNCs, the lysozyme could be detected.

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All the electrochemical experiments were carried out at room temperature. Cyclic voltammetry (CV) was performed in 0.1 M PBS (pH 7.4) within the potential range from 0 to -0.4 V using scan rate 100 mV/s. Electrochemical impedance spectra (EIS) was performed in 1/15 M PBS (pH 7.4) containing 5 mM Fe (CN)63-/Fe(CN)64- and 0.1 M KCl in the frequency range from 0.1 Hz to 100 kHz with 5 mV as the amplitude at a polarization potential of -0.18 V.

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immobilization buffer (0.05 M PBS, pH 7.4 with 20 mM MgCl and 20 mM KCl) was heated to 90 ◦C for 5 min and then gradually cooled to room temperature, maintaining the structural flexibility of the aptamer for binding lysozyme.40 Then, the precleaned electrode was incubated in the resulting DNA duplex solution for 10 h at room temperature, then the gold electrode was rinsed with ultrapure water and incubated in 2 mM 6mercaptohexanol (MCH) in PBS buffer for 30 min to remove non-specific aptamer adsorption on the gold electrode surface. After being thoroughly rinsed with ultrapure water and dried under a N2 stream, the S1 and probe modified gold electrode (S2S1/GE) was stored at 4 ◦C in HEPES buffer for further use. For lysozyme assays, the obtained S2-S1/GE was incubated in buffer solution (0.05 M PBS, pH 7.4 containing 20 mM MgCl2 and 20 mM KCl) with various concentrations of lysozyme, and kept for 30 min at 35◦C. Unbinding DNA duplex was removed by rinsing with wash buffer and ultrapure water. The resulting electrode was named as the aptasensor and then employed in this work. The aptasensor was then incubated in the hybridization buffer solution (0.05 M PBS, pH 7.4) containing 3 µM HP1 and HP2 for 10 h at 35 ◦C to form the supersandwich structure modified electrode (HP2-HP1-Aptasensor). According to previous reports,41 owing to HP2 was ingeniously designed cytosine-rich at 3’-termini, the oligonucleotide-stabilized Ag nanoclusters could be synthesized on the electrode surface. In brief, AgNO3 in a molar ratio of 6 : 1 was added to the 3 µM HP1 and HP2 mixture hybridization buffer solution, followed by the vigorous shaking of the solution for 30 sec and reacting for 1 h in the dark. Subsequently, freshly prepared NaBH4 (10 µL) with the same concentration (18 µM) was added into this mixture for reduction followed by vigorous shaking of the mixture for 30 sec and the reduced oligonucleotide-Ag solution was incubated at 4 °C and allowed to react for 1 h in the dark. Silver ions bound with cytosine42 were reduced to form nanoclusters. Once DNA/AgNCs/HP2 was synthesized, obtaining the supersandwich structure and DNA/AgNCs/HP2 modified electrode (regarded as DNA/AgNCs/HP2-HP1-Aptasensor). The modified electrode was thoroughly rinsed, dried with N2, and then incubated in 1 M NaClO4 before be utilized for further electrochemical measurements. In contrast, HP3 substituting HP2 was prepared for no DNA/AgNCs contained in DNA structure (HP3-HP1Aptasensor) modified gold electrode under the same conditions as DNA/AgNCs/HP2-HP1-Aptasensor. Likely, we designed S3 to replace HP2, and then S3 and HP1 were prepared for traditional sandwich DNA structure (DNA/AgNCs/S3-HP1-Aptasensor) modified electrode.

