- Email: [email protected]
A novel label free electrochemiluminescent aptasensor for the detection of lysozyme
Accepted Manuscript A novel label free electrochemiluminescent aptasensor for the detection of lysozyme
Yasamin Nasiri Khonsari, Shiguo Sun PII: DOI: Reference:
S0928-4931(17)34590-3 https://doi.org/10.1016/j.msec.2018.11.016 MSC 9036
To appear in:
Materials Science & Engineering C
Received date: Revised date: Accepted date:
29 November 2017 10 September 2018 13 November 2018
Please cite this article as: Yasamin Nasiri Khonsari, Shiguo Sun , A novel label free electrochemiluminescent aptasensor for the detection of lysozyme. Msc (2018), https://doi.org/10.1016/j.msec.2018.11.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A novel Label free electrochemiluminescent aptasensor for the detection of lysozyme Yasamin Nasiri Khonsaria, Shiguo Sun b *1
a
IP
T
State Key Laboratory of Fine Chemicals, Dalian University of Technology, No.2, Linggong Road, Ganjingzi District, Dalian 116023, China b
AC
CE
PT
ED
M
AN
US
CR
Key Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education, School of Pharmacy, Shihezi University, Shihezi 832000, China
* Corresponding author:
E-mail address: [email protected]
1
ACCEPTED MANUSCRIPT GRAPHICAL ABSTRACT
ED
M
AN
US
CR
IP
T
Representation of the fabrication process for electrochemiluminescent aptasensor
The bare indium tin oxide (ITO) has no Electrochemiluminescence (ECL) signal. When the Nitrogen-doped
PT
graphene quantum dot-chitosan/gold nanoparticles (NGQD-chitosan/AuNPs) composite film was electrodeposited on the ITO, the ECL intensity enhanced greatly. The ECL intensity decreased after
CE
aptamer and Bovine serum albumin (BSA) was dropped onto the electrode (curve a). However the ECL intensity further decreased after addition of lysozyme (curve b). The reason for this is that high specific
AC
affinity to lysozyme (Lys), hindered the diffusion of the ECL reagents towards the electrode surface and therefore signal further decreased after addition Lys.
2
ACCEPTED MANUSCRIPT ABSTRACT In this paper we describe a novel ultrasensitive electrochemiluminescent (ECL) aptasensor based on the quenching effect of aptamer and lysozyme incubation on co-reactant ECL mechanism of nitrogen-doped graphene quantum dots and persulfate. Incorporation of gold nanoparticles let to enhancement of electron transfer process of co-reactant mechanism species. The electrochemical behavior of each step modification of aptasensor was investigated using electrochemical impedance spectroscopy and verified by ECL responses. The aptasensor showed high stability,
T
sensitivity, reliable reproducibility, wide linear range (10 fM to 10 nM) with a low detection limit of 0.8 fM (at an
IP
S/N ratio of 3, n=10) can be successively applied to the human serum samples analysis. Recoveries ranged between
CR
91 and 104% (with RSDs of <3.1%).
Keywords: Nitrogen-doped graphene quantum dots; Electrochemiluminescence; Aptamer; Lysozyme; Gold
US
nanoparticle; Electrochemical behavior.
AN
1. Introduction
Lysozyme (Lys), also called muramidase or peptidoglycan N-acetylmuramoyl hydrolase [1], is an abundant protein
M
widely distributed in nature such as bacteria, bacteriophages, fungi, plants, and animals [2, 3]. Lys, contains 129 amino acid residues with a molecular weight of approximately 14,400 Da [4]. Increased concentration of Lys in serum is
ED
related to many diseases, such as cancer, HIV, leukemia, renal diseases, and meningitis [5, 6]. Therefore change in Lys amount can be a former marker for diagnosis, treatment and monitoring the progression of related diseases [7]. A variety of techniques have been used for Lys protein assay, including but not limited to fluorescence detection [8],
PT
electrochemical detection [9], Marty and coworkers reported a novel electrochemical aptamer–antibody based sandwich biosensor for the detection of Lys. In the sensing strategy, an anti-lysozyme aptamer was immobilized onto
CE
the carbon electrode surface by covalent binding via diazonium salt chemistry. After incubating with a target protein (Lys), a biotinylated antibody was used to complete the sandwich format. The results showed that the biosensor had good specificity, stability and reproducibility for Lys analysis. In addition, the biosensor was applied for detecting Lys
AC
in spiked wine samples, and very good recovery rates were obtained in the range from 95.2 to 102.0% for Lys detection. This implies that the sandwich biosensor is a promising analytical tool for the analysis of Lys in real samples [10]. Marty and coworkers described a comparison of two different aptamers (Apt) (COX and TRAN) for the detection of a ubiquitous protein Lys using Apt-based biosensors. The detection is based on the specific recognition by the Apt immobilized on screen printed carbon electrodes (SPCE) via diazonium coupling reaction. The results showed that the aptasensors exhibit good specificity, stability and reproducibility for Lys detection. For real application, the aptasensors were tested in wine samples and good recovery rates were recorded in the range from 94.2 to 102% for Lys detection. The recovery rates confirm the reliability and suitability of the method in wine matrix. The method can be a useful and promising platform for detection of Lys in different applications [11]. electrochemiluminescence [12], molecular imprinting [13], surface enhanced Raman scattering [14], series piezoelectric quartz crystal sensor [15] and
3
ACCEPTED MANUSCRIPT colorimetric assay [16]. Marty and coworkers developed a competitive aptamer based assay for detection of Lys by employing carboxylated magnetic beads as a support to immobilize the target molecule Lys. The used aptamer sequence was biotinylated which further binds with streptavidin-alkaline phosphatase in the micro wells. The assay displayed good recoveries of Lys in the range 99.00-99.27% and was demonstrated for the detection of Lys in wine samples [17]. Tseng and coworkers reported an aqueous solution of 13-nm gold nanoparticles (AuNPs) covalently bonded with human serum albumin (HSA) was used for sensing Lys. HSA molecules were good stabilizing agents for AuNPs in high-salt solution and exhibited the ability to bond with Lys electrostatically. It was found that the sensitivity
T
of HSA-AuNPs for Lys was highly dependent on the HSA concentration. They improved the selectivity of the probe
IP
by adjusting the pH solution to 8.0. Under the optimum conditions, the selectivity of this system for Lys over other proteins in high-salt solutions was remarkably high, even when their pI was very close to the Lys. The lowest
CR
detectable concentration of Lys in this approach was 50 nM. The applicability of the method was validated through the analyses of Lys in chicken egg white [18]. Tang’s group devised a new signal-enhanced fluorescence aptasensing
US
platform for quantitative screening of lysozyme by coupling with rolling circle amplification and strand hybridization reaction, accompanying the assembly of CdTe/CdSe quantum dots (QDs) and hemin/G-quadruplex DNzyme. Under optimal conditions, the fluorescent signal decreased with the increasing target Lys within the dynamic range from 5.0
AN
to 500 nM with a detection limit of 2.6 nM at the 3 sblank criterion. Intra-assay and inter-assay coefficients of variation were below 8.5% and 11.5%, respectively. Finally, the system was applied to analyze spiked human serum samples,
M
and the recoveries in all cases were 85-111.9% [19]. Therefore, detection of Lys has been getting importance and developing new, rapid, cheap and effective biosensors have been under investigation. An alternative method can be
ED
the use of ECL which is based on the emission of lights caused by high-energy electron-transfer reactions that happen on the surface of working electrodes. The advent of this technique can be traced back to the work of Tokel and Bard [20], who developed ECL systems based on the application of Ru(bpy)32+. Todays, ECL is an attractive and powerful
PT
detection tool in biosensing and bioassay [21, 22], because of its high sensitivity, and selectivity, relatively wide linear range, and low equipment cost [23]. Semiconductor nanocrystals and QDs have been extensively studied due to their
CE
unique size-dependent electronic, magnetic, optical and electrochemical properties. Since Bard’s group reported the ECL of Si QDs, QDs because of unique property have become one of the most popular ECL emitter and used to biomolecular determination [24]. However, compared with conventional ECL emitter such as Ru(bpy)32+ or luminol,
AC
QDs ECL suffer from some disadvantages such as weak ECL emissions [25]. Graphene quantum dots (GQDs), fewlayered graphene sheets with sizes smaller than 10 nm, are a novel type QDs in ECL [26]. Compared with the traditional QDs, GQDs have attracted great attention due to their robust biological and chemical inertness, low cytotoxicity and good biocompatibility [27]. Doping carbon nanomaterials with heteroatoms can effectively improve GQDs intrinsic properties, including electronic characteristics, surface and local chemical features. Nitrogen-doped graphene quantum dots (N-GQDs) with more active sites and more effective optical property rather than GQD S [27], exhibited excellent ECL performances and enhanced ECL intensity. Roushani and Abdi described a novel and sensitive electrochemical sensor based on graphene quantum dots/riboflavin modified glassy carbon electrode was constructed and utilized to determine persulfate (S2O82−). GQDs were expected to have the significant properties of graphene materials as well as new functions resulting from their quantum confinement and edge effects. The modified
4
ACCEPTED MANUSCRIPT electrode showed stable and well-defined redox couples at a wide pH range (1–10), with surface confined characteristics. This catalytic reduction allows an amperometric detection of S2O82− at a potential of -0.1 V with detection limit of 0.2 μM, concentration calibration range of 1.0 μM to 1 mM and sensitivity of 4.7 nA μM −1 [28]. Roushani and Valipour reported a facile green approach to employ silver nanoparticle (AgNPs) and thiol graphene quantum dots (GQD-SH) as the nanomaterial for ultrasensitive and selective detection of hepatitis C virus core antigen (HCV). AgNPs/GQD-SH was utilized as a substratum to load antibody for detection of hepatitis C virus core antigen. AgNPs have been immobilized on SH groups of GQDs via bonding formation of Ag-S and anti-HCV have been
T
loaded on the electrode surface via the interaction between –NH2 group of antibody and AgNPs. Using the
IP
nanocomposite provides a specific platform with increased surface which is capable of loading more antibodies to entrap the antigen. This novel immunosensor was used to analyze the serum sample. The immunosensor provides a
CR
convenient, low-cost and simple method for HCV core antigen detection and new horizons for quantitative detection of antigen in the clinical diagnosis [29]. Roushani and Fard described a highly sensitive and low-cost electrochemical
US
aptasensor was fabricated based on a Ag NPs/ GQD-SH nanocomposite for the measurement of 2, 4, 6-Trinitrotoluen (TNT) as a nitroaromatic explosive. For the first time Rutin as a biological molecule with inherent properties was used as the redox probe in the development of the TNT aptasensor was used. The system was based on a TNT-binding
AN
aptamer which is covalently attached onto the surface of a glassy carbon electrode modified with the nanocomposite for the formation of a sensing layer and improving the performance of the aptasensor. Applicability of the aptasensor
M
to easily detect TNT in real samples was evaluated. It seems that the strategy can be expanded to other nanoparticles and is expected to have promising implications in the design of electrochemical sensors or biosensors for the detection
ED
of various targets [30]. Roushani and coworkers described a novel electrochemical aptasensor for ultrasensitive detection of HCV core antigen by using the aptamer proximity binding assay strategy. The fabricated aptasensor can accurately detect HCV core antigen concentration in human serum samples. Such an aptasensor opens a rapid,
PT
selective and sensitive route for HCV core antigen detection and provides a promising strategy for potential applications in clinical diagnostics [31]. Roushani and coworkers reported a novel and sensitive method for detection
CE
of streptomycin (STR), to develop electrochemical sensing system based on GQDs functionalized with amine and thiol groups as the promising newest carbon-based nanomaterial. STR, an aminoglycoside antibiotic, was used in human and veterinary to treat gram-negative infections. STR residue causes serious side effects on human health. The
AC
system, in the presence of different interferences, displayed good selectivity in detecting STR. The aptasensor showed excellent results in quantitatively the detection of STR in serum samples [32]. Aptamer is a kind of synthetic oligonucleotides that engineered through repeated rounds of screening in vitro [33], a process called systematic evolution of ligands by exponential enrichment [34], which can be specifically recognized various molecular targets such as small molecules, proteins, nucleic acids, cells and even organisms. Compared with antibody, aptamer has similar high affinity and selectivity and because of their low cost, high affinity and simplicity, aptamer-based approaches has attracted much attention from researchers and has been widely used in the determination of various biomolecule [34]. Therefore, aptamer has been extensively applied as recognition element for the design of biosensors, especially ECL biosensors [35].
