Ultrasensitive Determination of Copper in Complex Biological Media

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Ultra-sensitive determination of copper in complex biological media based on modulation of plasmonic properties of gold nanorods Shenna Chen, Qian Zhao, Fang Liu, Haowen Huang, Linqian Wang, Shoujun Yi, Yunlong Zeng, and Yi Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac401789n • Publication Date (Web): 30 Aug 2013 Downloaded from http://pubs.acs.org on September 2, 2013

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Analytical Chemistry

Ultra-sensitive determination of copper in complex biological media based on modulation of plasmonic properties of gold nanorods †

†

†

†*

‡

Shenna Chen , Qian Zhao , Fang Liu , Haowen Huang , Linqian Wang , Shoujun †

†

Yi , Yunlong Zeng , Yi Chen

†

§*

Laboratory of Theoretical Chemistry and Molecular Simulation of Ministry of

Education. School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan, China ‡

Department of Laboratory, Hunan Provincial Tumor Hospital, the Affiliated Tumor

Hospital of Xiangya Medical School of Central South University, Changsha, Hunan Province, China §

Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of

Chemistry, Chinese Academy of Sciences, Beijing, China

* Corresponding authors: Haowen Huang, Tel: 86-731-58290045. E-mail: [email protected] Yi Chen,

Tel: 86-10-62618240. E-mail: [email protected]

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ABSTRACT：： Accurate determination of copper in complex biological media such as cells is quite difficult and an analytical strategy based on copper-modulated formation of core-shell gold nanorods is described.

Selective and label-free sensing can be

achieved by measuring the change in the localized surface plasmon resonance absorption.

The technique can determine trace amounts of copper in human serum,

urine, and red blood cells without or with minimal sample pretreatment.

The Cu

detection limits are 20.67 µM in human serum, 0.193 µM in human urine, and 3.09×10-16 g in a single cell.

The advantages of the technique are the high selectivity,

simple or no sample pretreatment, and label free.

Boasting a practical detection limit

down to 2 fM, only 103 red blood cells are needed to conduct the analysis and the technique may be extended to the detection of trace amounts of copper in a single cell.

Keywords: Localized surface plasmon resonance; Gold nanorods; biosensor; copper; Bio-fluidic samples; red blood cells

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INTRODUCTION Gold nanorods (GNRs) exhibit localized surface plasmon resonance (LSPR) and are attractive nanomaterials in analytical chemistry. Boasting unique physical, chemical, electronic, and catalytic properties,1,2 they are useful in photothermal therapy,3,4 molecular imaging,5 biosensing,6,7 gene delivery,8 and data storage.9

LSPR can

theoretically be modulated by geometric parameters of the GNRs such as shape, size, and length-to-width ratio and the dielectric environment.10,11

Recently, it has been

demonstrated that modulation could be achieved by adjusting and/or tailoring the plasmonic nanostructure

12

and composition of the nanorods thus providing a

convenient means to expand the applications of GNRs.

A common way is to utilize

core-shell GNRs which can easily be prepared by bioconjugation or on-surface self-assembly of functionalized

substances.13-15

However,

applications to

ultra-sensitive sensing are still lacking. Herein, an analytical strategy based on copper-modulated formation of core-shell GNRs for ultra-sensitive and label-free determination of trace amounts of copper in biological samples is described.

Copper modulates the formation of core-shell

GNRs and is an essential trace element responsible for maintaining homeostasis in the human body.

However, an excess amount of copper is toxic and/or cytotoxic to

living organisms16 and can cause liver damage17 and neurodegenerative diseases.18 Elevated copper concentrations have been found in sera from patients with various malignant tumors19-21 and correlated to cancer progression.

It is thus crucial to

monitor the variation of copper concentrations in the human body and fluorescent

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techniques,22-24 electrochemical methods,25,26 atomic absorption, and emission spectrometry27,28 have been applied to the detection of trace copper amounts in biological systems.

However, these conventional suffer from various disadvantages

including tedious sample preparation, complicated steps for separation and detection, and high cost.

On the other hand, the technique based on core-shell GNRs can

directly detect copper in complicated matrices without or with minimal sample pretreatment, because of the copper-specific reaction in the preparation of the core-shell GNRs.