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As Fig. 2A showed there was no characteristic CV peak corresponding to DNA/AgNCs on the bare GE (curve a). While it was obviously noticed that an oxidation peak at about 0.37 V and a reduction peak at 0 V nearby had been obtained by CV (curve c of Fig. 2A), which was the proof of the existence of metal-based oxidation and reduction activity of the nanoclusters labeled onto the HP2-HP1-Aptasensor. However, a negligible characteristic CV peak (curve b of Fig. 2A) corresponding to DNA/AgNCs was observed on the Ag nanoclusters modified electrode with traditional sandwich structure (DNA/AgNCs-S3-HP1Aptasensor). It demonstrated that the signal of DNA/AgNCs in traditional sandwich structure is less than those in the supersandwich structure (DNA/AgNCs-HP2-HP1-Aptasensor). To verify our proposal, electrochemical impedance measurements were first performed to study the modified electrodes with different interface properties. In the investigation, the impedance spectra were obtained in the form of a Nyquist plot in which Ret indicated the surface electron transfer electric resistance of the electrode. One observed that the bare GE exhibited a definitely small semicircular domain (curve a of Fig. 2B), illustrating a very low electron transfer resistance. After the immobilization of DNA duplex, the Ret gave a rise obviously on S2-S1/GE (curve b of Fig. 2B) due to the formation of a negatively charged interface, which may severely hinder the electron transfer toward the electrode surface. When S2-S1/GE was incubated with 10-5 M lysozyme, an obvious decrease of the semicircle diameter was observed on the Aptasensor (curve c of Fig. 2B). This may disclose the fact that S2 binded to lysozyme with high affinity and lysozyme-binding S2 was liberated into the solution. Then the EIS of the obtained HP2-HP1-Aptasensor was also studied. A greatly increased semicircle was observed (curve d of Fig. 2B), which may be ascribed to the inhibition effect of the further 4 | Journal Name, [year], [vol], 00–00

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loading of negatively charged DNA strands. As shown in curve e of Fig. 2B, the Ret gave a marginal reduce on DNA/AgNCs-HP2HP1-Aptasensor. The reason for this was that the formation of DNA/AgNCs. All these indicated that the supersandwich DNA structure was likely formed on the electrode as what we predicted before. We also investigated the electrocatalytic of different modified electrodes to prove our design. As we can see in curve a of Fig. 2C, a negligible CV signal on bare GE was obtained in PBS (pH 7.4) with 100 µM H2O2, suggesting that there was no catalytic activity. After the bare GE was modified with DNA duplex (curve b of Fig. 2C) or the S2-S1/GE was incubated in lysozyme solution (curve c of Fig. 2C), negligible changes were also found from CV responses, implying that DNA strands on the electrode surface cannot catalyze H2O2. However, a large elecatrocatalytic response was obtained on DNA/AgNCs-HP2-HP1-Aptasensor shown as curve d in Fig. 2C, which may be resulted from the intrinsic enzyme mimetic activity of DNA/AgNCs loaded on the electrode surface successfully. In order to study the signal amplification effect, the DNA/AgNCs-S3-HP1-Aptasensor with traditional sandwich DNA structure was discussed. As shown in curve e of Fig. 2C, the signal based on traditional sandwich DNA structure is remarkably less than that of supersandwich DNA structure, which may be due to the supersandwich structure with larger units of DNA/AgNCs. In the control experiments, the cyclic voltammograms response of HP3-HP1-Aptasensor without DNA/AgNCs was also investigated. No obvious elecatrocatalytic response could be observed (curve f of Fig. 2C), implying that the system hadn’t the ability to catalyze H2O2 without the formation of DNA/AgNCs. In addition, the intrinsic enzyme mimetic activity of DNA/AgNCs in DNA/AgNCs-HP2-HP1-Aptasensor was studied by the colorimetric method in TMB system. As shown in Fig. 2D, it was observed that the formation of DNA/AgNCs in TMB/H2O2 solution showed blue (the inset, a) and the maximal absorption wavelength at 370 nm and 650 nm of the solution (curve a of Fig.2D) was higher than that in the TMB/H2O2 solution without DNA/AgNCs (curve b of Fig.2D), confirming that the DNA/AgNCs possesses the intrinsic enzyme mimetic activity.

