Electronic Supporting Information · 2011-09-07 · 1 Electronic Supporting Information: Enzyme-free colorimetric assay of serum uric acid Raj Kumar Bera, Anakuthil Anoop and C. Retna

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Electronic Supporting Information:

Enzyme-free colorimetric assay of serum uric acid

Raj Kumar Bera, Anakuthil Anoop and C. Retna Raj*

Department of Chemistry, Indian Institute of Technology, Kharagpur 721308, India

[email protected]

Experimental Section

Materials. HAuCl4, NaBH4, 2-TU, and UA were purchased from Sigma-Aldrich. All

other chemicals used in this investigation were of analytical grade and used without

further purification.

Instrumentation. Electronic absorption spectra were recorded with CARY 5000 UV-

visible-NIR spectrophotometer. Fourier transform infrared spectroscopic (FTIR)

measurements were performed with Perkin Elmer FTIR spectrophotometer RX1. TEM

measurements were performed using JEOL Model JEM-2010 microscope with an

operating voltage of 200 kV.

Synthesis of 2-TU functionalized Au nanoparticles. In a typical synthesis, 40 µL of

HAuCl4 (30 mM) was mixed with 90 µL of 2-TU (1 mM) and 2.8 mL of water. The

reducing agent NaBH4 (70 µL of 22 mM) was added to the mixture under vigorous

stirring. The total volume of the sample was 3 mL and the final concentration of HAuCl4,

2-TU and NaBH4 was 0.4 mM, 30 µM and 0.51 mM, respectively. The stirring was

continued for 40 min. and the resulting wine red colored 2-TU functionalized Au

nanoparticle solution was stored at room temperature. The final concentration of HAuCl4,

2-TU and NaBH4 are 0.4, 0.03, and 0.51 mM, respectively. The molar ratio of 2-TU to

HAuCl4 is 0.075:1. The pH of the colloidal nanoparticles was measured to be 5.6.

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2011

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It should be mentioned here that the nanoparticles synthesized at higher

concentration (>30 M) were not stable, presumably due to the self-aggregation (please

refer the spectra given in ESI15 on page no 18) and they cannot be effectively used for

the sensing of UA.

Synthesis of unfunctionalized Au nanoparticles: In a typical synthesis, 70 µL of

NaBH4 (22 mM) was added to an aqueous solution containing 0.4 mM of HAuCl4 under

vigorous stirring and the stirring was continued for 40 min.

Synthesis of citrate-stabilized Au nanoparticles: Citrate-stabilized Au nanoparticles

were prepared according to the literature procedure with slight modification.1 Briefly, 0.9

mL of 39 mM of sodium citrate dihydrate was added to the boiling solution of HAuCl4

(9.1 mL, 0.9 mM) and stir for 30 min and the resulting wine red colored citrate-stabilized

Au nanoparticle stored at room temperature.

Sensing of UA. Typically, an aqueous solution of UA (5 L of 150 µM) was gradually

added to the colloidal 2-TU functionalized Au nanoparticle (3 mL) and the spectrum was

recorded after 1 min.

Real sample analysis. In order to minimize the matrix effect due to high molecular

weight proteins, the serum samples were diluted by 10 times and filtered using 3 kDa

molecular weight cut off (MWCO) micro-centrifuge filter. The filtered serum samples

(25 µL) were injected into 2-TU functionalized nanoparticles and the optical spectrum

was recorded after 1 min. After registering the spectrum, the sample was spiked with

known concentration of UA and recovery was calculated from the spectral response after

the spike.

1. G. Frens, Nature Phys. Sci., 1973, 241, 20.


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DFT calculations : The density functional calculation in gas phase were performed using

model system; 2-(methylthio)pyrimidine-4(3H)-one (MeTU) was chosen to represent 2-

TU functionalized nanoparticles. Because AA exist as a monoionic species at pH>4.2

(first pKa = 4.17, J. Am. Chem. Soc. 1935, 57, 1929), the ascorbate anion was considered

in the calculation. All geometris were fully optimized using TURBOMOLE program

package (TURBOMOLE V6.2 2010, a development of University of Karlsruhe and

Forschungszentrum Karlsruhe GmbH, 1989-2007, TURBOMOLE GmbH, 1989-2007,

TURBOMOLE GmbH, since 2007 available from http://www.turbomole.com). B97-D

functional in conjunction with def2-SVP bais set was used.


http://www.turbomole.com/

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Figure ESI1

(a) UV-vis spectrum, (b) TEM image and (c) particle size distribution plot of 2-TU

functionalized Au nanoparticles

360 480 600 720 840

0.2

0.4

0.6

0.8

1.0 (a)

Wavelength(nm)

Ab

sorb

an

ce

0 5 10 15 20 25 30 35 40 450

8

16

24

32

40(c)

Particle size, nm

Ab

un

dan

ce(%

)


5

Figure ESI2

FTIR spectra of (a) 2-TU and (b) 2-TU functionalized Au nanoparticles.

