A sensitive plasmonic copper(II) sensor based on gold nanoparticles deposited on ITO glass substrate

Author’s Accepted Manuscript A sensitive plasmonic copper(II) sensor based on gold nanoparticles deposited on ITO glass substrate Lijun Ding, Yan Gao, Junwei Di www.elsevier.com/locate/bios

PII: DOI: Reference:

S0956-5663(16)30277-9 http://dx.doi.org/10.1016/j.bios.2016.04.002 BIOS8589

To appear in: Biosensors and Bioelectronic Received date: 15 February 2016 Revised date: 22 March 2016 Accepted date: 4 April 2016 Cite this article as: Lijun Ding, Yan Gao and Junwei Di, A sensitive plasmonic copper(II) sensor based on gold nanoparticles deposited on ITO glass substrate Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.04.002 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 galley proof before it is published in its final citable 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.

A sensitive plasmonic copper(II) sensor based on gold nanoparticles deposited on ITO glass substrate Lijun Ding, Yan Gao, Junwei Di* College of Chemistry, Chemical Engineering and Material Science, Soochow University, Suzhou, 215123, PR China ABSTRACT: Gold nanoparticles (Au NPs) based plasmonic probe was developed for sensitive and selective detection of Cu2+ ions. The Au NPs were self-assembled on transparent indium tin oxide (ITO) film coated glass substrate using poly dimethyl diallyl ammonium chloride (PDDA) as a linker and then calcined at 400°C to obtain pure Au NPs on ITO surface (ITO/Au NPs). The probe was fabricated by functionalizing L-cysteine (Cys) on to gold surface (ITO/Au NPs/Cys). The strong chelation of Cu2+ with Cys formed a stable Cys-Cu complex, and resulted in the red-shift of localized surface plasmon resonance (LSPR) peak of the Au NPs. The introduction of bovine serum albumin (BSA) as the second complexant could form complex of Cys-Cu-BAS and further markedly enhanced the red-shift of the LSPR peak. This plasmonic probe provided a highly sensitive and selective detection towards Cu2+ ions, with a wide linear detection range (10-11–10-5M) over 6 orders of magnitude. The simple and cost-effective probe was successfully applied to the determination of Cu2+ in real samples.

* Corresponding author. E-mail address: [email protected] 1

Keywords: Gold nanoparticle arrays; self-assembly; calcination; plasmonic sensor; Cu(II) ions

1. Introduction It is essential in many life processes when the copper ion concentration is less than 1 μM. Copper element involves in the synthesis of hemoglobin, a variety of enzymes, and the metabolism of the body, so it plays an important role in human health. When its concentration in the organism is too high, it will inhibit some necessary enzymes and make biological oxidation/reduction process abnormal. It can pose serious adverse effects in the liver and gall bladder (Harrison et al., 2007). Therefore, it is important to develop a method for the highly sensitive, selective, and efficient detection of copper in environmental analysis, food testing and biomedical assay. The classic methods of detecting copper ions include atomic absorption spectrometry（AAS）(Ghaedi et al., 2007; Chen and Teo, 2001), inductively coupled plasma atomic emission spectrometry (ICP-AES) (Huber, 1999; Song et al., 2004), spectrophotometry (Chaisuksant, et al., 2000; Winkler and Arenhövel-Pacuła, 2000), and fluorescence (Li et al., 2012; Tsai and Lin, 2013) etc., but they have strong disturbance, low sensitivity, reagent toxicity, expensive instruments or other shortcomings. In particular, they are limit in the application of on-site rapid detection. Noble metal nanoparticles, such as gold and silver nanoparticles, have unique localized surface plasmon resonance (LSPR) characteristics, which are closely related

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with their composition, morphology, size and the surrounding media, etc. This feature can be used for preparing cheap, portable, multi-function, high sensitive LSPR sensors (Niemeyer, 2001; Shipway and Katz, 2000; Wohltjen and Snow, 1998; Bailey et al., 2003). There are two main strategies for detection of Cu2+ ions: One is based on that Cu2+ ions could induce the aggregation/anti-aggregation of functionalization of gold nanoparticles which result in the change of the absorption peaks (Chen et al., 2014; Ye et al., 2015). The other is LSPR probe based on the shift of peak wavelength induced by the adsorption of Cu2+ ions on the surface of gold nanoparticles (Au NPs). Liu et al. developed polyamine-capped gold nanorodes as probes for detection of Cu2+ ions (Liu et al., 2015). However, when the gold nanomaterials suspended in liquid solution were used as probe, it is difficult to avoid uncertain changes for nanoparticles (Huang et al., 2009). Therefore, it is benefit that the probes of metal nanomaterials are immobilized on transparent solid substrate (Huang et al., 2009; Deng et al., 2010; Marinakos et al., 2007) Several methods, such as Vacuum evaporation, nanosphere lithography, and self-assembly, have been developed to fabricate Au NP array on glass surface (Shon et al., 2009; Xu et al., 2005; Tan et al., 2005). Among them, self-assembly method is attracted greatly attention because of its simplicity and convenience. Au NPs can be immobilized on glass surface using organic adhesion layers as linkers. However, the particle surfaces are also covered with the capping regent or stabilizers. This makes the Au NP surfaces somewhat difficult for further functionalization with specific molecules.