The spectroscopic variation of the longitudinal plasmon

wavelength (LPW) of copper-modulated core-shell GNRs, which depends on the copper content, is monitored.

Compared to pure GNRs, core-shell GNRs give a

red-shift of LPW due to geometric elongation and the red-shift is very sensitive to the copper content offering a detection limit of 2×10-15 M and applicable to the determination of copper concentration from about 103 red blood cells.

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EXPERIMENTAL SECTION Chemicals.

Cetyltrimethylammonium bromide (CTAB), sodium borohydride,

HAuCl4·3H2O, ascorbic acid, sodium thiosulfate, silver nitrate, zinc nitrate, calcium nitrate, ferric chloride, manganese sulfate, copper nitrate, cobalt sulfate, nickel sulfate, aluminum sulfate, sodium chloride, chromium sulfate, mercuric chloride were purchased from Sinopharm Chmical Reagent Co., Ltd. (Shanghai, China).

All the

chemicals were of analytical reagent grade, unless mentioned otherwise, and used as received.

The aqueous solutions were prepared with doubly distilled water.

Synthesis of GNRs.

The GNRs were synthesized using a seed-mediated

silver-assisted growth method29, 30 in two steps, namely preparation of gold seeds and synthesis of GNRs.

The gold seeds were prepared by addition of 100 µL of HAuCl4

(0.02 M) and 100 µL of ice-cold NaBH4 (0.01 mM) into 1.5 mL of CTAB (0.1 M) under stirring.

After a yellow brown solution was obtained, the seed solution was

ready for use but must be used within 2-5 h.

The GNRs were synthesized by

addition of 1.5 mL of HAuCl4 (0.02 M) into 30 mL of CTAB (0.1 M), followed by addition of 0.8 mL of ascorbic acid (0.08 M) which served as a mild reducing agent. After the dark yellow solution became colorless, 70 µL of the seed solution were added and the reaction proceeded for 30 min. Preparation of copper-based core-shell GNRs.

20 µL of Cu(NO3)2 (0.01 M), 20

µL of HgCl2 (0.01 M), 10 µL of NaBH4 (0.01 M), and 50 µL Na2S2O3 (0.05M) were added to 2 mL of the GNR suspension at room temperature.

The LSPR absorption

spectra were monitored at different time intervals until the spectra became stable, after

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which the final spectrum was recorded. Preparation of biological samples.

Urine samples were collected from healthy

human volunteers, put in centrifuge tube, and refrigerated until use. samples and red blood cells were obtained from the hospital.

All the serum

The serum samples

were stored at 4 ºC until use while the whole vein blood samples (2 mL each) were separately put into heparinized containers, diluted with 5 mL of phosphate buffer saline (PBS) containing 0.9% NaCl, and centrifuged at 2,500 rpm for 5 min. washing three times with PBS, the cells were re-suspended in PBS.

After

The size

distributions of the RBCs was measured on the Sysmex XE 5000 (Sysmex Corporation, Kobe, Japan), a fully automated blood cell counter on which the RBCs were detected at the center of the aperture employing a Sheath Flow DC detection method.

Because a high concentration of Hg2+ ions was present in the assay, the

necessary safety precautions were exercised.

As soon as the Hg2+ solution was

added to the solution, the cuvette was covered. Instrumentation.

Transmission electron microscopy (TEM) was performed on a

Tecnai G2 20 (USA) transmission electron microscope (TEM) operating at 100 kV. The nanorod solution (1-2 µL) was placed on a carbon coated copper grid and dried at room temperature.

Absorption spectra were acquired from the GNR suspensions on

the Lambda 35 (PerkinElmer, USA).

The copper concentration in the RBCs was

also determined by atomic absorption using a Perkin Elmer atomic absorbance spectrophotometer (PEAAnalyst 300).

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RESULTS AND DISCUSSION The GNRs are first synthesized with AgNO3 in the presence of CTAB according to previous reports.29, 30

The TEM image of the GNRs in Figure 1a indicates that the

transverse width and axis length are 9±1 and 37±2 nm, respectively.