5
ACCEPTED MANUSCRIPT In this work, we introduced a novel electrochemiluminescent biosensor based on quenching effect of lysozyme on NGQD-S2O82– co-reactant mechanism of the NGQD-chitosan/AuNPs aptasensor. Immobilization of aptamer was successfully done by using –SH group as the binder of aptamer on AuNPs. The incorporation of AuNPs also enhanced electrochemical behavior of aptasensor resulting enhancement of NGQD-S2O82– ECL signals and higher sensitivity of aptasensor. This aptasensor showed excellent properties including high sensitivity and selectivity while using low cost
T
instrument setup. It can be nominated as the alternative method for clinical and medical diagnosis.
IP
2. Experimental
CR
2.1. Materials and reagents
Sulfuric acid (95%), hydrogen peroxide (30%), graphite powder (99.995%), potassium permanganate (99%),
US
potassium nitrate (99%), ammonium hydroxide (25%), hydrazine monohydrate (98%), chloroauric acid (HAuCl4) (98%), trisodium citrate dehydrate (99%) and sodium persulfate (98%) purchased from Sigma Aldrich Co. (St. Louis,
AN
MO, USA, https: //www.sigmaaldrich.com/chemistry.html). Piranha solution was prepared by mixing mentioned sulfuric acid and hydrogen peroxide (3:1).
The chitosan solution prepared by dispersing 25 mg of chitosan in 5 mL of acetic acid (3%). The lysozyme aptamer
Chemical
Reagent
Factory.
(https://www.chemicalregister.com/Tianjin_Chemical_Reagent_Factory/
ED
Tianjin
M
5′-HS-(CH2)6-ATC TAC GAA TTC ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG-3′ was prepared from
Supplier/sid21665. html).
The phosphate buffer was used at pH 7.4 and potassium chloride was added to increase ionic strength to adopt
PT
biological environment. All other chemical were reagent grade and used without any treatment.
CE
2.2. Apparatus
MPI-B ECL analyzer was used to monitor ECL signals. (Xi’an Remax Electronic Science Tech. Co., Ltd., China,
AC
http://www.chinaremax.com/). Electrochemical experiments such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were carried out with AUTOLAB PGSTAT 30 potentiostat/galvanostate (Eco Chemie BV, Utrecht, The Netherlands, http://www.ecochemie.nl/Company/Countries/Netherlands.html). Three electrode system including NGQD-chitosan/AuNPs aptasensor modified low resistance indium tin oxide (ITO) plate (1 cm2 active surface) as the working electrode, platinum (Pt) rode and silver/silver chloride (Ag/AgCl) as counter and reference electrode, respectively, were used in ECL and electrochemical studies. Size and dispersion of nanomaterial was investigated by transmission electron microscopy (TEM, Phillips EM 2085, https://www.dotmed.com/AnalyticalLab/Electron-Microscope/Models/Phillips/Em-208/11522). Optical properties of nitrogen-doped graphene quantum dots
and
gold
nanoparticles
were
studied
by using
UV-vis
spectroscopy
(PerkinElmer,
http://www.perkinelmer.com.cn/) and the photoluminescence experiments carried on a LS50 Perkin Elmer.
6
America,
ACCEPTED MANUSCRIPT 2.3. Preparation of N-GQDs N-GQDs were synthesized by pyrolyzing citric acid (CA) [36]. Briefly, 2 g of CA was heated to 200 °C in a 10 mL beaker on a heater for approximately 30 minutes until CA changed to orange liquid. The resulting liquid pH were adjusted to 8 with sodium hydroxide (NaOH) under stirring. N-GQDs were prepared by hydrothermal method. 20 mL of N-GQDs treated with 0.3 mL hydrazine. The mixture transferred into 25 mL autoclave and heated at 175 °C for 12 h. The final solution were centrifuged at 9,500 rpm in aqueous ethanol solution (3:1) and then evaporated by rotary to
IP
T
collect N-GQDs.
CR
2.4. Au NPs synthesis
US
AuNPs is prepared by using Turkevich method [37]. Briefly, 20 mL of 1.0 mM HAuCl4 was added to a 50 mL beaker and reflux for heated to boiling point while stirring. After, 2 mL of 1% solution of trisodium citrate dehydrate quickly added to the reaction, a deep red color solution was observed. After a defined time (normally 15 min), the final solution
AN
cooled to room temperature.
M
2.5. Fabrication of electrochemiluminescent aptasensor
ED
The aptasensor was made. First, ITO plate were pretreated by ultrasonication in ethanol and deionized water then rinsed with deionized water and dried under nitrogen gas. The surface of pretreated ITO activated by rinsing in piranha
PT
solution for 1 min to obtain hydroxyl group on the surface of ITO. A mixture of N-GQDs and chitosan solution (1:3) was prepared by ultrasonication for about 1 hour. 0.5 mL of NGQD-chitosan mixture drop casted on activated ITO
CE
plate and a galvanostatic reaction was carried out at 25 Am-2 then 20 µL of AuNPs solution added on the NGQDchitosan/ITO and rinsed in deionized water. The prepared Au NPs/NGQD-chitosan/ITO immersed in mixture of 5 wt % glutaric dialdehyde and aptamer solution for 6 hours at room temperature. To bond chitosan to fabric chemically,
AC
glutaric dialdehyde is chosen as the crosslinking agent. Finally, the aptasensor AuNPs/NGQD-chitosan/ITO was rinsed with 0.1 M phosphate buffer (pH 7.3) and nonspecific sites of mention sensor was block by incubation with 20 µL 1% bovine serum albumin (BSA) for 1 hour and then rinsed with deionized water and stored in refrigerator.
3. Results and discussion 3.1. The characterization of the prepared AuNPs and N-GQDs
7
ACCEPTED MANUSCRIPT The morphologies of the N-GQDs were studied by using TEM. Fig. 1 shows the TEM images of N-GQDs. N-GQDs particles were randomly selected to measure the particle size and the particle size distribution was shown in Fig. S1. It can be seen that average size of N-GQDs is 5.1± 0.65 nm. Fig. 1 N-GQDs were water-dispersible due to the presence of hydroxyl and carboxylic functional groups on the surface, which was confirmed by FT-IR measurement. The FT-IR spectrum of N-GQDs are shown in Fig. S2. In the spectrum
T
of N-GQDs, the weak peak located at 1723 cm-1 assigned to the stretching vibration of C ═ O groups. The absorption
IP
band around 1160 and 1032 cm-1 are ascribed to C-C and C-O stretching, respectively. More importantly, the peak at
CR
1556 cm-1 corresponding to the superposition of the vibrations of C ═ C and C ═ N, and the absorption peaks at 3434, 3087cm-1 can be ascribed to the stretching vibration of O-H and N-H which gives the evidence for the embedding of nitrogen-containing groups [38].
US
Optical properties of aqueous dispersed N-GQDs also investigated by UV-vis absorption and photoluminescence spectroscopy. Fig. S3 (A), it can be seen that UV-vis spectrum of N-GQDs showed a broadened absorption band
AN
centered at 332 nm [38]. Fig. S3 (B), presents the photoluminescence (PL) emission spectrum of the aqueous solution of N-GQDs with excitation at 370 nm; the N-GQDs shows emission at 445 nm. The quantum yield of N-GQDs was calculated to be 25 % which is far better than previously reported N-GQDs. The higher PL emission quantum yield
ED
electronic characteristics of the N-GQDs [39].
M
possibly arise from the oxygen rich groups in N-GQDs and N-doping-induced modulation of the chemical and
PT
3.2. Electrochemical and ECL behavior of the aptasensor Electrochemical behavior of an electrochemiluminescent aptasensor is the most important factor which indicates
CE
aptasensor performance. Furthermore, EIS test was carried out in 0.1M KCl solution with 10 mM Fe(CN) 63−/ Fe(CN)64−, frequency range of 10 mHz to 1 MHz and the direct current (DC) bias range was 10 mV. The EIS response
AC
of aptasensor in each modification step is shown in Nyquist plot in Fig. 2. Diameter of semicircle in high frequency zone indicates the charge transfer resistance of the electrochemical reaction. The Warburg resistance in lower frequencies shows the diffusion control electrochemical reaction on the surface of the modified aptasensor. EIS spectrum for the bare electrode exhibited small semicircle domain, at high frequencies (curve a), and its electron transfer resistance is small, indicating a fast electron-transfer process of [Fe(CN)6]3−/4− . After casting NGQD-chitosan on the ITO plate (curve b), the EIS response showed a high imaginary resistance indicating increase of active surface of the electrode due to the N-GQDs higher surface area, and high electron transfer resistance can be explained by less conductivity of chitosan compared to the bare ITO plate. Gold nanoparticles addition on NGQD-chitosan/ITO lowered electron transfer resistance due to high conductivity of AuNPs which results in fast electron-transfer process of [Fe(CN)6]3−/4− (curve c). HS-aptamer increased the electron transfer resistance
8
ACCEPTED MANUSCRIPT which demonstrate successively immobilization of the HS-aptamer (curve d). Finally, BSA and lysozyme also increased electron transfer resistance indicating adsorption of lysozyme was done on the immobilized aptamer (curve e, f). N-GQDs, along with chitosan added to the electrode surface and chitosan increases surface resistant due to its low conductivity. N-GQDs is luminophore in this work. Fig. 2 CV measurements were used to characterize the process of electrochemiluminescent aptasensor modification. CV
T
curves of stepwise modification of SPCE in 10 mM Fe(CN)63−/ Fe(CN)64− containing 0.1 M potassium chloride (KCl)
IP
solution were shown in Fig. 3. SPCE is screen printed carbon electrode, it’s used to apply a potential to the
CR
luminophore and co-reactant in ECL mechanisms. SPCE possess excellent biocompatibility, a wide pH range and high sensitivity. The SPCE is of particular interest due to its easy fabrication, mass productivity and low cost. And the carbon ink for SPCEs often contains some mineral binders or insulating polymers, which cover at the surface of
US
graphite particles to form the thin insulating layer for cathodic ECL. A well-defined redox peak of Fe(CN)63−/ Fe(CN)64− was observed at the bare ITO (curve a). When NGQD-chitosan were coated on the ITO, the peak current decreased (curve b), however modification of ITO by AuNPs (curve c) the peak current increase, due to the increase
AN
in the electrode surface area and the excellent conductivity of AuNPs. While, the electrode modified with aptamer exhibited a decrease peak current (curve d). Also the modified electrode was blocked with BSA (curve e) and high
M
specific affinity of aptamer and successfully bound to Lys (curve f) on electrode surface, the CV responses significantly declined. For ECL measurement cyclic voltammetry was done in the range of 0 to -1.8 V in phosphate
ED
buffer (pH 7.4) containing 0.1 M Na2S2O8.