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In this work, we used self-assembled method to immobilize Au NPs onto a transparent indium tin oxide (ITO) film coated glass surface. Then it was calcined at high temperature to remove organic linkers and stabilizers. Therefore, the pure Au NP arrays on ITO substrate were obtained. The clean gold surface was functionalized by self-assembly monolayer of L-cysteine (Cys), which can selectively react with Cu2+ to form chelation. Therefore, this LSPR probe could be applied to detect Cu2+ ions. The detection sensitivity was further improved greatly by introduction of bovine serum albumin (BSA) as the second complexant.

2. Experimental 2.1 Materials and equipment Sodium citrate tribasic dihydrate (C6H5Na3O7·2H2O), tetrachloroauric acid (HAuCl4·4H2O), disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), citric acid monohydrate (C6H8O7·H2O), poly dimethyl diallyl ammonium chloride (PDDA),

L-Cysteine

(Cys),

copper

(II)

chloride

dihydrate

hydrochloride

(CuCl2·2H2O), bovine serum albumin (BSA), and copper reagent (CR, sodium diethyldithiocarbamate) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Besides Cys and BSA are biological reagents, other reagents are analytical grade. 0.1% CR solution was prepared by dissolving 0.1 g CR in 25 mL absolute ethanol and then diluting to 100mL with ultrapure water. ITO glass (1.1 mm) was purchased from Suzhou NSG Electronics Co. Ltd. (Suzhou, China). Milli-Q water (18.25 MΩ cm−1) was used to prepare all the solutions in this study.

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The absorption

spectra were measured with a Cary 60 spectrometer (Agilent, Australia). Transmission electron microscopy (TEM) image was obtained from Tecnai G20 (FEI, U.S.A.). Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) were performed using an S-4700 SEM (Hitachi, Japan).

2.2 Preparation of Au NP arrays onto ITO glass surface Au NPs were prepared by sodium citrate reduction method (Frens, 1973). Briefly, the 20 mL aqueous solution of tetrachloroauric acid (1mM) and 35mL ultrapure water were added in a 100 mL three-necked flask. The mixture was stirred with a magnetic stirrer and heated to 100ºC, then added 10 mL sodium citrate solution (10mM) to heat and reflux 30min. Au NPs with diameter about 15 nm were obtained after stopping heating and cooling to room temperature. The prepared colloidal Au NPs were centrifuged at 10 000 rpm for 30 min to remove the excess sodium citrate, and redispersed in water to obtain concentrated Au sol. The transparent ITO glass was used as solid substrate for deposition of Au NP arrays. Firstly, the ITO glass (0.6 × 4.0 cm2) was cleaned by using dilute NH3·H2O, water, ethanol and water for 10 min sequentially in an ultrasonic bath. Then, the clean ITO glasses were soaked in 1:40 (PDDA: H2O) PDDA solution for six hours and removed to dry. Next, they were immersed in the Au NP solution overnight, and then removed to rinse and dry. Finally, they were calcined in tube furnace from room temperature to 400 °C at 10 °C/min and then kept at 400°C for 30 min. The product of ITO substrate immobilized with Au NPs (defined as ITO/Au NPs) can be long-term

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preservation in 4°C.

2.3 Detection of copper ions with gold nanoparticle probe The prepared ITO/Au NPs stripes were immersed in 10 mM cysteine solution for 20 min at room temperature. Then, they were soaked in the phosphate-citrate buffer solution (pH 5.0) containing a certain concentration of CuCl2 kept at 30 °C for 20 min. Finally, they were incubated in BSA solution (8 mg/L BSA in the phosphate buffer solution, pH=7) kept at 30 °C for 15min measured. The UV-vis absorption peak wavelength was recorded at each step after the strip was rinsed with water and dried with N2 gas. The change of peak wavelength was calculated for determination of Cu2+ ion concentration.