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DNA/AgNCs. The characterization of formed DNA/AgNCs was shown as Fig. 1A with the typical HRTEM images. As we can see from the HRTEM image, the DNA/AgNCs was individual spherical and uniform. In some cases, a small number of Ag nanoparticles for larger or aggregated clusters are detected as well. The gel-electrophoresis results confirmed the formation of the supersandwich DNA structure. As we can see from Fig. 1B, it produces a ladder of different lengths of the supersandwich structure, with the maximum in the range 700 to 750 base pairs.

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3.3. The electrochemical catalysis toward various H2O2 concentrations Based on the results of cyclic voltammogram in Fig. 1C, the supersandwich DNA structure based aptasensor was further studied its response toward different concentrations of H2O2. As shown in Fig. 3A, the electrocatalytic currents intensity increased linearly with the concentration of H2O2 increasing from 0 to 100 µM. The calibration curve of the CV responses on the aptasenor at different H2O2 concentrations was shown in Fig. 3B. The obtained regression equation was expressed as y = 3.641 + 0.1182x, R2=0.9908, where y is the electrocatalytic signal, x is the concentration of H2O2 and R is the regression coefficient. The detection limit was calculated to be 1.71 µM (3 n/s, where n was the standard deviation of the intercept and s was the slope of the calibration curve). 14

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In order to achieve high assay sensitivity, a number of experimental conditions were optimized in this paper. The optimization of the variables of the system was shown as Fig. S4, including pH value, incubation time, incubation temperature in lysozyme solution, and hybridization time for HP1 and HP2 in the hybridization buffer solution. Fig. 4A depicted the effect of pH value in lysozyme solution the of the system of the DNA/AgNCs-HP2-HP1-Aptasensor over the pH range 4.0-9.0. Obviously, the reduction current increased with the pH value increasing from 4.0 to 7.5. The suggested reason for this is that This journal is © The Royal Society of Chemistry [year]

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the protonation of the nitrogen atoms of DNA bases reduces its affinity with lysozyme.43 However, when the pH is higher than 7.5, the current decreased rapidly. The suggested reason for this is that silver may be complexed by OH- once the pH value is relatively higher, which, in turn, reduces its binding with cytosine bases.44 The effect of incubation time was investigated by incubating DNA/AgNCs-HP2-HP1-Aptasensor in PBS buffer for different time periods ranging from 5 min to 45 min. As we can see from Fig. 4B, the electrocatalytic response intensified with the incubation time increasing and kept constant to a saturation value after about 30 min, indicating that a 30 min incubation time was efficient for the specific binding of lysozyme to S2. The effect of incubation temperature in lysozyme solution the of the system of the DNA/AgNCs-HP2-HP1-Aptasensor was studied at varying temperature ranging from 5 to 45 °C shown as Fig. 4C. One observes that, the currents response enlarged with the incubation temperature rising from 5 to 35 °C but then decreased gradually with the incubation temperature increasing from 35 to 45 °C. Fig. 4D displayed the the effect of hybridization time for HP1 and HP2 in the hybridization buffer solution at varying hybridization time ranging from 2 to 10 h. It was obvious that the currents response of the sensor increased with the increase of the hybridization time for HP1 and HP2. While a negligible increase or slight decrease was observed at the hybridization time of 10 h, which suggested that 10 h hybridization time HP1 and HP2 was efficient for the fabricating of a supersandwich DNA structure on the gold electrode. Thus, 35°C of incubation temperature, 0.05 M PBS buffer (pH 7.4), 30 min incubation time in lysozyme solution and 10 h hybridization time for HP1 and HP2 in the hybridization buffer solution were chosen as the optimized experiment conditions in subsequent experiments.

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Fig 2. (A) CV of DNA/AgNCs on bare GE (curve a), DNA/AgNCs-S3-HP1-Aptasensor (curve b) and DNA/AgNCsHP2-HP1-Aptasensor (curve c) in PBS (pH 7.4); (B) Nyquist plot for EIS measurements for different modified electrodes: bare GE (a), S2-S1/GE (b), Aptasensor (S2-S1/GE after incubation with 10-5 M lysozyme) (c), HP2-HP1-Aptasensor (d), DNA/AgNCsHP2-HP1-Aptasensor (e); (C) CV of different modified eletrodes: bare GE (a); S2-S1/GE (b); Aptasensor (c); DNA/AgNCs-HP2HP1-Aptasensor (d); DNA/AgNCs-S3-HP1-Aptasensor (e); HP3HP1-Aptasensor (f) in PBS (pH 7.4) with 100 µM H2O2; (D) UV–vis absorption spectra with photographs in the inset of H2O2mediated oxidation of TMB by DNA/AgNCs in PBS (pH 7.0) in the presence (a) and absence (b) of the DNA/AgNCs.