Fig. 3
PT
For each step of modification, ECL response recorded. Fig. S4, however, no ECL signals were observed for bare ITO. Modification of ITO plate with NGQD-chitosan results a high ECL signal. Moreover, addition of AuNPs greatly
CE
increase ECL signal due to the improvement of electron transfer resistance which verifies the EIS results. The ECL intensity slightly decreased after addition of aptamer and BSA and addition of lysozyme greatly quenched the ECL
AC
signals. Lysozyme and aptamer act as blocking layer for aptasensor and lowering active sites of electron transfer reaction, resulting in low efficient ECL process [40]. The ECL mechanism of N-GQDs and sodium persulfate (Na2S2O8) can be described as below: [40] eq1
S2O8 2– + e–
SO42– + SO4– •
eq2
NGQDs + e–
NGQDs– •
eq3
NGQDs– • + SO4– •
eq4
NGQDs*
NGQDs* + SO42– NGQDs + hν
9
ACCEPTED MANUSCRIPT The negative potential scanning results in electrochemically generation of SO4– • and N-GQDs– • which then undergo a high energy electron transfer reaction by injection of a hole into the N-GQDs, resulting excited state N-GQDs which emits light.
3.3. Optimization of the detection conditions Experimental parameters, such as pH, Na2S2O8 and incubation time were optimized in order to obtain best performance
T
of aptasensor. pH of the working solution, as one of the most effective parameters, was optimized in the range between
IP
5 to 9 Fig. S5 (A). pH 7.5 showed the highest ECL signal, however, pH value of 7.4 is for the common biological matrixes, pH 7.4 selected for further experiments.
CR
Concentration of Na2S2O8 as the co-reactant for ECL enhancement also optimized. Increasing Na2S2O8 concentration from 0.05 to 0.1 M resulted in enhancement of ECL signals and further increasing the Na2S2O8 concentration
US
decreased ECL efficiency. Thus 0.1 M Na2S2O8 was selected as the optimum concentration and used for further experiments Fig. S5 (B).
AN
Incubation time of aptamer and lysozyme was studied and in the range between 10 min to 2 h, the quenching effect increased with incubation time and reached a plateau at 1 h Fig. S5 (C). Therefore, pH value of 7, 0.1 M Na2S2O8 and
ED
M
1 h incubation time between aptamer and lysozyme was selected as the optimum experiment condition.
3.4. Analytical performance of the electrochemiluminescent aptasensor
PT
Fig. 4 shows the response of AuNPs/NGQD-chitosan/ITO aptasensor before (curve a) and after (curves a–i) the reaction with different concentrations of lysozyme, in 4 mL 0.1 M phosphate buffer (pH 7.4) containing 0.1 M
CE
Na2S2O8. The Fig. 5 shows the typical calibration curve of lysozyme detection. The limit of detection was found to be 0.8 fM with the relative standard deviation (RSD) of 2.9% in 10 times analysis of blank solution (S/N = 3). RSD of 5 pM lysozyme in 10 times also determined to be 3.1%. These results show highly sensitive response of aptasensor Au
AC
NPs/NGQD-chitosan/ITO in presence of lysozyme. Fig. 4 Fig. 5
3.5. Stability, selectivity and reproducibility of the aptasensor Stability of AuNPs/NGQD-chitosan/ITO aptasensor also investigated, results is shown in Fig. 6. Under consecutive cyclic potential scan for 10 cycles, the ECL signals are very high and the RSD for the change in the ECL intensity is less than 3.4%, revealing good stability of the aptasensor.
10
ACCEPTED MANUSCRIPT Fig. 6 Reproducibility of aptasensor Au NPs/NGQD-chitosan/ITO, 10 aptasensors were fabricated in 10 individual days then signals were measured from optimum experiment condition in presence of 5 pM lysozyme. The RSD of the 10 individually fabricated aptasensors AuNPs/NGQD-chitosan/ITO to be found 4.2, indicating high reproducibility of the aptasensor. The selectivity of the aptasensor was also studied by choosing HSA, Hemoglobin (Hb), Immunoglobulin G (IgG) and
T
thrombin (Thr) as interferents, and the results are shown in Fig. 7. The concentrations of HSA, Hb, IgG, alpha-
IP
fetoprotein (AFP) were 10-fold (10-11 M) than target lysozyme (5×10-12 M). The results showed negligible ECL with interference proteins compared with that of presence of the target lysozyme. Also the response of the aptasensor in a
CR
mixture containing of HSA, Hb, IgG, AFP and Lys was examined. The ∆I of the mixture had little difference compared with that in only the lysozyme solution. The results demonstrate that the aptasensor has good selectivity for the
US
detection of Lysozyme.
AN
Fig. 7
3.6. Application of the electrochemiluminescent aptasensor
M
For real sample testing of AuNPs/NGQD-chitosan/ITO aptasensor, human serum were examined. The Spiking method was chosen to establish the sensor performance in complex matrixes and results are shown in Table 1. The high
ED
recovery and repeatability of aptasensor would make it a great alternative method for lysozyme detection in human
AC
4. Conclusions
Table 1
CE
PT
serum.
In summary, an electrochemiluminescent aptasensor fabricated by using AuNPs/NGQD-chitosan/ITO composite. The ECL signals generated by co-reactant ECL mechanism of N-GQDs and Na2S2O8 and the lysozyme measurement was based on quenching effect lysozyme aptamer incubation, which result to low electron transfer process between both N-GQDs and Na2S2O8. Au NPs was very effective in increasing ECL signals due to the high conductivity and surface area which results in higher sensitivity of aptasensor. Application of AuNPs/NGQD-chitosan/ITO aptasensor in biological matrix leaves no doubts about applicability of aptasensor as a high selective and interference free detection system of lysozyme. The bare ITO has no ECL signal. When the NGQD-chitosan/AuNPs composite film was electrodeposited on the ITO, the ECL intensity enhanced greatly. The ECL intensity decreased after aptamer and BSA was dropped onto the electrode. The ECL intensity further decreased after addition of lysozyme. The reason for this
11
ACCEPTED MANUSCRIPT is that high specific affinity to Lys, hindered the diffusion of the ECL reagents towards the electrode surface and therefore signal further decreased after addition Lys.
Acknowledgements This work was financially supported by State Key Laboratory of Fine Chemicals, the National Natural Science
CR
IP
T
Foundation of China (No. 21272030, 21472016, 21306019, 21576042).
References
D. M. Donovan, S. Dong, W. Garrett, G. M. Rousseau, S. Moineau, D. G. Pritchard, Peptidoglycan Hydrolase
US
1.
Fusions Maintain Their Parental Specificities, Appl. Environment. Microbiol. 72 (2006) 2988-2996. D. M. Donovan, Google Patents, 2011.
3.
P. Markart, N. Faust, T. Graf, C.-L. Na, T. E. Weaver, H. T. Akinbi, Comparison of the microbicidal and
AN
2.
muramidase activities of mouse lysozyme M and P, Biochem. J. 380 (2004) 385-392. A. Amit, R. Mariuzza, S. Phillips, R. Poljak, Three-dimensional structure of an antigen-antibody complex at
M
4.
2.8 a resolution, Science. 233 (1986) 747-753.
D. Canet, M. Sunde, A. M. Last, A. Miranker, A. Spencer, C. V. Robinson, C. M. Dobson, Mechanistic
ED
5.
Studies of the Folding of Human Lysozyme and the Origin of Amyloidogenic Behavior in Its Disease-Related
6.
PT
Variants, Biochem. 38 (1999) 6419-6427.
S. L.-Huang, P. L. Huang, Y. Sun, P. L. Huang, H.-F. Kung, D. L. Blithe, H.-C. Chen, Lysozyme and R Nases as anti-HIV components in β-core preparations of human chorionic gonadotropin, Proc. Natl. Acad.
7.
CE
Sci. 96 (1999) 2678-2681.
B. Schmekel, P. Blomstrand, P. Venge, Serum lysozyme – a surrogate marker of pulmonary microvascular
8.
AC
injury in smokers, Clin Physiol Funct Imaging. 33 (2013) 307-312. S. Liu, W. Na, S. Pang, F. Shi, X. Su, A label-free fluorescence detection strategy for lysozyme assay using CuInS2 quantum dots, The Analyst. 139 (2014) 3048-3054. 9.
Q. Wang, H. Zheng, X. Gao, Z. Lin, G. Chen, A label-free ultrasensitive electrochemical aptameric recognition system for protein assay based on hyperbranched rolling circle amplification, Chem. Commun. 49 (2013) 11418-11420.
10. C. Ocaña, A. Hayat, R. Mishra, A. Vasilescu, M. D. Valle, J. L. Marty, A novel electrochemical aptamer– antibody sandwich assay for lysozyme detection, Analyst. 140 (2015) 4148-4153. 11. C. Ocaña, A. Hayat, R. K. Mishra, A. Vasilescu, M. D. Valle, J. L. Marty, Label free aptasensor for Lysozyme detection: A comparison of the analytical performance of two aptamers, Bioelectrochem. 105 (2015) 72-77.
12
ACCEPTED MANUSCRIPT 12. H. Xia, L. Li, Z. Yin, X. Hou, J. J. Zhu, Biobar-Coded Gold Nanoparticles and DNAzyme-Based Dual Signal Amplification Strategy for Ultrasensitive Detection of Protein by Electrochemiluminescence, ACS Appl. Mater & Inter. 7 (2014) 696-703. 13. S. Khurshid, L. Govada, H. F. El.-Sharif, S. M. Reddy, N. E. Chayen, Automating the application of smart materials for protein crystallization, Acta. Crystall. Sec D: Biology. Crystall. 71 (2015) 534-540. 14. L.-J. Xu, C. Zong, X.-S. Zheng, P. Hu, J.-M. Feng, B. Ren, Label-Free Detection of native proteins by surface-enhanced Raman Spectroscopy using lodide-Modified nanoparticles, Anal. Chem. 86 (2014) 2238-
T
2245.
IP
15. Y. Lian, F. He, X. Mi, F. Tong, X. Shi, Lysozyme aptamer biosensor based on electron transfer from SWCNTs to SPQC-IDE, Sens. Actuator B: Chem. 199 (2014) 377-383.
CR
16. Y. Zeng, Y. Wan, D. Zhang, Lysozyme as sensitive reporter for fluorometric and PCR based detection of E. coli and S. aureus using magnetic microbeads, Microchim. Acta. 183 (2016) 741-748.
US
17. R. K. Mishra, A. Hayat, G. K. Mishra, G. Catanante, V. Sharma, J. L. Marty, A novel colorimetric competitive aptamer assay for lysozyme detection based on superparamagnetic nanobeads, Talanta. 165 (2017) 436-441.
AN
18. Y. M. Chen, C. J. Yu, T. L. Cheng, W. L. Tseng, Colorimetric Detection of Lysozyme Based on Electrostatic Interaction with Human Serum Albumin-Modified Gold Nanoparticles, Langmuir. 24 (2008) 3654-3660.