3. Result and discussion 3.1. Fabrication and characterization of ITO/Au NPs The Au NPs were synthesized by chemical method with chloride acid and sodium citrate. A typical TEM image is shown in Fig. 1A. The spherical Au NPs were distributed uniformly. The average particle size was about 16 nm. The UV-vis absorption peak of Au NPs in aqueous solution appeared at about 520 nm (Figure 1B), which was consistent with the size obtained from TEM image. PDDA is a positive-charged polyelectrolyte (Chen et al., 2004), and it can be adsorbed on the surface of ITO. Gold nanoparticles are negatively charged due to adsorption of citrate (Yuan and He, 2013). Therefore, we can assemble the Au NPs on the ITO surface

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using PDDA as a linker (Liu et al., 2003; Zhang et al., 2016). Figure 1C shows the SEM image of Au NPs self-assembled on the ITO substrate. It can be seen that the nanoparticles were sparsely and randomly distributed on the surface. Size distribution of the nanoparticles gives a diameter of 18±4 nm (Figure 1D). Figure 1

PDDA is an organic matter. It may wrap on the surface of the Au NPs and affect the further fictionalization of the gold surface. Thus, high temperature calcination method is selected to remove PDDA macromolecules and obtained pure gold surface. Fig. 1E exhibits the typical SEM images of Au NPs deposited on ITO substrate after calcination. Comparing with the SEM images before and after calcination, they showed little change. Size distribution of the Au NPs after calcination (Fig. 1F) gives an average diameter of 20±3 nm, which was a little larger than that of before calcination. This is attributed to some transmutation after spherical Au NPs on solid substrate suffered from high temperature. Since the LSPR of Au NPs is influenced on size, shape, and surrounding medium, the self-assembly and calcination step can be monitored by spectrometry. Figure 1B shows the UV-vis absorption spectra of Au NPs at different steps. As Au NPs were self-assembled on to the ITO surface, the LSPR peak of the Au NPs shifted from 520 nm to 526 nm due to adsorption on ITO/PDDA surface. The change of environmental medium results in red-shifted peak. After calcination at 400°C for 30 min, the LSPR peak of ITO/Au NPs was blue-shifted to ~524 nm. This comes from the combination

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of two opposite change direction: One is the blue-shift due to the elimination of organic PDDA molecules surrounded it; the other is the red-shift due to the transmutation of spherical Au NPs at high temperature. The temperature of calcination was very important in the treatment process. When we calcined the ITO/PDDA/Au NPs stripes at 300°C for 30 min, the organic materials could not be deleted completely. As the temperature increasing to 500°C, the LSPR peak of ITO/Au NPs located at 526 nm (Figure S1). Further increasing to 600°C, the LSPR peak was largely red-shifted to 553 nm due to marked distortion of spherical Au NPs. Figure S2 shows the SEM images and their size distribution of ITO/PDDA/Au NPs stripes after calcination at different temperatures. The SEM image after calcination of 500 °C was similar to that of 400 °C and the average diameter of nanoparticles was increased to 22.8±3.3 nm. However, the particle size in SEM image after calcination of 600 °C was markedly increased to 39.4±10.2 nm. This is attributed to the some aggregation of nanoparticles at high temperatures. Therefore, we selected 400°C for 30 min as the calcination conditions in this experiment.

3.2. Sensing mechanism for the detection of copper(II) ions The reaction mechanism of the whole process was shown in Figure 2. It is well known that Cys molecules can self-assembled on gold surface forming Au–S bond due to active thiol group (-SH) (Teranishi et al., 1998; Corbierre et al., 2001; Shan et al., 2003). The Cys modified Au NPs could selectively complex with Cu2+ ions by basic (-NH2) and acidic (-COOH) functional groups (Liu et al., 1991; Uvdal et al.,