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3.5. Detection of lysozyme After the discussion of optimization conditions and the effect of supersandwich DNA structure on the electrochemical Journal Name, [year], [vol], 00–00 | 5

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DOI: 10.1039/C4NR06664B

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DOI: 10.1039/C4NR06664B

25

30

Current / µΑ

35

14

A

16

i

12 10

a

8 6 4 2

45

10 8 6 4

a

b

c

d

e

-0.1

-0.2

-0.3

Potential / V (vs. SCE)

-0.4

0

100

200

75

80

85

h

f

300

70

90

400

95

Time/s

C

14 12

100

10 8 6 4

y = 25. 49 + 2.188logx 2 R = 0.9946 105

log([lysozyme]/ M)

55

16 14 12 10 8 6 4 2 0

A

Fig. 5 (A) CV on DNA/AgNCs-HP2-HP1-Aptasensor with various concentr- ations of lysozyme: (a – i) 0, 10-10, 5×10-10, 10-9, 5×10-9, 10-8, 10-7, 10-6, 10-5 M in PBS (pH 7.4) in the presence of 100 µM H2O2; (B) Steady state amperometric current at –0.4V on DNA/AgNCs-HP2-HP1-Aptasensor with various concentrations of lysozyme: (a – h) 10-10, 5×10-10, 10-9, 5×10-9, 10-8, 10-7, 10-6, 10-5 M in PBS (pH 7.4) in the presence of 100 µM H2O2; (C) The corresponding calibration curve of current vs. the logarithmic concentration of lysozyme.

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110

120

B

100 80 60 40 20 0

lysozyme

2 -10 -9 -8 -7 -6 -5 1x10 1x10 1x10 1x10 1x10 1x10

50

The aptasensor was sensitive to different concentrations of the lysozyme that had been demonstrated. The specificity of the sensor was also investigated by challenging it with other common proteins: Thrombin, BHb and BSA. When 10-5 M each lysozyme, Thrombin, BHb or BSA was incubated and detected individually with the aptasensor, as we can see from Fig. 6A, the interference proteins exhibited almost negligible responses except for lysozyme. Therefore, the proposed aptasensor showed remarkably high sensitivity and selectivity with no obvious interference from other three proteins. The storage ability of the aptasensor was also checked. It was found that the current response to lysozyme was no apparent change in the first continuous seven days by everyday use. Fig. 6B depicted the time dependence of the signal change relative to the original signal of 10−6 M lysozyme in the 1st-day measurement. From Fig. S5B, it was obvious that only 15.12% leakage was found after 1-month storage in buffer solution. This result suggested that the aptasensor showed a good and long-term stability. In addition, the practicality of the proposed method was applied to analyze lysozyme in human urine samples, in which the detection of lysozyme was carried out in 100-fold diluted human urine samples and the detection procedure was the same as the aforementioned detection of lysozyme in clean PBS buffer solution. As can be seen from Table 2, all the results obtained indicated a satisfactory application of the system. In order to further confirm the reliability of the proposed sensor, we compared our sensor with the currently available method of High Performance Liquid Chromatography (HPLC) to detect lysozyme.54 The results were shown in Table 3. It shows that the proposed method can be efficiently used for the determination of lysozyme.