M
19. Z. Qiu, J. Shu, Y. He, Z. Lin, K. Zhang, S. Lv, D. Tang, CdTe/CdSe quantum dot-based fluorescent aptasensor with hemin/G-quadruplex DNzyme for sensitive detection of lysozyme using rolling circle
ED
amplification and strand hybridization, Biosens. Bioelectron. 87 (2017) 18-24. 20. M. Hesari, Z. Ding, Review: Electrogenerated Chemiluminescence: Light Years Ahead, J. Electrochem. Soc. 163 (2016) 3116-3131.
PT
21. M. Hosseini, M. R. Moghaddam, F. Faridbod, P. Norouzi, M. R. K. Pur, M. R. Ganjali, A novel solid-state electrochemiluminescence sensor based on a Ru(bpy)32+/nano Sm2O3 modified carbon paste electrode for the
CE
determination of L-proline, RSC Adv. 5 (2015) 64669-64674. 22. A. Zhang, H. Xiang, X. Zhang, W. Guo, E. Yuan, C. Huang, N. Jia, A novel sandwich electrochemiluminescence immunosensor for ultrasensitive detection of carbohydrate antigen 19-9 based on
AC
immobilizing luminol on Ag@BSA core/shell microspheres, Biosens. Bioelectron. 75 (2016) 206-212. 23. W. Miao, Electrogenerated Chemiluminescence and Its Biorelated Applications, Chem. Rev. 108 (2008) 2506-2553.
24. Z. Ding, B. M. Quinn, S. K. Haram, L. E. Pell, B. A. Korgel, A. J. Bard, Electrochemistry and Electrogenerated Chemiluminescence from Silicon Nanocrystal Quantum Dots, Science. 296 (2002) 12931297. 25. Y. Xu, J. Liu, C. Gao, E. Wang, Applications of carbon quantum dots in electrochemiluminescence: A mini review, Electrochem. Commun. 48 (2014) 151-154. 26. H. Sun, L. Wu, W. Wei, X. Qu, Recent advances in graphene quantum dots for sensing, Mater. Today. 16 (2013) 433- 442.
13
ACCEPTED MANUSCRIPT 27. Q. Liu, B. Guo, Z. Rao, B. Zhang, J. R. Gong, Strong Two-Photon-Induced Fluorescence from Photostable, Biocompatible Nitrogen-Doped Graphene Quantum Dots for Cellular and Deep-Tissue Imaging, Nano Lett. 13 (2013) 2436-2441. 28. M. Roushani, Z. Abdi, Novel electrochemical sensor based on graphene quantum dots/riboflavin nanocomposite for the detection of persulfate, Sens. Actuator B: Chem. 201 (2014) 503-510. 29. A.Valipour, M. Roushani, Using silver nanoparticle and thiol graphene quantum dots nanocomposite as a substratum to load antibody for detection of hepatitis C virus core antigen: Electrochemical oxidation of
T
riboflavin was used as redox probe, Biosens. Bioelectron. 89 (2017) 946-951.
IP
30. F. S. Fard, M. Roushani, Designing an ultra-sensitive aptasensor based on an AgNPs/thiol-GQD
redox probe, Biosens. Bioelectron. 87 (2017) 724-731.
CR
nanocomposite for TNT detection at femtomolar levels using the electrochemical oxidation of Rutin as a
31. K. Ghanbari, M. Roushani, A. Azadbakht, Ultra-sensitive aptasensor based on a GQD nanocomposite for
US
detection of hepatitis C virus core antigen, Anal. Biochem. 534 (2017) 64-69. 32. M. Roushani, K. Ghanbari, S. J. Hoseini, Designing an electrochemical aptasensor based on immobilization of the aptamer onto nanocomposite for detection of the streptomycin antibiotic, Microchem. J. 141 (2018)
AN
96-103.
33. W. Zhou, P.-J. J. Huang, J. Ding, J. Liu, Aptamer-based biosensors for biomedical diagnostics, The Analyst.
M
139 (2014) 2627-2640.
34. N. Duan, S. Wu, X. Chen, Y. Huang, Y. Xia, X. Ma, Z. Wang, Selection and Characterization of Aptamers
ED
against Salmonella typhimurium Using Whole-Bacterium Systemic Evolution of Ligands by Exponential Enrichment (SELEX), J. Agricul. Food. Chem. 61 (2013) 3229-3234. 35. E. M. Gross, S. S. Maddipati, S. M. Snyder, A review of electrogenerated chemiluminescent biosensors for
PT
assays in biological matrices, Bioanalysis. 8 (2016) 2071-2089. 36. J. Ju, W. Chen, Synthesis of highly fluorescent nitrogen-doped graphene quantum dots for sensitive, label-
CE
free detection of Fe (III) in aqueous media, Biosens. Bioelectron. 58 (2014) 219-225. 37. J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot, A. Plech, Turkevich Method for Gold Nanoparticle Synthesis Revisited, J. Physic. Chem B. 110 (2006) 15700-15707.
AC
38. J. Ju, R. Zhang, S. He, W. Chen, Nitrogen-doped graphene quantum dots-based fluorescent probe for the sensitive turn-on detection of glutathione and its cellular imaging, RSC Adv. 4 (2014) 52583-52589. 39. L. Lin, X. Song, Y. Chen, M. Rong, T. Zhao, Y. Jiang, Y. Wang, X. Chen, One-pot synthesis of highly Greenish-yellow fluorescent nitrogen-doped graphene quantum dots for pyrophosphate sensing via competitive coordination with Eu 3+ ions, Nanoscale. 7 (2015) 15427-15433. 40. X. Du, D. Jiang, Q. Liu, G. Zhu, H. Mao, K. Wang, Fabrication of graphene oxide decorated with nitrogendoped graphene quantum dots and its enhanced electrochemiluminescence for ultrasensitive detection of pentachlorophenol, Analyst. 140 (2015) 1253-1259.
14
ACCEPTED MANUSCRIPT Figures Legend
Fig. 1. TEM images of N-GQDs. Fig. 2. EIS behaviors of (a) bare ITO (b) NGQD-Chitosan (c) AuNPs /NGQD-Chitosan/ITO (d) aptamer/AuNPs/NGQD-Chitosan/ITO
(e)
BSA/aptamer/AuNPs/NGQD-Chitosan/ITO.
(f)
T
Lys/BSA/aptamer/AuNPs/NGQD-Chitosan/ITO in Fe(CN)63−/ Fe(CN)64– (10 mM) (1:1)
IP
containing KCl (0.1 M) and EIS in the frequency range (0.01 Hz to 100 kHz) and signal amplitude
CR
(5 mV).
Fig. 3. CV behaviors of (a) bare ITO (b) NGQD-Chitosan (c) AuNPs /NGQD-Chitosan/ITO (d) (e)
BSA/aptamer/AuNPs/NGQD-Chitosan/ITO.
(f)
US
aptamer/AuNPs/NGQD-Chitosan/ITO
Lys/BSA/aptamer/ AuNPs/NGQD-Chitosan/ITO in Fe(CN)63−/ Fe(CN)64– (10 mM) (1:1)
AN
containing KCl (0.1 M) and EIS in the frequency range (0.01 Hz to 100 kHz) and signal amplitude (5 mV).
M
Fig. 4. ECL-potential signals of the aptasensor with different concentrations of Lysozyme samples. (a–i, respectively, C (Lys)= 0, 0.01, 0.05, 0.5, 5, 50, 500, 5000, 50000 pM and wavelength of
ED
fluorescence emission = 445 nm; potential = 0 to -1.8 V).
PT
Fig. 5. The calibration plot against the corresponding log C (Lys). Fig. 6. Stability of ECL signal from aptasensor in phosphate buffer 0.1 M (pH 7.4), containing
CE
Na2S2O8 (0.1M) under ten cycles of CV scan.
AC
Fig. 7. The selectivity of the electrochemiluminescent aptasensor towards different targets.
15
Fig. 1
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Fig. 2 16
AC
CE
PT
ED
M
AN
Fig. 3
US
CR
IP
T
ACCEPTED MANUSCRIPT
Fig. 4
17
ACCEPTED MANUSCRIPT 3500
y = 476.49x + 6784.9 R² = 0.995
3000
∆IECL
2500 2000
T
1500
IP
1000
0 -15
-13
-11
CR
500
US
LogC (M)
AC
CE
PT
ED
M
AN
Fig. 5
Fig. 6
18
-9
-7
US
CR
IP
T
ACCEPTED MANUSCRIPT
ED
M
AN
Fig. 7
PT
Table 1 Recovery and relative standard deviation of Lys in human serum samples.
2 3
AC
1
Spiked (pM) 1
CE
Serum
Found (pM) 0.91± 0.65
Recovery () 91
RSD (, n=3) 3.1
5
4.8± 0.4
96
2.6
25
26± 0.55
104
2.9
Mean value ± SD of three measurements
19
ACCEPTED MANUSCRIPT Supplementary data
Fig. S1. Size distribution of N-GQDs. Fig. S2. FT-IR spectrum of N-GQDs.
IP
T
Fig. S3 (A). UV-vis spectrum of the N-GQDs. (B). Fluorescence spectrum of the N-GQDs. Fig. S4. ECL behavior of (a) bare ITO. (b) NGQD-Chitosan. (c) AuNPs /NGQD-Chitosan/ITO.
CR
(d) aptamer/AuNPs/NGQD-Chitosan/ITO. (e) BSA/aptamer/AuNPs/NGQD-Chitosan/ITO. (f) Lys/BSA/aptamer/ AuNPs/NGQD-Chitosan/ITO.
US
Fig. S5 (A). ECL signal from aptasensor in phosphate buffer (0.1M) with different pH containing
AN
Na2S2O8 (0.1M). (B). ECL signal from aptasensor with different concentration of Na2S2O8 in
AC
CE
PT
ED
M
phosphate buffer 0.1 M (pH 7.4). (C). Effect of the incubation time on ECL peak intensity.
20
US
CR
IP
T
ACCEPTED MANUSCRIPT
M
AN
Fig. S1.
ED
0.35
PT
0.3
0.15 0.1
0.05
CE
0.2
AC
Absorbance (a.u.)
0.25
0
500
1000
1500
2000
2500
3000
wavenumber (cm-1) Fig. S2. 21
3500
4000
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
Fig. S3 (A).
Fig. S3 (B).
22
ACCEPTED MANUSCRIPT
6000
c b d e
4000
IP
T
f
CR
3000
US
2000
1000
AN
a 0 -1.5
-1
M
-2
Fig. S4.
CE
PT
ED
Potential / V
AC
ECL Intensity
5000
23
-0.5
0
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
Fig. S5 (A).
24
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Fig. S5 (B).
Fig. S5 (C).
25
ACCEPTED MANUSCRIPT
IP
T
HIGHLIGHTS
CR
►A novel ultrasensitive electrochemiluminescent (ECL) aptasensor based on the quenching effect of aptamer and lysozyme incubation on co-reactant ECL
US
mechanism of nitrogen-doped graphene quantum dots and persulfate was developed.
range (10 fM to 10 nM) were reached.
AN
► A low detection limit of 0.8 fM (at an S/N ratio of 3, n=10) and a wide linear
M
►The aptasensor applied to the human serum samples analysis.