Figure 1c

depicts the absorption spectrum and the bands at 518 nm and 826 are the transverse and longitudinal plasmon absorptions, respectively. The as-synthesized GNRs do not response to Cu(NO3)2 and hence, copper-based core-shell GNRs (Figure 1b) are synthesized and experimented. response to Cu2+ at room temperature.

They also show no

The LPW exhibits a slight blue-shift after

Na2S2O3 addition but the optical intensity decreases when Na2S2O3 is added together with HgCl2 and NaBH4. Figure 2.

A significant red-shift of the LPW occurs as shown in

This phenomenon provides the foundation for selective and label-free

determination of Cu2+. The core-shell structure is characterized by TEM (Figure 1b) and XRD (Figure 3). Cu (in the form of Cu2S) is present but at a small concentration. LPW is due to the increase in the shell stems.

The red-shift in the

The associated reactions include

attachment of Na2S2O3 onto the GNRs to generate AuAgS as well as formation and deposition of HgS and Cu2S onto the surface from the solution composed of Cu(NO3)2, Na2S2O3, and HgCl2 in the presence of NaBH4.

The reactions for the

GNRs, Na2S2O3, Cu(NO3)2, HgCl2, and NaBH4 are shown in equations 1 to 3: 2Ag(S2O3)23-+2Au(S2O3)23-+8H2O = 4AuAgS+8H2SO4+4S

(1)

2Cu2+ + 2S2O32- + 2H2O = Cu2S + S + 2SO42- + 4H+

(2)

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2Cu2++2S2O32-+2H2O+NaBH4+3H2O=Cu2S+S2-+2SO42-+6H++Na++H2BO3-+3H2 (3). As previously reported, 31 Na2S2O3 is found to induce leaching of Au or/and Ag from ores forming Au(S2O3)23- and Ag(S2O3)23-, which may coexist stably in the solution.

However, Ag(S2O3)23- reacts with Au(S2O3)23- producing AuAgS in the

presence of CTAB.32 Cu2+ and S2O32- react to produce Cu2S and S as shown in equation 2,33,34 but NaBH4 being a stronger reduction reagent changes the products slightly according to equation 3 generating S2- in place of S.

The added Hg2+ reacts

with S2- to form insoluble HgS (Ksp = 2×10-52) as one of the components of the shell around the GNR core.

An extra shell layer with multiplex components and larger

refractive indices will further form around the nanorods.

It results in a change of

plasmon resonance frequency and red-shift of LPW because both the radii and length of the GNRs and dielectric constant of the medium in the vicinity of the nanorods increase after they are covered by a thin shell.

Both longitudinal and transverse

red-shifts occur here, but the longitudinal shift is more significant than the transverse one which is almost negligible (Figure 2). The LPW shift can be adjusted by addition of four chemicals, Cu(NO3)2, Na2S2O3, HgCl2, and NaBH4.

The LPW of the nanorods can be maintained at a certain value

if the GNRs co-exist only with HgCl2 or with Na2S2O3 and NaBH4 despite a small decrease in the adsorption intensity.

A significant LPW red-shift is observed when

the GNRs co-exist with all four chemicals [Cu(NO3)2, Na2S2O3, HgCl2, and NaBH4] as aforementioned and it serves as the foundation for the analytical method. phenomenon is observed when Cu(NO3)2 and Na2S2O3 are taken away.

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A new

That is, a

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significant LPW blue-shift occurs when the GNRs are mixed with HgCl2 and NaBH4 (Figures S1 and S2 in supporting information) thereby enabling the determination of Hg2+.

The comparison in Figure S3 illustrates that coexistence of these chemicals

leads to the formation of specific nanostructures and consequently LPW red-shift. Hence, the GNRs can either be formed as core-shell nanostructures by Cu2+ or shortened by Hg2+ with the assistance of NaBH4. Na2S2O3 is able to initiate the formation of core-shell nanorods and at the same time turn off the shortening reaction. Using the core-shell GNRs, Hg2+ can be selectively detected in the presence of a fixed concentration of Cu2+ to a limit of detection (LOD) of 10 nM with a linear relationship in the range between 10 nM and 13 µM as shown in Figure S4.