Current / µΑ

0.0

16

g

2 0

0

40

B

14 12

65

3.6. The selectivity, storage stability and analytical applications of the sensor

BSA

BHb

Thrombin

0

5

10

15 20 Days

25

30

Fig. 6 (A) Selectivity of the system for lysozyme analysis in PBS (pH 7.4) with 100 µM H2O2 (The concentration of lysozyme or the interference proteins was 10-5 M and the incubation time for proteins was 30 min.); (B)The time dependence of the signal change relative to the original signal of 10−6 M lysozyme in the 1st-day measurement.

This journal is © The Royal Society of Chemistry [year]

Nanoscale Accepted Manuscript

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Relative signal change /%

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Current / µΑ

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Current / µΑ

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amplification, we further studied the response of the system relative to the concentration of lysozyme. Fig. 5A showed the CV responses of the DNA/AgNCs-HP2-HP1-Aptasensor after reacting with various concentrations of lysozyme ranging from 0 to 10-5 M. It was observed that a series of dynamically increased CV peak currents in response to lysozyme with increasing concentrations as a consequence of the efficient capture of the lysozyme by the aptasensor. With the proposed lysozyme aptasensor, chronoamperometry was also investigated to assess the quantitative response range of lysozyme. Fig. 5B depicted the amperometric currentwhich was recorded from the sensor, reached a steady state response rapidly with each addition of H2O2. From Fig. 5C, it could be seen that amperometric current increased linearly with the logarithm of lysozyme concentration between 10−10 and 10−5 M. From the calibration curve of the electrocatalytic responses on the aptasenor to different lysozyme concentrations, the obtained regression equation was expressed as y = 25. 49 + 2.188 logx, R2= 0.9946, where y is the electrocatalytic signal, x is the concentration of lysozyme and R is the regression coefficient. A detection limit of 42 pM was then calculated using a signal three-fold the background noise. The relative standard deviation (RSD) for 3 successive determinations was 4.28%, suggesting a good reproducibility. Furthermore, a comparison table of our proposed sensor with the previous lysozyme aptasensors was shown as Table 1. It could be seen that our sensor had remarkable advantages over most other analogical aptasensors.

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DOI: 10.1039/C4NR06664B

Table 1. Comparison of the proposed technique with other reported assays for the detection of lysozyme. Method

Technique

Optical

plasmon

Detection limit

References

0.5 nM

[45]

35 nM

[46]

20

25

resonance surfaceOptical

enhanced Raman

30

scattering surfaceOptical

enhanced Raman

3.5 nM

[47]

2 µM

[48]

7 nM

[49]

36 nM

[50]

scattering Optical

Fluorescence

Electrochemilu-

Electrochemilu

minescence

-minescence

Electrochemical

Electrochemical

Electrochemical

square-wave voltammogram square-wave voltammogram Stripping potentiometry

0.45 nM

[51]

7 nM

[52]

35

This work was financially supported by the projects (No. 21371007) from National Natural Science Foundation of China, Anhui Provincial Natural Science Foundation (1208085QB28), Anhui Provincial Natural Science Foundation for Distinguished Youth (1408085J03), Natural Science Foundation of Anhui (KJ2012A139) and the Program for Innovative Research Team at Anhui Normal University.

40

Notes and references

45

Electrochemi Electrochemical

-cal impedance

0.35 µM

[53]

42 pM

This paper

spectroscopy Electrochemical

5

CV

50

Table 2. Determination of lysozyme added in diluted human urine samples 55

Number 1 2 3 4 5

10

Added (nM) 5 10 20 40 100

Founded(n=3; nM) 4.65+0.6 9.25+0.9 18.57+3 42.13+4 98.54+7

Recovery 0.930 0.925 0.928 1.053 0.985

Table 3. Comparison of the proposed technique with the HPLC method to detect lysozyme added in clean PBS buffer solution Sample Added (µM) A 5 B 10 C 20 D 50

By HPLC (n=3; µM) 4.74+0.4 9.56+0.7 18.25+4 48.86 +8

By this method (n=3; µM ) 4.67+0.5 9.35+0.6 18.93+3 50.37+6

60

65

RSD (%) 70

-1.5 -2.2 3.7 3.1 75

15

Conclusions In summary, the present study proved the novel peroxidase-like character of DNA/AgNCs and introduced a “turn-on”, label-free, sensitive electrochemical aptasensor based on the amplification This journal is © The Royal Society of Chemistry [year]