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1992). Since the complexation constant of the Cys with Cu2+ ion is 4 orders of magnitude higher than with other metal ions, the modified stripe was used to sensitive and selective determination of Cu2+ ions using voltammetric method (Zhu et al., 2013). According to this selective reaction, we expect that Cys modified Au NPs can be used to plasmonic probe for detection of Cu2+ due to change the surrounding environment. Figure 3 shows the UV-vis absorption spectra for ITO/Au NPs stripe at different modification steps. When Cys molecules were self-assembled on the Au NP surface through the Au-S bond, the LSPR peak of the Au NPs was red-shifted from 524 nm to 535 nm. This phenomenon was consistent with previous reports (Hostetler et al., 1996; Johnson et al., 1998; Patil et al., 1999). After further incubated in 0.1 µM Cu2+ solution, the LSPR peak of Au NPs was further red-shifted of 5.1 nm. The EDX spectrum was demonstrated that Cu2+ ions were adsorbed on the probe surface (Figure S3). This result indicated that the Cys modified Au NPs could selectively complex with Cu2+ ions by basic (-NH2) and acidic (-COOH) functional groups and lead to a red-shift of the LSPR absorption band. The change in the LSPR peak is the basis of the detection of the Cu (II) concentration. It was known that one Cu2+ ion can coordinate with two Cys molecules because of its four chelation bands (Weng et al., 2013). However, in this case, the absorbed Cu2+ ion on ITO/Au NPs/Cys surface complexed with an amino group and a carboxyl group in only one Cys molecule. Therefore, the Cu2+ ion could complex with another chelating reagent due to the two chelation bands remained. Then we examined CR as the second complexant in this system because CR is a common chromogenic agent for

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determination of Cu2+ ions. We found the introduction of CR in this system for 0.1 µM Cu2+ resulted in red-shift of ~6 nm compared to ITO/Au NPs/Cys (Figure 3), indicating a little higher sensitivity obtained. Next, we further examine BSA as the second complexant in this system because of application in the fluorescence analysis (Durgadas et al., 2011). It was found that the red-shift reached 12.8 nm for 0.1 µM Cu2+ (Figure 3) compared with ITO/Au NPs/Cys, indicating that the sensitivity for the determination of Cu2+ ions was enhanced greatly. This is attributed to the adsorption of macromolecular of BSA which induces large changes of refractive index surrounded Au NPs. This concept is similar to that the second target (antibody) was introduced to increase the red-shift in ammunoassays (Guo and Kim, 2012; Haes et al., 2005). However, antibodys are commonly very expensive and poor stable. Figure 2 Figure 3

3.3. Optimization of the experiments Since the complex reaction was heterophase process, the reaction time and temperature were important factors. Figure 4A displays the effect on the different reaction times and temperatures in the presence of 0.1 μM Cu2+ ions. Under all reaction temperatures, the LSPR peak of the sensor was gradually red-shifted with the increasing incubating time and then reached the plateau. Furthermore, the higher reaction temperature, the shorter reaction time needed for complete reaction. Therefore, 30 ºC and 20 min were selected in the next experiments. We also examined

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the reaction times and temperatures in incubation of BSA solution (Figure 4B). The similar results were obtained. We selected 30ºC and 15 min as the experimental conditions. For comparison, the control experiments were carried out. The LSPR peak wavelength was almost no changes in the absence of Cu2+ ions in the system. Figure 4

3.4. Detection of Cu2+ ions Under the optimized experimental conditions, this plasmonic sensor was applied to detect Cu2+ ions. Figure 5 shows the linear plots of the LSPR peak red-shift at variable concentration of Cu2+ ions. The red-shift of the LSPR peak shows a good linear with the concentration of Cu2+ ions in the range of 10-11 – 10-5 M (R2=0.9937), with the detection limit of 5×10-12 M (a signal-to-noise ratio of 3). For comparison, the linear relationships between peak red-shift and the concentration of Cu2+ ions were also exhibited in the absence and presence CR as the second complexant (Figure 5). In the absence of the second complexant, the sensor showed a linear in the Cu2+ concentration in the range of 2×10-8 ~ 10-3 M. In the presence of CR as the second complexant, a linear relationship was achieved in the concentration range of 10-8 ~ 10-3 M. It indicated that the ITO/Au NPs/Cys sensor coupled with BSA as the second complexant can be used to detect Cu2+ with a very low minimum detection concentration of 10-11 M. This method has an obvious advantage over many representative methods (Table S1). Figure 5

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To examine the effect of nanoparticle size, we also fabricated large Au NPs by the same method with different volume ratio of the reactants. The average size of large Au NPs was about 25 nm observed in the TEM image (Fig S4). Using the same procedure, the prepared sensor with the large Au NPs could also be used to detect Cu2+ ions. Compared with the sensor based on16 nm of Au NPs, it exhibited a narrower linear range of 10-10 ~ 10-5 M with a higher slope (Figure S5). The high sensitivity (slope) is attributed to the high refractive index sensitivity for the large nanoparticles. The selectivity of the assay for Cu2+ was investigated by testing the LSPR peak change of the film sensor combined with BSA to copper (0.01 µM) and other metal ions (1 µM), including K+, Fe2+, Pb2+,Co2+, Zn2+, Ni2+, Cd2+, Ag+ and Hg2+ ions. The peak wavelength changes of the film sensor to various cations were illustrated in Figure 6. The concentration of interfering ions is equivalent to 100 times to the concentration of copper ions (0.01 µM). The response of the ITO/Au NPs/Cys sensor is very small under such high concentrations, so these ions do not interfere with the determination of copper ions. Figure 6

The reproducibility was investigated from the response to the 1µM Cu2+ ions at five different LSPR probes. An acceptable relative standard deviation (RSD) of 2.6% was obtained, demonstrating that the construction method was highly reproducible.