AC
CE
PT
ED
► Range of recoveries were between 91 and 104% (with RSDs of <3.1%).
26
Yasamin Nasiri Khonsari, Shiguo Sun PII: DOI: Reference:
S0928-4931(17)34590-3 https://doi.org/10.1016/j.msec.2018.11.016 MSC 9036
To appear in:
Materials Science & Engineering C
Received date: Revised date: Accepted date:
29 November 2017 10 September 2018 13 November 2018
Please cite this article as: Yasamin Nasiri Khonsari, Shiguo Sun , A novel label free electrochemiluminescent aptasensor for the detection of lysozyme. Msc (2018), https://doi.org/10.1016/j.msec.2018.11.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A novel Label free electrochemiluminescent aptasensor for the detection of lysozyme Yasamin Nasiri Khonsaria, Shiguo Sun b *1
a
IP
T
State Key Laboratory of Fine Chemicals, Dalian University of Technology, No.2, Linggong Road, Ganjingzi District, Dalian 116023, China b
AC
CE
PT
ED
M
AN
US
CR
Key Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education, School of Pharmacy, Shihezi University, Shihezi 832000, China
* Corresponding author:
E-mail address: [email protected]
1
ACCEPTED MANUSCRIPT GRAPHICAL ABSTRACT
ED
M
AN
US
CR
IP
T
Representation of the fabrication process for electrochemiluminescent aptasensor
The bare indium tin oxide (ITO) has no Electrochemiluminescence (ECL) signal. When the Nitrogen-doped
PT
graphene quantum dot-chitosan/gold nanoparticles (NGQD-chitosan/AuNPs) composite film was electrodeposited on the ITO, the ECL intensity enhanced greatly. The ECL intensity decreased after
CE
aptamer and Bovine serum albumin (BSA) was dropped onto the electrode (curve a). However the ECL intensity further decreased after addition of lysozyme (curve b). The reason for this is that high specific
AC
affinity to lysozyme (Lys), hindered the diffusion of the ECL reagents towards the electrode surface and therefore signal further decreased after addition Lys.
2
ACCEPTED MANUSCRIPT ABSTRACT In this paper we describe a novel ultrasensitive electrochemiluminescent (ECL) aptasensor based on the quenching effect of aptamer and lysozyme incubation on co-reactant ECL mechanism of nitrogen-doped graphene quantum dots and persulfate. Incorporation of gold nanoparticles let to enhancement of electron transfer process of co-reactant mechanism species. The electrochemical behavior of each step modification of aptasensor was investigated using electrochemical impedance spectroscopy and verified by ECL responses. The aptasensor showed high stability,
T
sensitivity, reliable reproducibility, wide linear range (10 fM to 10 nM) with a low detection limit of 0.8 fM (at an
IP
S/N ratio of 3, n=10) can be successively applied to the human serum samples analysis. Recoveries ranged between
CR
91 and 104% (with RSDs of <3.1%).
Keywords: Nitrogen-doped graphene quantum dots; Electrochemiluminescence; Aptamer; Lysozyme; Gold
US
nanoparticle; Electrochemical behavior.
AN
1. Introduction
Lysozyme (Lys), also called muramidase or peptidoglycan N-acetylmuramoyl hydrolase [1], is an abundant protein
M
widely distributed in nature such as bacteria, bacteriophages, fungi, plants, and animals [2, 3]. Lys, contains 129 amino acid residues with a molecular weight of approximately 14,400 Da [4]. Increased concentration of Lys in serum is
ED
related to many diseases, such as cancer, HIV, leukemia, renal diseases, and meningitis [5, 6]. Therefore change in Lys amount can be a former marker for diagnosis, treatment and monitoring the progression of related diseases [7]. A variety of techniques have been used for Lys protein assay, including but not limited to fluorescence detection [8],
PT
electrochemical detection [9], Marty and coworkers reported a novel electrochemical aptamer–antibody based sandwich biosensor for the detection of Lys. In the sensing strategy, an anti-lysozyme aptamer was immobilized onto
CE
the carbon electrode surface by covalent binding via diazonium salt chemistry. After incubating with a target protein (Lys), a biotinylated antibody was used to complete the sandwich format. The results showed that the biosensor had good specificity, stability and reproducibility for Lys analysis. In addition, the biosensor was applied for detecting Lys
AC
in spiked wine samples, and very good recovery rates were obtained in the range from 95.2 to 102.0% for Lys detection. This implies that the sandwich biosensor is a promising analytical tool for the analysis of Lys in real samples [10]. Marty and coworkers described a comparison of two different aptamers (Apt) (COX and TRAN) for the detection of a ubiquitous protein Lys using Apt-based biosensors. The detection is based on the specific recognition by the Apt immobilized on screen printed carbon electrodes (SPCE) via diazonium coupling reaction. The results showed that the aptasensors exhibit good specificity, stability and reproducibility for Lys detection. For real application, the aptasensors were tested in wine samples and good recovery rates were recorded in the range from 94.2 to 102% for Lys detection. The recovery rates confirm the reliability and suitability of the method in wine matrix. The method can be a useful and promising platform for detection of Lys in different applications [11]. electrochemiluminescence [12], molecular imprinting [13], surface enhanced Raman scattering [14], series piezoelectric quartz crystal sensor [15] and
3
ACCEPTED MANUSCRIPT colorimetric assay [16]. Marty and coworkers developed a competitive aptamer based assay for detection of Lys by employing carboxylated magnetic beads as a support to immobilize the target molecule Lys. The used aptamer sequence was biotinylated which further binds with streptavidin-alkaline phosphatase in the micro wells. The assay displayed good recoveries of Lys in the range 99.00-99.27% and was demonstrated for the detection of Lys in wine samples [17]. Tseng and coworkers reported an aqueous solution of 13-nm gold nanoparticles (AuNPs) covalently bonded with human serum albumin (HSA) was used for sensing Lys. HSA molecules were good stabilizing agents for AuNPs in high-salt solution and exhibited the ability to bond with Lys electrostatically. It was found that the sensitivity
T
of HSA-AuNPs for Lys was highly dependent on the HSA concentration. They improved the selectivity of the probe
IP
by adjusting the pH solution to 8.0. Under the optimum conditions, the selectivity of this system for Lys over other proteins in high-salt solutions was remarkably high, even when their pI was very close to the Lys. The lowest
CR
detectable concentration of Lys in this approach was 50 nM. The applicability of the method was validated through the analyses of Lys in chicken egg white [18]. Tang’s group devised a new signal-enhanced fluorescence aptasensing
US
platform for quantitative screening of lysozyme by coupling with rolling circle amplification and strand hybridization reaction, accompanying the assembly of CdTe/CdSe quantum dots (QDs) and hemin/G-quadruplex DNzyme. Under optimal conditions, the fluorescent signal decreased with the increasing target Lys within the dynamic range from 5.0
AN
to 500 nM with a detection limit of 2.6 nM at the 3 sblank criterion. Intra-assay and inter-assay coefficients of variation were below 8.5% and 11.5%, respectively. Finally, the system was applied to analyze spiked human serum samples,
M
and the recoveries in all cases were 85-111.9% [19]. Therefore, detection of Lys has been getting importance and developing new, rapid, cheap and effective biosensors have been under investigation. An alternative method can be
ED
the use of ECL which is based on the emission of lights caused by high-energy electron-transfer reactions that happen on the surface of working electrodes. The advent of this technique can be traced back to the work of Tokel and Bard [20], who developed ECL systems based on the application of Ru(bpy)32+. Todays, ECL is an attractive and powerful
PT
detection tool in biosensing and bioassay [21, 22], because of its high sensitivity, and selectivity, relatively wide linear range, and low equipment cost [23]. Semiconductor nanocrystals and QDs have been extensively studied due to their
CE
unique size-dependent electronic, magnetic, optical and electrochemical properties. Since Bard’s group reported the ECL of Si QDs, QDs because of unique property have become one of the most popular ECL emitter and used to biomolecular determination [24]. However, compared with conventional ECL emitter such as Ru(bpy)32+ or luminol,
AC
QDs ECL suffer from some disadvantages such as weak ECL emissions [25]. Graphene quantum dots (GQDs), fewlayered graphene sheets with sizes smaller than 10 nm, are a novel type QDs in ECL [26]. Compared with the traditional QDs, GQDs have attracted great attention due to their robust biological and chemical inertness, low cytotoxicity and good biocompatibility [27]. Doping carbon nanomaterials with heteroatoms can effectively improve GQDs intrinsic properties, including electronic characteristics, surface and local chemical features. Nitrogen-doped graphene quantum dots (N-GQDs) with more active sites and more effective optical property rather than GQD S [27], exhibited excellent ECL performances and enhanced ECL intensity. Roushani and Abdi described a novel and sensitive electrochemical sensor based on graphene quantum dots/riboflavin modified glassy carbon electrode was constructed and utilized to determine persulfate (S2O82−). GQDs were expected to have the significant properties of graphene materials as well as new functions resulting from their quantum confinement and edge effects. The modified
4
ACCEPTED MANUSCRIPT electrode showed stable and well-defined redox couples at a wide pH range (1–10), with surface confined characteristics. This catalytic reduction allows an amperometric detection of S2O82− at a potential of -0.1 V with detection limit of 0.2 μM, concentration calibration range of 1.0 μM to 1 mM and sensitivity of 4.7 nA μM −1 [28]. Roushani and Valipour reported a facile green approach to employ silver nanoparticle (AgNPs) and thiol graphene quantum dots (GQD-SH) as the nanomaterial for ultrasensitive and selective detection of hepatitis C virus core antigen (HCV). AgNPs/GQD-SH was utilized as a substratum to load antibody for detection of hepatitis C virus core antigen. AgNPs have been immobilized on SH groups of GQDs via bonding formation of Ag-S and anti-HCV have been
T
loaded on the electrode surface via the interaction between –NH2 group of antibody and AgNPs. Using the
IP
nanocomposite provides a specific platform with increased surface which is capable of loading more antibodies to entrap the antigen. This novel immunosensor was used to analyze the serum sample. The immunosensor provides a
CR
convenient, low-cost and simple method for HCV core antigen detection and new horizons for quantitative detection of antigen in the clinical diagnosis [29]. Roushani and Fard described a highly sensitive and low-cost electrochemical
US
aptasensor was fabricated based on a Ag NPs/ GQD-SH nanocomposite for the measurement of 2, 4, 6-Trinitrotoluen (TNT) as a nitroaromatic explosive. For the first time Rutin as a biological molecule with inherent properties was used as the redox probe in the development of the TNT aptasensor was used. The system was based on a TNT-binding
AN
aptamer which is covalently attached onto the surface of a glassy carbon electrode modified with the nanocomposite for the formation of a sensing layer and improving the performance of the aptasensor. Applicability of the aptasensor
M
to easily detect TNT in real samples was evaluated. It seems that the strategy can be expanded to other nanoparticles and is expected to have promising implications in the design of electrochemical sensors or biosensors for the detection
ED
of various targets [30]. Roushani and coworkers described a novel electrochemical aptasensor for ultrasensitive detection of HCV core antigen by using the aptamer proximity binding assay strategy. The fabricated aptasensor can accurately detect HCV core antigen concentration in human serum samples. Such an aptasensor opens a rapid,
PT
selective and sensitive route for HCV core antigen detection and provides a promising strategy for potential applications in clinical diagnostics [31]. Roushani and coworkers reported a novel and sensitive method for detection
CE
of streptomycin (STR), to develop electrochemical sensing system based on GQDs functionalized with amine and thiol groups as the promising newest carbon-based nanomaterial. STR, an aminoglycoside antibiotic, was used in human and veterinary to treat gram-negative infections. STR residue causes serious side effects on human health. The
AC
system, in the presence of different interferences, displayed good selectivity in detecting STR. The aptasensor showed excellent results in quantitatively the detection of STR in serum samples [32]. Aptamer is a kind of synthetic oligonucleotides that engineered through repeated rounds of screening in vitro [33], a process called systematic evolution of ligands by exponential enrichment [34], which can be specifically recognized various molecular targets such as small molecules, proteins, nucleic acids, cells and even organisms. Compared with antibody, aptamer has similar high affinity and selectivity and because of their low cost, high affinity and simplicity, aptamer-based approaches has attracted much attention from researchers and has been widely used in the determination of various biomolecule [34]. Therefore, aptamer has been extensively applied as recognition element for the design of biosensors, especially ECL biosensors [35].