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a Key Laboratory of Chem-Biosensing, Anhui province; Key Laboratory of Functional Molecular Solids, Anhui province; College of Chemistry and Materials Science, Center for Nano Science and Technology, Anhui Normal University, Wuhu 241000, PR China b State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR China E-mail: [email protected], [email protected] 1 J. Sharma, R. C. Rocha, M. L.Phipps, H.mYeh, K. A. Balatsky, D. M. Vu, A. P. Shreve, J. H. Werner and J. S. Martinez, Nanoscale, 2012, 4, 4107−4110. 2 W. Lesniak, A. U. Bielinska, K. Sun, K. W. Janczak, X. Shi, J. R. Baker and L. P. Balogh, Nano Lett. 2005, 5, 2123−2130. 3 X. Q. Liu, F. A. Wang, R. Aizen, O. Yehezkeli and I. Willner, J. Am. Chem. Soc. 2013, 135, 11832−11839. 4 Y. T. Su, G. Y. Lan, W. Y. Chen and H. T. Chang, Anal. Chem. 2010, 82, 8566−8572. 5 W. Y. Chen, G. Y. Lan and H. T. Chang, Anal. Chem. 2011, 83, 9450−9455. 6 X. Liu, F. Wang, A. Niazov-Elkan, W. Guo and I. Willner, Nano Lett. 2013, 13, 309−314. 7 J. Sharma, H. C. Yeh, H. Yoo, J. H. Werner and J. S. Martinez, Chem. Commun. 2011, 47, 2294−2296. 8 H. C. Yeh, J. Sharma, I. M. Shih, D. M. Vu, J. S. Martinez and J. H. Werner, J. Am. Chem. Soc. 2012, 134, 11550−11558. 9 J. Li, X. Zhong, F. Cheng, J. R. Zhang, L. P. Jiang and J. J. Zhu, Anal. Chem. 2012, 84, 4140−4146. 10 C. I. Richards, S. Choi, J. C. Hsiang, Y. Antoku, T. Vosch, A. Bongiorno, Y. L. Tzeng and R. M. Dickson, J. Am. Chem. Soc. 2008, 130, 5038−5039. 11 L. B. Zhang, J. B. Zhu, S. J. Guo, T. Li, J. Li and E. K. Wang, J. Am. Chem. Soc. 2013, 135, 2403−2406. 12 S. B. Xie, Y. Q. Chai, Y. L. Yuan and R. Yuan, Chem. Commun. 2014, 50, 7169-7172; G. L. Wang, X. F. Xu, L. H. Cao, C. H. He, Z. J. Li and C. Zhang, RSC Adv. 2014, 4, 5867−5872. 13 J. S. Mu, Y. Wang, M. Zhao and L. Zhang, Chem. Commun. 2012, 48, 2540−2542; Y. Song, K. Qu, C. Zhao, J. Ren and X. Qu, Adv. Mater. 2010, 22, 2206−2210. 14 H. Wei and E. Wang, Chem. Soc. Rev. 2013, 42, 6060−6093; J. Xie, X. Zhang, H. Wang, H. Zheng and Y. Huang, TrAC, Trends Anal. Chem. 2012, 39, 114−129.

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Nanoscale Accepted Manuscript

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Surface

of HCR and the electrocatalytic character of DNA/AgNCs. In the sensing system, the target, lysozyme, induced the specific binding of lysozyme and its aptamer, which in turn triggered the formation of the supersandwich DNA structure based on HCR. The wide linear range, low detection limits and operational stability for the analysis of lysozyme were satisfactory. Moreover, the developed method achieves comparable or even better sensitivity against some other common amplified detection. In addition, the proposed electrochemical aptasensor could be a universal and promising platform for other small molecule detection and medical diagnostics, which expanded the electrochemical application of DNA/AgNCs.

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