This is a

promising method and worthu of further exploration, but the focus of this work is determination of Cu2+ and associated mechanism. For a fixed Hg2+ concentration, significant LPW red-shift is observed even for a trace amount of Cu2+.

The LOD is 2×10-15 M under optimized experimental

conditions as determined from sequential dilution of a stock standard solution of Cu(NO3)2. ones.22-26

This is thus an ultra-sensitive sensing method compared to current The shift of LPW is also found to depend on the concentration of Cu2+.

Figure 4a shows an upward asymptote and a good calibration curve can be derived for the concentration range covering about 2 orders of magnitude.

For a wider range,

for example, from 2 fM to 2.56 pM as shown in inset figure of Figure 4a, the logarithm of the copper concentration can be used as the variable.

In addition, the

range of Cu2+ concentration can be extended to µM in the regression equation ∆λ =

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85.6989 + 5.6787logc with only small deviation.

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As discussed above, the degree of

red-shift depends on the formation of the core-shell nanostructure.

In the process of

forming the core-shell structure capsulated by CTAB, a preferential shell is formed on the ends of the nanorods because the CTAB concentration on the nanorod ends is less than that on the transverse surface.35 Subsequent formation of the shell on the surface of the nanorods in the transverse and longitudinal directions is not proportional to the aspect ratio of GNRs, resulting in the LPW red-shift deviation from the calibration curve.

In practice, in order to determine trace amounts of copper in complex

samples, the standard addition method is more precise and this will be discussed later. The selectivity of the method is high because the four reactants, HgCl2, NaBH4, Na2S2O3, Cu(NO3)2, and GNRs are closely correlated and none can be deleted in the preparation of the specific core-shell GNRs.

This is validated experimentally.

Cu2+

is selectively determined in the presence of other metal ions such as Ca2+, Na+, Mg2+, Pb2+, K+, Co2+, Mn2+, Ni2+, Pb2+, Hg2+, Cd2+, Zn2+, Fe3+, and Al3+ at a concentration of 10-6 M.

The influence of these interfering ions is less than 8% as determined from

the variation in the LPW absorption wavelength (Figure 4b).

This suggests that in

this technique, sample pretreatment can be substantially simplified or even omitted. To demonstrate the simplicity and applicability of the method to determine copper in complex media, human serum samples are used.

No appreciable LPW change is

observed by simply adding serum into the GNR dispersion.

However, if HgCl2,

NaBH4, and Na2S2O3 are added to the mixture, a significant LPW red-shift emerges and the copper content can be determined and no sample pretreatment is required

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compared to other current methods.27,28

Coupled with direct and label-free detection

capability, this technique is the simplest one while offering ultrahigh sensitivity and selectivity.

Human serum is first added to the GNR dispersion at room temperature.

After sonication for 2 min to disperse the serum, HgCl2, Na2S2O3, and NaBH4 under are introduced to obtain final concentrations of 9.5×10-5, 1.25×10-3 and 4.76×10-5 M, respectively in the pH range of 3 to 7. Similar condition would be utilized in the following samples including human urine and RBC. The absorption spectra in Figure 5a show the gradual LPW red-shift.

Reduced absorption intensity is observed for 10

min before no further shift can be seen.

The measurement takes less than 15 min.

In order to attain better precision, the standard addition method is adopted.

Copper

concentrations in the range between 8.21 and 28.38 µM are indicated by Figure 5b. The average is 20.67 µM and in line with reported results (5.9 ~ 84 µM) provided by other techniques.36,37 The human body contains an estimated amount of 100 to 150 mg of total copper and the major portion exists in the present in the liver and central nervous system, whereas the copper concentration is much smaller in the spleen and bones.

Copper

also exists in blood plasma in which 95% bond tightly to alpha-2 globulin while the remaining 5% bond loosely with other proteins such as albumin.38,39 The copper content determined by this method indicates total copper in the serum, that is, not only the free but also the bonded copper species. Another advantage of this method is that only a very small sample size of less than 20 20 µL is needed in one measurement while ultra-high sensitivity (LOD) of 2 fM is

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achieved.