5
ACCEPTED MANUSCRIPT In this work, we introduced a novel electrochemiluminescent biosensor based on quenching effect of lysozyme on NGQD-S2O82– co-reactant mechanism of the NGQD-chitosan/AuNPs aptasensor. Immobilization of aptamer was successfully done by using –SH group as the binder of aptamer on AuNPs. The incorporation of AuNPs also enhanced electrochemical behavior of aptasensor resulting enhancement of NGQD-S2O82– ECL signals and higher sensitivity of aptasensor. This aptasensor showed excellent properties including high sensitivity and selectivity while using low cost
T
instrument setup. It can be nominated as the alternative method for clinical and medical diagnosis.
IP
2. Experimental
CR
2.1. Materials and reagents
Sulfuric acid (95%), hydrogen peroxide (30%), graphite powder (99.995%), potassium permanganate (99%),
US
potassium nitrate (99%), ammonium hydroxide (25%), hydrazine monohydrate (98%), chloroauric acid (HAuCl4) (98%), trisodium citrate dehydrate (99%) and sodium persulfate (98%) purchased from Sigma Aldrich Co. (St. Louis,
AN
MO, USA, https: //www.sigmaaldrich.com/chemistry.html). Piranha solution was prepared by mixing mentioned sulfuric acid and hydrogen peroxide (3:1).
The chitosan solution prepared by dispersing 25 mg of chitosan in 5 mL of acetic acid (3%). The lysozyme aptamer
Chemical
Reagent
Factory.
(https://www.chemicalregister.com/Tianjin_Chemical_Reagent_Factory/
ED
Tianjin
M
5′-HS-(CH2)6-ATC TAC GAA TTC ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG-3′ was prepared from
Supplier/sid21665. html).
The phosphate buffer was used at pH 7.4 and potassium chloride was added to increase ionic strength to adopt
PT
biological environment. All other chemical were reagent grade and used without any treatment.
CE
2.2. Apparatus
MPI-B ECL analyzer was used to monitor ECL signals. (Xi’an Remax Electronic Science Tech. Co., Ltd., China,
AC
http://www.chinaremax.com/). Electrochemical experiments such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were carried out with AUTOLAB PGSTAT 30 potentiostat/galvanostate (Eco Chemie BV, Utrecht, The Netherlands, http://www.ecochemie.nl/Company/Countries/Netherlands.html). Three electrode system including NGQD-chitosan/AuNPs aptasensor modified low resistance indium tin oxide (ITO) plate (1 cm2 active surface) as the working electrode, platinum (Pt) rode and silver/silver chloride (Ag/AgCl) as counter and reference electrode, respectively, were used in ECL and electrochemical studies. Size and dispersion of nanomaterial was investigated by transmission electron microscopy (TEM, Phillips EM 2085, https://www.dotmed.com/AnalyticalLab/Electron-Microscope/Models/Phillips/Em-208/11522). Optical properties of nitrogen-doped graphene quantum dots
and
gold
nanoparticles
were
studied
by using
UV-vis
spectroscopy
(PerkinElmer,
http://www.perkinelmer.com.cn/) and the photoluminescence experiments carried on a LS50 Perkin Elmer.
6
America,
ACCEPTED MANUSCRIPT 2.3. Preparation of N-GQDs N-GQDs were synthesized by pyrolyzing citric acid (CA) [36]. Briefly, 2 g of CA was heated to 200 °C in a 10 mL beaker on a heater for approximately 30 minutes until CA changed to orange liquid. The resulting liquid pH were adjusted to 8 with sodium hydroxide (NaOH) under stirring. N-GQDs were prepared by hydrothermal method. 20 mL of N-GQDs treated with 0.3 mL hydrazine. The mixture transferred into 25 mL autoclave and heated at 175 °C for 12 h. The final solution were centrifuged at 9,500 rpm in aqueous ethanol solution (3:1) and then evaporated by rotary to
IP
T
collect N-GQDs.
CR
2.4. Au NPs synthesis
US
AuNPs is prepared by using Turkevich method [37]. Briefly, 20 mL of 1.0 mM HAuCl4 was added to a 50 mL beaker and reflux for heated to boiling point while stirring. After, 2 mL of 1% solution of trisodium citrate dehydrate quickly added to the reaction, a deep red color solution was observed. After a defined time (normally 15 min), the final solution
AN
cooled to room temperature.
M
2.5. Fabrication of electrochemiluminescent aptasensor
ED
The aptasensor was made. First, ITO plate were pretreated by ultrasonication in ethanol and deionized water then rinsed with deionized water and dried under nitrogen gas. The surface of pretreated ITO activated by rinsing in piranha
PT
solution for 1 min to obtain hydroxyl group on the surface of ITO. A mixture of N-GQDs and chitosan solution (1:3) was prepared by ultrasonication for about 1 hour. 0.5 mL of NGQD-chitosan mixture drop casted on activated ITO
CE
plate and a galvanostatic reaction was carried out at 25 Am-2 then 20 µL of AuNPs solution added on the NGQDchitosan/ITO and rinsed in deionized water. The prepared Au NPs/NGQD-chitosan/ITO immersed in mixture of 5 wt % glutaric dialdehyde and aptamer solution for 6 hours at room temperature. To bond chitosan to fabric chemically,
AC
glutaric dialdehyde is chosen as the crosslinking agent. Finally, the aptasensor AuNPs/NGQD-chitosan/ITO was rinsed with 0.1 M phosphate buffer (pH 7.3) and nonspecific sites of mention sensor was block by incubation with 20 µL 1% bovine serum albumin (BSA) for 1 hour and then rinsed with deionized water and stored in refrigerator.
3. Results and discussion 3.1. The characterization of the prepared AuNPs and N-GQDs
7
ACCEPTED MANUSCRIPT The morphologies of the N-GQDs were studied by using TEM. Fig. 1 shows the TEM images of N-GQDs. N-GQDs particles were randomly selected to measure the particle size and the particle size distribution was shown in Fig. S1. It can be seen that average size of N-GQDs is 5.1± 0.65 nm. Fig. 1 N-GQDs were water-dispersible due to the presence of hydroxyl and carboxylic functional groups on the surface, which was confirmed by FT-IR measurement. The FT-IR spectrum of N-GQDs are shown in Fig. S2. In the spectrum
T
of N-GQDs, the weak peak located at 1723 cm-1 assigned to the stretching vibration of C ═ O groups. The absorption
IP
band around 1160 and 1032 cm-1 are ascribed to C-C and C-O stretching, respectively. More importantly, the peak at
CR
1556 cm-1 corresponding to the superposition of the vibrations of C ═ C and C ═ N, and the absorption peaks at 3434, 3087cm-1 can be ascribed to the stretching vibration of O-H and N-H which gives the evidence for the embedding of nitrogen-containing groups [38].
US
Optical properties of aqueous dispersed N-GQDs also investigated by UV-vis absorption and photoluminescence spectroscopy. Fig. S3 (A), it can be seen that UV-vis spectrum of N-GQDs showed a broadened absorption band
AN
centered at 332 nm [38]. Fig. S3 (B), presents the photoluminescence (PL) emission spectrum of the aqueous solution of N-GQDs with excitation at 370 nm; the N-GQDs shows emission at 445 nm. The quantum yield of N-GQDs was calculated to be 25 % which is far better than previously reported N-GQDs. The higher PL emission quantum yield
ED
electronic characteristics of the N-GQDs [39].
M
possibly arise from the oxygen rich groups in N-GQDs and N-doping-induced modulation of the chemical and
PT
3.2. Electrochemical and ECL behavior of the aptasensor Electrochemical behavior of an electrochemiluminescent aptasensor is the most important factor which indicates
CE
aptasensor performance. Furthermore, EIS test was carried out in 0.1M KCl solution with 10 mM Fe(CN) 63−/ Fe(CN)64−, frequency range of 10 mHz to 1 MHz and the direct current (DC) bias range was 10 mV. The EIS response
AC
of aptasensor in each modification step is shown in Nyquist plot in Fig. 2. Diameter of semicircle in high frequency zone indicates the charge transfer resistance of the electrochemical reaction. The Warburg resistance in lower frequencies shows the diffusion control electrochemical reaction on the surface of the modified aptasensor. EIS spectrum for the bare electrode exhibited small semicircle domain, at high frequencies (curve a), and its electron transfer resistance is small, indicating a fast electron-transfer process of [Fe(CN)6]3−/4− . After casting NGQD-chitosan on the ITO plate (curve b), the EIS response showed a high imaginary resistance indicating increase of active surface of the electrode due to the N-GQDs higher surface area, and high electron transfer resistance can be explained by less conductivity of chitosan compared to the bare ITO plate. Gold nanoparticles addition on NGQD-chitosan/ITO lowered electron transfer resistance due to high conductivity of AuNPs which results in fast electron-transfer process of [Fe(CN)6]3−/4− (curve c). HS-aptamer increased the electron transfer resistance
8
ACCEPTED MANUSCRIPT which demonstrate successively immobilization of the HS-aptamer (curve d). Finally, BSA and lysozyme also increased electron transfer resistance indicating adsorption of lysozyme was done on the immobilized aptamer (curve e, f). N-GQDs, along with chitosan added to the electrode surface and chitosan increases surface resistant due to its low conductivity. N-GQDs is luminophore in this work. Fig. 2 CV measurements were used to characterize the process of electrochemiluminescent aptasensor modification. CV
T
curves of stepwise modification of SPCE in 10 mM Fe(CN)63−/ Fe(CN)64− containing 0.1 M potassium chloride (KCl)
IP
solution were shown in Fig. 3. SPCE is screen printed carbon electrode, it’s used to apply a potential to the
CR
luminophore and co-reactant in ECL mechanisms. SPCE possess excellent biocompatibility, a wide pH range and high sensitivity. The SPCE is of particular interest due to its easy fabrication, mass productivity and low cost. And the carbon ink for SPCEs often contains some mineral binders or insulating polymers, which cover at the surface of
US
graphite particles to form the thin insulating layer for cathodic ECL. A well-defined redox peak of Fe(CN)63−/ Fe(CN)64− was observed at the bare ITO (curve a). When NGQD-chitosan were coated on the ITO, the peak current decreased (curve b), however modification of ITO by AuNPs (curve c) the peak current increase, due to the increase
AN
in the electrode surface area and the excellent conductivity of AuNPs. While, the electrode modified with aptamer exhibited a decrease peak current (curve d). Also the modified electrode was blocked with BSA (curve e) and high
M
specific affinity of aptamer and successfully bound to Lys (curve f) on electrode surface, the CV responses significantly declined. For ECL measurement cyclic voltammetry was done in the range of 0 to -1.8 V in phosphate
ED
buffer (pH 7.4) containing 0.1 M Na2S2O8.