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In order to prove accuracy and precision of the method for measurement of copper, we examined the actual samples of tap water, river water, and milk. Tap water and lake water samples were collected from our laboratory and a lake of Suzhou university. Milk was purchase from a supermarket. Before analysis, milk was first boiling and centrifugal (12000 r/min) for 10 min, then supernatant fluid was taken for analysis. Test results were shown in Table S2. The recoveries for the assay of Cu2+ were between 103 and 85% after addition of 10-6M Cu (II).

4. Conclusion Gold nanoparticles were self-assembled on the ITO glass surface using PDDA as a linker. Pure gold nanoparticle arrays were obtained through high temperature calcination to remove PDDA macromolecules. Cys molecules can easily be adsorbed on the surface of gold particles and form a monolayer. Then the Cys can react with Cu2+ ions with amino nitrogen and carbonyl oxygen. The Cu2+ ions adsorbed on the sensor surface induced a red-shift of the LSPR peak wavelength. Especially, BSA is a micromolecule which induces a great change of refraction index at environment of Au NPs. The sensitivity of the sensor can be further improved by addition of BSA. This method shows the following advantages: (1) High sensitivity: it is sensitive enough for detecting Cu2+ ions in tap water, environmental water, clinical diagnostic and food safety. (2) Simplicity: it is very simple in the sensor preparation, detection procedure, and instrumentation. (3) Reproducibility: it is very good in terms of reproducibility. Therefore, we expect that this method is useful for application in real samples for

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field analysis.

Acknowledgment This work was financially supported by the National Natural Science Foundation of China (No. 21475092) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Captions of the Figures Fig. 1. A TEM image of the Au NPs (A), UV-vis absorption spectra of Au NPs in solution, assembly on ITO substrate with PDDA before (ITO/PDDA/Au NPs) and after (ITO/Au NPs) calcination at 400 °C for 0.5 h (B), a typical SEM image of ITO/PDDA/Au NPs (C) and its size distribution (D), a typical SEM image of ITO/Au NPs (E) and its size distribution (F).

Fig. 2. The mechanism for LSPR-based detection of Cu2+ ions using ITO/Au NPs.

Fig. 3. UV–visible absorption spectra of the AuNPs, modification of Cys, incubation of 10-5M Cu2+ in phosphate–citric acid buffer solution, and after reaction with the second complexant of CR or BSA.

Fig. 4. The effect of temperature and time on the wavelength shift of absorption peaks for the ITO/Au NPs/Cys in the presence of 10-5M Cu2+ ions (A) and after incubation in 8 mg/L BSA solution (B)

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Fig. 5. Calibration curves for the detection of Cu2+ ions.

Fig. 6. Comparison of the LSPR peak change for copper and different metal ions. The concentration of metal ions is 0.01 µM for Cu2+ ions, but 1 µM for the other metal ions.

6 5

/nm

4 3 2 1 0 Pb

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2+

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Zn

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+

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2+

Metal ions

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Fe

2+

Hg

+

Ag

Cu

2+

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0.12

AuNPs in solution ITO/PDDA/AuNPs ITO/AuNPs

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/nm

D 0.20

d=184nm

Count

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0.00 8

10 12 14 16 18 20 22 24 26 28 30 32

Cross-sectional diameter/nm 0.25

F 0.20

d=20 3nm

Count

0.15

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0.05

0.00 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Cross-sectional diameter/nm

19

20

AuNPs

0.12

AuNPs/Cys

0.10

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2+ 2+

AuNPs/Cys+Cu +CR

Abs

0.08

2+

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600

/nm

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700

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4

20 30 40 50

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0 0

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t/min 14 12

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20 30 40 50

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t/min

22

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8 6 4 2 0 -12

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logC(mol/L)

Highlights > The Au NPs onto ITO solid substrate was fabricated by self-assembly method and calcination. > The ITO/Au NPs/Cys probe can be used to plasmonic detection of Cu2+ ions. > The introduction of BSA can greatly improve the sensitivity of detection. > The plasmonic sensor was of high sensitivity and wide linear range.

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