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It is thus suitable for the determination of trace amounts of copper in

human urine.

As shown in Figure 6, the range is between 0.169 and 0.226 µM,

which is 10 times smaller than that for sera, and the average is 0.193 µM which again is consistent with previous reports.40,41 Excellent results are obtained in the determination of total copper in human RBCs. Copper exists in RBCs in two forms.

The first one is loosely bonded copper species

in equilibrium with other copper species in the plasma and the second one involves bonded species formed in 24 h after the copper ions are transported to the cells.39 To ensure precise determination, the cells after collection are immediately separated from the blood sample by centrifugation and all the erythrocytic copper is made to react completely with the GNRs by rupturing the cells ultrasonically.

The LSPR signal of

the GNR dispersion containing RBCs in the absence of HgCl2, Na2S2O3, and NaBH4 is measured at different time points such as 5, 10, 30, and 240 min and no detectable change in the LPW of GNRs is observed.

The copper content in the RBCs is thus

determined by measuring the LSPR shift from samples containing 6.7×106, 3350, and 1675 cells.

The data are quite close with an average concentration at 1.049 mg/L,

which again agrees with reported data.42-45 RBC is calculated to be 3.09×10-16 g.

The copper concentration in a single

The validity is corroborated by flame atomic

absorption spectrometry (AAS) as shown in Figure S5.

The results further disclose

that the method is valid even down to 103 RBCs, thereby suggesting that it may be possible to determine the amount of Cu in a single RBC and further experiments are being conducted in our laboratory.

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CONCLUSION A LPW-based analytical method which is label-free, selective and ultra-sensitive is applied to the determination of trace amounts of copper in biological media.

The

key to the success is the ability to form core-shell GNRs so that the longitudinal LSPR wavelength can be tuned by or respond to the concentration of analyte which is copper in this study.

This strategy is universally applicable.

In this study, a

specific reaction system composed of 4 reactants of GNR, HgCl2, NaBH4, and Na2S2O3 is described and it offers a LOD of 2 fM of Cu.

Total copper

concentrations 20.67 µM and 0.193 µM can be determined from human serum and urine samples, respectively.

The method can also be used to determine the amount

of copper in human red blood cells up to 3.09×10-16 g per cell as confirmed by AAS. The technique is so sensitive that only 1,000 cells are needed in the measurement and offers the possibility of detecting the Cu concentration in a single cell.

ACKNOWLEDGMENTS This work was supported by Natural Science Foundation of China (Nos. 21075035 and 21235007) and Aid Program for Science and Technology Innovative Research Team in Higher Educational Institution of Hunan Province.

Supporting Information: This material is available free of charge via the Internet at http://pubs.acs.org.

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(27) Brandao, G. C.; Aureliano, M. D.; Sauthier, M. C. D.; dos Santos, W. N. L. Anal. Meth. 2012, 4, 855-858. (28) Shoaee, H.; Roshdi, M.; Khanlarzadeh, N.; Beiraghi, A. Spectrochim. Acta. A. 2012, 98, 70-75. (29) Murphy, C. J.; San, T. K.; Gole, A. M.; Orendorff, C. J.; Gao, J.; Gou, L.; Hunyad, S. E.; Li, T. J. Phys Chem. B. 2005, 109, 13857-13870. (30) Nikoobakht, B.; El-Sayed, M. A. Chem. Mater. 2003, 15, 1957-1962. (31) Aylmore, M. G.; Muir, D. M. Miner. Eng. 2001, 14, 135-174. (32) Huang, H.; Qu, C.; Liu, X.; Huang, S.; Xu, Z.; Liao, B.; Zeng, Y.; Chu, P. K. ACS Appl. Mater. Interfaces 2011, 3, 183-190. (33) Beijing Normal University et al. Inorganic Chemistry; 4th Ed; Higher Education Press: Beijing, 2003; p 706. (34) Hiskey, J. B.; Lee, J. Hydrometallurgy 2003, 69, 45–56. (35) Yu, C.; Irudayaraj，J. Anal. Chem. 2007, 79, 572-579. (36) Arnaud, J.; Weber, J. P.; Weykamp, C. W. ; Parsons, P. J. ; Angerer, J.; Mairiaux, E.; Mazarrasa, O. ; Valkonen, S.; Menditto, A.; Patriarca, M.; Taylor, A. Clin. Chem. 2008, 54, 1892-1899. (37) Wang, J.; Zhao, M. Chinese J. Anal. Chem. 2006, 34, 355-358. (38) Gubler, C. J. JAMA. 1956, 161, 530- 535. (39) William, B. H.; Leavell, B. S.; Paixao, L. M.; John, H. Y. American Journal of Clinical Nutrition. 1960, 8, 846-854.