Fig. 3
PT
For each step of modification, ECL response recorded. Fig. S4, however, no ECL signals were observed for bare ITO. Modification of ITO plate with NGQD-chitosan results a high ECL signal. Moreover, addition of AuNPs greatly
CE
increase ECL signal due to the improvement of electron transfer resistance which verifies the EIS results. The ECL intensity slightly decreased after addition of aptamer and BSA and addition of lysozyme greatly quenched the ECL
AC
signals. Lysozyme and aptamer act as blocking layer for aptasensor and lowering active sites of electron transfer reaction, resulting in low efficient ECL process [40]. The ECL mechanism of N-GQDs and sodium persulfate (Na2S2O8) can be described as below: [40] eq1
S2O8 2– + e–
SO42– + SO4– •
eq2
NGQDs + e–
NGQDs– •
eq3
NGQDs– • + SO4– •
eq4
NGQDs*
NGQDs* + SO42– NGQDs + hν
9
ACCEPTED MANUSCRIPT The negative potential scanning results in electrochemically generation of SO4– • and N-GQDs– • which then undergo a high energy electron transfer reaction by injection of a hole into the N-GQDs, resulting excited state N-GQDs which emits light.
3.3. Optimization of the detection conditions Experimental parameters, such as pH, Na2S2O8 and incubation time were optimized in order to obtain best performance
T
of aptasensor. pH of the working solution, as one of the most effective parameters, was optimized in the range between
IP
5 to 9 Fig. S5 (A). pH 7.5 showed the highest ECL signal, however, pH value of 7.4 is for the common biological matrixes, pH 7.4 selected for further experiments.
CR
Concentration of Na2S2O8 as the co-reactant for ECL enhancement also optimized. Increasing Na2S2O8 concentration from 0.05 to 0.1 M resulted in enhancement of ECL signals and further increasing the Na2S2O8 concentration
US
decreased ECL efficiency. Thus 0.1 M Na2S2O8 was selected as the optimum concentration and used for further experiments Fig. S5 (B).
AN
Incubation time of aptamer and lysozyme was studied and in the range between 10 min to 2 h, the quenching effect increased with incubation time and reached a plateau at 1 h Fig. S5 (C). Therefore, pH value of 7, 0.1 M Na2S2O8 and
ED
M
1 h incubation time between aptamer and lysozyme was selected as the optimum experiment condition.
3.4. Analytical performance of the electrochemiluminescent aptasensor
PT
Fig. 4 shows the response of AuNPs/NGQD-chitosan/ITO aptasensor before (curve a) and after (curves a–i) the reaction with different concentrations of lysozyme, in 4 mL 0.1 M phosphate buffer (pH 7.4) containing 0.1 M
CE
Na2S2O8. The Fig. 5 shows the typical calibration curve of lysozyme detection. The limit of detection was found to be 0.8 fM with the relative standard deviation (RSD) of 2.9% in 10 times analysis of blank solution (S/N = 3). RSD of 5 pM lysozyme in 10 times also determined to be 3.1%. These results show highly sensitive response of aptasensor Au
AC
NPs/NGQD-chitosan/ITO in presence of lysozyme. Fig. 4 Fig. 5
3.5. Stability, selectivity and reproducibility of the aptasensor Stability of AuNPs/NGQD-chitosan/ITO aptasensor also investigated, results is shown in Fig. 6. Under consecutive cyclic potential scan for 10 cycles, the ECL signals are very high and the RSD for the change in the ECL intensity is less than 3.4%, revealing good stability of the aptasensor.
10
ACCEPTED MANUSCRIPT Fig. 6 Reproducibility of aptasensor Au NPs/NGQD-chitosan/ITO, 10 aptasensors were fabricated in 10 individual days then signals were measured from optimum experiment condition in presence of 5 pM lysozyme. The RSD of the 10 individually fabricated aptasensors AuNPs/NGQD-chitosan/ITO to be found 4.2, indicating high reproducibility of the aptasensor. The selectivity of the aptasensor was also studied by choosing HSA, Hemoglobin (Hb), Immunoglobulin G (IgG) and
T
thrombin (Thr) as interferents, and the results are shown in Fig. 7. The concentrations of HSA, Hb, IgG, alpha-
IP
fetoprotein (AFP) were 10-fold (10-11 M) than target lysozyme (5×10-12 M). The results showed negligible ECL with interference proteins compared with that of presence of the target lysozyme. Also the response of the aptasensor in a
CR
mixture containing of HSA, Hb, IgG, AFP and Lys was examined. The ∆I of the mixture had little difference compared with that in only the lysozyme solution. The results demonstrate that the aptasensor has good selectivity for the
US
detection of Lysozyme.
AN
Fig. 7
3.6. Application of the electrochemiluminescent aptasensor
M
For real sample testing of AuNPs/NGQD-chitosan/ITO aptasensor, human serum were examined. The Spiking method was chosen to establish the sensor performance in complex matrixes and results are shown in Table 1. The high
ED
recovery and repeatability of aptasensor would make it a great alternative method for lysozyme detection in human
AC
4. Conclusions
Table 1
CE
PT
serum.
In summary, an electrochemiluminescent aptasensor fabricated by using AuNPs/NGQD-chitosan/ITO composite. The ECL signals generated by co-reactant ECL mechanism of N-GQDs and Na2S2O8 and the lysozyme measurement was based on quenching effect lysozyme aptamer incubation, which result to low electron transfer process between both N-GQDs and Na2S2O8. Au NPs was very effective in increasing ECL signals due to the high conductivity and surface area which results in higher sensitivity of aptasensor. Application of AuNPs/NGQD-chitosan/ITO aptasensor in biological matrix leaves no doubts about applicability of aptasensor as a high selective and interference free detection system of lysozyme. The bare ITO has no ECL signal. When the NGQD-chitosan/AuNPs composite film was electrodeposited on the ITO, the ECL intensity enhanced greatly. The ECL intensity decreased after aptamer and BSA was dropped onto the electrode. The ECL intensity further decreased after addition of lysozyme. The reason for this
11
ACCEPTED MANUSCRIPT is that high specific affinity to Lys, hindered the diffusion of the ECL reagents towards the electrode surface and therefore signal further decreased after addition Lys.
Acknowledgements This work was financially supported by State Key Laboratory of Fine Chemicals, the National Natural Science
CR
IP
T
Foundation of China (No. 21272030, 21472016, 21306019, 21576042).
References
D. M. Donovan, S. Dong, W. Garrett, G. M. Rousseau, S. Moineau, D. G. Pritchard, Peptidoglycan Hydrolase
US
1.
Fusions Maintain Their Parental Specificities, Appl. Environment. Microbiol. 72 (2006) 2988-2996. D. M. Donovan, Google Patents, 2011.
3.
P. Markart, N. Faust, T. Graf, C.-L. Na, T. E. Weaver, H. T. Akinbi, Comparison of the microbicidal and
AN
2.
muramidase activities of mouse lysozyme M and P, Biochem. J. 380 (2004) 385-392. A. Amit, R. Mariuzza, S. Phillips, R. Poljak, Three-dimensional structure of an antigen-antibody complex at
M
4.
2.8 a resolution, Science. 233 (1986) 747-753.
D. Canet, M. Sunde, A. M. Last, A. Miranker, A. Spencer, C. V. Robinson, C. M. Dobson, Mechanistic
ED
5.
Studies of the Folding of Human Lysozyme and the Origin of Amyloidogenic Behavior in Its Disease-Related
6.
PT
Variants, Biochem. 38 (1999) 6419-6427.
S. L.-Huang, P. L. Huang, Y. Sun, P. L. Huang, H.-F. Kung, D. L. Blithe, H.-C. Chen, Lysozyme and R Nases as anti-HIV components in β-core preparations of human chorionic gonadotropin, Proc. Natl. Acad.
7.
CE
Sci. 96 (1999) 2678-2681.
B. Schmekel, P. Blomstrand, P. Venge, Serum lysozyme – a surrogate marker of pulmonary microvascular
8.
AC
injury in smokers, Clin Physiol Funct Imaging. 33 (2013) 307-312. S. Liu, W. Na, S. Pang, F. Shi, X. Su, A label-free fluorescence detection strategy for lysozyme assay using CuInS2 quantum dots, The Analyst. 139 (2014) 3048-3054. 9.
Q. Wang, H. Zheng, X. Gao, Z. Lin, G. Chen, A label-free ultrasensitive electrochemical aptameric recognition system for protein assay based on hyperbranched rolling circle amplification, Chem. Commun. 49 (2013) 11418-11420.
10. C. Ocaña, A. Hayat, R. Mishra, A. Vasilescu, M. D. Valle, J. L. Marty, A novel electrochemical aptamer– antibody sandwich assay for lysozyme detection, Analyst. 140 (2015) 4148-4153. 11. C. Ocaña, A. Hayat, R. K. Mishra, A. Vasilescu, M. D. Valle, J. L. Marty, Label free aptasensor for Lysozyme detection: A comparison of the analytical performance of two aptamers, Bioelectrochem. 105 (2015) 72-77.
12
ACCEPTED MANUSCRIPT 12. H. Xia, L. Li, Z. Yin, X. Hou, J. J. Zhu, Biobar-Coded Gold Nanoparticles and DNAzyme-Based Dual Signal Amplification Strategy for Ultrasensitive Detection of Protein by Electrochemiluminescence, ACS Appl. Mater & Inter. 7 (2014) 696-703. 13. S. Khurshid, L. Govada, H. F. El.-Sharif, S. M. Reddy, N. E. Chayen, Automating the application of smart materials for protein crystallization, Acta. Crystall. Sec D: Biology. Crystall. 71 (2015) 534-540. 14. L.-J. Xu, C. Zong, X.-S. Zheng, P. Hu, J.-M. Feng, B. Ren, Label-Free Detection of native proteins by surface-enhanced Raman Spectroscopy using lodide-Modified nanoparticles, Anal. Chem. 86 (2014) 2238-
T
2245.
IP
15. Y. Lian, F. He, X. Mi, F. Tong, X. Shi, Lysozyme aptamer biosensor based on electron transfer from SWCNTs to SPQC-IDE, Sens. Actuator B: Chem. 199 (2014) 377-383.
CR
16. Y. Zeng, Y. Wan, D. Zhang, Lysozyme as sensitive reporter for fluorometric and PCR based detection of E. coli and S. aureus using magnetic microbeads, Microchim. Acta. 183 (2016) 741-748.
US
17. R. K. Mishra, A. Hayat, G. K. Mishra, G. Catanante, V. Sharma, J. L. Marty, A novel colorimetric competitive aptamer assay for lysozyme detection based on superparamagnetic nanobeads, Talanta. 165 (2017) 436-441.
AN
18. Y. M. Chen, C. J. Yu, T. L. Cheng, W. L. Tseng, Colorimetric Detection of Lysozyme Based on Electrostatic Interaction with Human Serum Albumin-Modified Gold Nanoparticles, Langmuir. 24 (2008) 3654-3660.