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Figures and Captions a 0.8

c

0.7

Absorbance

0.6 0.5 0.4 0.3 0.2

b

0.1 400

500

600

700

800

900

1000

1100

Wavelength (nm)

Figure 1. TEM images: (a) As-synthesized GNRs, (b) Cu2+-based core-shell GNRs formed by mixing GNRs with Cu2+ in the presence of Na2S2O3, HgCl2, and NaBH4, and (c) Absorption spectrum acquired from the as-synthesized GNRs.

0.8

t im

0.6

Absorbance

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

Page 18 of 21

ei nc re as e

0.4

0.2

0.0 400

500

600

700

800

900

1000

1100

Wavelength (nm)

Figure 2. Absorption spectra acquired from GNRs reacted with Cu(NO3)2 (9.5×10-5 M) in the presence of 1.25×10-3 M Na2S2O3, 9.5×10-5 M HgCl2, and 4.76×10-5 M NaBH4 using visible-NIR spectroscopy at 3 min.

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Au/Ag AgAuS Cu2S HgS S

Intensity (cps)

20000

15000

10000

5000

0 10

20

30

40

50

60

70

80

2-theta (deg)

Figure 3. XRD patterns of the core-shell nanostructure obtained reacting the GNRs with Cu(NO3)2, Na2S2O3, HgCl2, and NaBH4.

25

a

50

b

40

20 16

30

15

∆λ

∆λ

12

10

∆λ

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


20 8

10

4

5

0 -15.0

0

-14.5

-14.0

-13.5

-13.0

0

-12.5

Logarithm of concentration (M)

0.0

-12

2+

Zn Ni2+ Co2+ -12

2.0x10

4.0x10

2+

2+

2+

Ca Fe3+ 3+ Cu2+Na+ Mn Cd2+ Mg Pb2+ + Al K

Metal ions

2+

Concentration of Cu (M)

Figure 4. a: Plot of LPW shift versus Cu2+ concentration after incubation for 10 min at room temperature.

The inset figure represents the relationship between the

logarithm of the Cu2+ concentration in the range between 2×10-15 and 2.56×10-13 M and the LPW change. The regression equation is ∆λ = 85.6989+5.6787logc, with a regression coefficient of R=0.9884.

b: LPW red-shift measured from the core-shell

GNR suspension containing various interfering metal ions in the presence of HgCl2, Na2S2O3, and NaBH4. -19-



a

30

Concentrations of copper (µM)

3.0 2.5

time

Absorbance

2.0

incre

ase

1.5 1.0 0.5

b

25 20 15 10 5 0

0.0 400

A

500

600

700

800

900

1000

1100

B

C

D

E

F

G

H um an seru m sam ples

H

I

J

Wavelength (nm)

Figure 5. a: Sequential visible-NIR absorption spectra measured every 3 min after the addition of 20 µL of human serum sample into 2 mL of the as-synthesized GNRs in the presence of HgCl2, Na2S2O3, and NaBH4. b: Copper concentrations determined from ten samples of human serum.

0.6

a b

0.5

Absorbance

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

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0.4 0.3 0.2 0.1 0.0

400

500

600

700

800

900

1000

Wavelength (nm)

Figure 6. Absorption spectra obtained before (a) and after (b) addition of 100 µL of human urine sample into 2 mL of the GNR suspension in the presence of HgCl2, Na2S2O3, and NaBH4.

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For TOC only

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Ultrasensitive Determination of Copper in Complex Biological Media

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