M
19. Z. Qiu, J. Shu, Y. He, Z. Lin, K. Zhang, S. Lv, D. Tang, CdTe/CdSe quantum dot-based fluorescent aptasensor with hemin/G-quadruplex DNzyme for sensitive detection of lysozyme using rolling circle
ED
amplification and strand hybridization, Biosens. Bioelectron. 87 (2017) 18-24. 20. M. Hesari, Z. Ding, Review: Electrogenerated Chemiluminescence: Light Years Ahead, J. Electrochem. Soc. 163 (2016) 3116-3131.
PT
21. M. Hosseini, M. R. Moghaddam, F. Faridbod, P. Norouzi, M. R. K. Pur, M. R. Ganjali, A novel solid-state electrochemiluminescence sensor based on a Ru(bpy)32+/nano Sm2O3 modified carbon paste electrode for the
CE
determination of L-proline, RSC Adv. 5 (2015) 64669-64674. 22. A. Zhang, H. Xiang, X. Zhang, W. Guo, E. Yuan, C. Huang, N. Jia, A novel sandwich electrochemiluminescence immunosensor for ultrasensitive detection of carbohydrate antigen 19-9 based on
AC
immobilizing luminol on Ag@BSA core/shell microspheres, Biosens. Bioelectron. 75 (2016) 206-212. 23. W. Miao, Electrogenerated Chemiluminescence and Its Biorelated Applications, Chem. Rev. 108 (2008) 2506-2553.
24. Z. Ding, B. M. Quinn, S. K. Haram, L. E. Pell, B. A. Korgel, A. J. Bard, Electrochemistry and Electrogenerated Chemiluminescence from Silicon Nanocrystal Quantum Dots, Science. 296 (2002) 12931297. 25. Y. Xu, J. Liu, C. Gao, E. Wang, Applications of carbon quantum dots in electrochemiluminescence: A mini review, Electrochem. Commun. 48 (2014) 151-154. 26. H. Sun, L. Wu, W. Wei, X. Qu, Recent advances in graphene quantum dots for sensing, Mater. Today. 16 (2013) 433- 442.
13
ACCEPTED MANUSCRIPT 27. Q. Liu, B. Guo, Z. Rao, B. Zhang, J. R. Gong, Strong Two-Photon-Induced Fluorescence from Photostable, Biocompatible Nitrogen-Doped Graphene Quantum Dots for Cellular and Deep-Tissue Imaging, Nano Lett. 13 (2013) 2436-2441. 28. M. Roushani, Z. Abdi, Novel electrochemical sensor based on graphene quantum dots/riboflavin nanocomposite for the detection of persulfate, Sens. Actuator B: Chem. 201 (2014) 503-510. 29. A.Valipour, M. Roushani, Using silver nanoparticle and thiol graphene quantum dots nanocomposite as a substratum to load antibody for detection of hepatitis C virus core antigen: Electrochemical oxidation of
T
riboflavin was used as redox probe, Biosens. Bioelectron. 89 (2017) 946-951.
IP
30. F. S. Fard, M. Roushani, Designing an ultra-sensitive aptasensor based on an AgNPs/thiol-GQD
redox probe, Biosens. Bioelectron. 87 (2017) 724-731.
CR
nanocomposite for TNT detection at femtomolar levels using the electrochemical oxidation of Rutin as a
31. K. Ghanbari, M. Roushani, A. Azadbakht, Ultra-sensitive aptasensor based on a GQD nanocomposite for
US
detection of hepatitis C virus core antigen, Anal. Biochem. 534 (2017) 64-69. 32. M. Roushani, K. Ghanbari, S. J. Hoseini, Designing an electrochemical aptasensor based on immobilization of the aptamer onto nanocomposite for detection of the streptomycin antibiotic, Microchem. J. 141 (2018)
AN
96-103.
33. W. Zhou, P.-J. J. Huang, J. Ding, J. Liu, Aptamer-based biosensors for biomedical diagnostics, The Analyst.
M
139 (2014) 2627-2640.
34. N. Duan, S. Wu, X. Chen, Y. Huang, Y. Xia, X. Ma, Z. Wang, Selection and Characterization of Aptamers
ED
against Salmonella typhimurium Using Whole-Bacterium Systemic Evolution of Ligands by Exponential Enrichment (SELEX), J. Agricul. Food. Chem. 61 (2013) 3229-3234. 35. E. M. Gross, S. S. Maddipati, S. M. Snyder, A review of electrogenerated chemiluminescent biosensors for
PT
assays in biological matrices, Bioanalysis. 8 (2016) 2071-2089. 36. J. Ju, W. Chen, Synthesis of highly fluorescent nitrogen-doped graphene quantum dots for sensitive, label-
CE
free detection of Fe (III) in aqueous media, Biosens. Bioelectron. 58 (2014) 219-225. 37. J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot, A. Plech, Turkevich Method for Gold Nanoparticle Synthesis Revisited, J. Physic. Chem B. 110 (2006) 15700-15707.
AC
38. J. Ju, R. Zhang, S. He, W. Chen, Nitrogen-doped graphene quantum dots-based fluorescent probe for the sensitive turn-on detection of glutathione and its cellular imaging, RSC Adv. 4 (2014) 52583-52589. 39. L. Lin, X. Song, Y. Chen, M. Rong, T. Zhao, Y. Jiang, Y. Wang, X. Chen, One-pot synthesis of highly Greenish-yellow fluorescent nitrogen-doped graphene quantum dots for pyrophosphate sensing via competitive coordination with Eu 3+ ions, Nanoscale. 7 (2015) 15427-15433. 40. X. Du, D. Jiang, Q. Liu, G. Zhu, H. Mao, K. Wang, Fabrication of graphene oxide decorated with nitrogendoped graphene quantum dots and its enhanced electrochemiluminescence for ultrasensitive detection of pentachlorophenol, Analyst. 140 (2015) 1253-1259.
14
ACCEPTED MANUSCRIPT Figures Legend
Fig. 1. TEM images of N-GQDs. Fig. 2. EIS behaviors of (a) bare ITO (b) NGQD-Chitosan (c) AuNPs /NGQD-Chitosan/ITO (d) aptamer/AuNPs/NGQD-Chitosan/ITO
(e)
BSA/aptamer/AuNPs/NGQD-Chitosan/ITO.
(f)
T
Lys/BSA/aptamer/AuNPs/NGQD-Chitosan/ITO in Fe(CN)63−/ Fe(CN)64– (10 mM) (1:1)
IP
containing KCl (0.1 M) and EIS in the frequency range (0.01 Hz to 100 kHz) and signal amplitude
CR
(5 mV).
Fig. 3. CV behaviors of (a) bare ITO (b) NGQD-Chitosan (c) AuNPs /NGQD-Chitosan/ITO (d) (e)
BSA/aptamer/AuNPs/NGQD-Chitosan/ITO.
(f)
US
aptamer/AuNPs/NGQD-Chitosan/ITO
Lys/BSA/aptamer/ AuNPs/NGQD-Chitosan/ITO in Fe(CN)63−/ Fe(CN)64– (10 mM) (1:1)
AN
containing KCl (0.1 M) and EIS in the frequency range (0.01 Hz to 100 kHz) and signal amplitude (5 mV).
M
Fig. 4. ECL-potential signals of the aptasensor with different concentrations of Lysozyme samples. (a–i, respectively, C (Lys)= 0, 0.01, 0.05, 0.5, 5, 50, 500, 5000, 50000 pM and wavelength of
ED
fluorescence emission = 445 nm; potential = 0 to -1.8 V).
PT
Fig. 5. The calibration plot against the corresponding log C (Lys). Fig. 6. Stability of ECL signal from aptasensor in phosphate buffer 0.1 M (pH 7.4), containing
CE
Na2S2O8 (0.1M) under ten cycles of CV scan.
AC
Fig. 7. The selectivity of the electrochemiluminescent aptasensor towards different targets.
15
Fig. 1
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Fig. 2 16
AC
CE
PT
ED
M
AN
Fig. 3
US
CR
IP
T
ACCEPTED MANUSCRIPT
Fig. 4
17
ACCEPTED MANUSCRIPT 3500
y = 476.49x + 6784.9 R² = 0.995
3000
∆IECL
2500 2000
T
1500
IP
1000
0 -15
-13
-11
CR
500
US
LogC (M)
AC
CE
PT
ED
M
AN
Fig. 5
Fig. 6
18
-9
-7
US
CR
IP
T
ACCEPTED MANUSCRIPT
ED
M
AN
Fig. 7
PT
Table 1 Recovery and relative standard deviation of Lys in human serum samples.
2 3
AC
1
Spiked (pM) 1
CE
Serum
Found (pM) 0.91± 0.65
Recovery () 91
RSD (, n=3) 3.1
5
4.8± 0.4
96
2.6
25
26± 0.55
104
2.9
Mean value ± SD of three measurements
19
ACCEPTED MANUSCRIPT Supplementary data
Fig. S1. Size distribution of N-GQDs. Fig. S2. FT-IR spectrum of N-GQDs.
IP
T
Fig. S3 (A). UV-vis spectrum of the N-GQDs. (B). Fluorescence spectrum of the N-GQDs. Fig. S4. ECL behavior of (a) bare ITO. (b) NGQD-Chitosan. (c) AuNPs /NGQD-Chitosan/ITO.
CR
(d) aptamer/AuNPs/NGQD-Chitosan/ITO. (e) BSA/aptamer/AuNPs/NGQD-Chitosan/ITO. (f) Lys/BSA/aptamer/ AuNPs/NGQD-Chitosan/ITO.
US
Fig. S5 (A). ECL signal from aptasensor in phosphate buffer (0.1M) with different pH containing
AN
Na2S2O8 (0.1M). (B). ECL signal from aptasensor with different concentration of Na2S2O8 in
AC
CE
PT
ED
M
phosphate buffer 0.1 M (pH 7.4). (C). Effect of the incubation time on ECL peak intensity.
20
US
CR
IP
T
ACCEPTED MANUSCRIPT
M
AN
Fig. S1.
ED
0.35
PT
0.3
0.15 0.1
0.05
CE
0.2
AC
Absorbance (a.u.)
0.25
0
500
1000
1500
2000
2500
3000
wavenumber (cm-1) Fig. S2. 21
3500
4000
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
Fig. S3 (A).
Fig. S3 (B).
22
ACCEPTED MANUSCRIPT
6000
c b d e
4000
IP
T
f
CR
3000
US
2000
1000
AN
a 0 -1.5
-1
M
-2
Fig. S4.
CE
PT
ED
Potential / V
AC
ECL Intensity
5000
23
-0.5
0
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
Fig. S5 (A).
24
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Fig. S5 (B).
Fig. S5 (C).
25
ACCEPTED MANUSCRIPT
IP
T
HIGHLIGHTS
CR
►A novel ultrasensitive electrochemiluminescent (ECL) aptasensor based on the quenching effect of aptamer and lysozyme incubation on co-reactant ECL
US
mechanism of nitrogen-doped graphene quantum dots and persulfate was developed.
range (10 fM to 10 nM) were reached.
AN
► A low detection limit of 0.8 fM (at an S/N ratio of 3, n=10) and a wide linear
M
►The aptasensor applied to the human serum samples analysis.
AC
CE
PT
ED
► Range of recoveries were between 91 and 104% (with RSDs of <3.1%).
26