Frequency Assignment

3350 cm-1

N–H stretching

3080 cm-1

C–H stretching

1700 cm-1

C=O stretching

1567 cm-1

N–H in plane bending

1000 2000 3000 4000

1567 cm-1

1567 cm-1

3080 cm-1

3080 cm-1

1700 cm-1

1700 cm-1

3350 cm-1

3350 cm-1

(b)

(a)

Wavenumber (cm-1

)

Tra

nsm

itta

nce

(%

T)


6

Figure ESI3

TEM image of 2-TU functionalized Au nanoparticles in presence of UA (4 µM).


7

Figure ESI4

UV-vis spectra of (a) citrate-stabilized and (b) unfunctionalized Au nanoparticles in

presence and absence of UA (10 µM). Inset shows TEM image Au nanoparticles. The

procedure for the synthesis of citrate-stabilized and unfunctionalized nanoparticles are

given on page 2 of ESI.

400 600 800 10000.0

0.5

1.0

1.5

2.0

2.5(a)

10 M

5 M

0 M

Wavelength(nm)

Ab

sorb

an

ce

480 600 720 840

0.2

0.4

0.6

0.8

1.0 (b)

Wavelength (nm)

Ab

sorb

an

ce

10 M

5 M0 M


8

Figure ESI5

UV-vis spectral response of 2-TU functionalized Au nanoparticles towards UA,

illustrating the concentration-dependent sensitivity and limit of detection of UA.

Concetration of nanoparticles: (a) 0.25 nM and (b) 0.06 nM. Concentration of 2-TU:

(a) 30 μM and (b) 10 μM. The cocnetration of the nanoparticles was calculated

according to the standard literature procedure (Anal. Chem. 2001, 73, 5220-5227).

The nanoparticles were synthesized according to the following procedures.

(a) Mixing an aqueous solution of 0.5 mM of HAuCl4, 30 μM of 2-TU, and 0.51

mM of NaBH4 and stirring in a magnetic stirrer for 40 min. The total volume

of the reaction mixture was 3 mL.

(b) Mixing an aqueous solution of 0.4 mM of HAuCl4, 10 μM of 2-TU, and 0.51

mM of NaBH4 and stirring in a magnetic stirrer for 40 min. The total volume

of the reaction mixture was 3 mL.

Analytical data obtained from the measurements:

(a) Concentration of nanoparticle: 0.25 nM

Concentration of 2-TU: 30 μM

LOD (3σ): 1 μM

Slope of the calibration plot (sensitivity): 0.06 μM-1

(Y=0.317+0.065 X)

(b) Concentration of nanoparticles: 0.06 nM

Concentration of 2-TU: 10 μM

LOD (3σ): 1.5 μM

Slope of the calibration plot (sensitivity): 0.08 μM-1

(Y= 0.359+0.081 X)

360 480 600 720 840

0.2

0.4

0.6

0.8

1.0

1.2

5 M4 M3 M2 M

1 M

0 M

(a)

Wavelength (nm)

Ab

sorb

an

ce

0 1 2 3 4 5

0.32

0.40

0.48

0.56

0.64

A6

55/A

53

0

Concentration (M)

Y = 0.317 + 0.065X

360 480 600 720 840

0.2

0.4

0.6

0.8

1.0

5.5 M

4.5 M

3.5 M2.5 M

1.5 M

0 M

(b)

Wavelength(nm)

Ab

sorb

an

ce 0 1 2 3 4 5 6

0.4

0.5

0.6

0.7

0.8

0.9

Y = 0.359 + 0.081X

Concentration (M)

A6

55/A

53

0


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Figure ESI6

UV-vis spectra of 2-TU functionalized Au nanoparticle in presence of different

concentrations of UA. Inst shows the calibration plot. Different concentrations of UA

were individually added to the sample vials of freshly prepared functionalized

nanoparticles.

480 600 720 840

0.2

0.4

0.6

0.8

1.0

Wavelength(nm)

Ab

sorb

an

ce

0 1 2 3 4

0.4

0.6

0.8

1.0Y=0.36+0.17x

Concentration (M)

A6

55/A

53

0


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Figure ESI7

UV-vis spectra of 2-TU functionalized nanoparticles in the presence of UA (2.5 µM) at

different time intervals.

480 600 720 840

0.2

0.4

0.6

0.8

1.0

10 min

5 min

1 min

Wavelength(nm)

Ab

sorb

an

ce


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Figure ESI8

(a) Absorption spectra illustrating the selectivity of the optical sensing method towards

UA using 2-TU functionalized Au nanoparticles and (b) Plot illustrating the selectivity

of UA assay. The ratio of A655/A530 is plotted against analyets. Concentration of the

analytes: 0.1 mM each.

400 600 800 10000.0

0.2

0.4

0.6

0.8

1.0

NaClGlucose

l-DOPA

APAA

DA

UA

Wavelength(nm)

Ab

sorb

an

ce

0.2

0.4

0.6

0.8

1.0

NaC

lA

AD

AUA

AP

L-D

OPA

Glu

cose

A6

55/A

53

0


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Figure ESI9

Structure of 2-(methylthio)pyrimidin-4(3H)-one and ascorbate anion.

HN N

S

CH3

O

2-(methylthio)pyrimidin-4(3H)-one

OO

HO

HO

O OH

Ascorbate


13

Figure ESI10

The hydrogen bonding interaction UA with the MeTU based on the DFT calculation. The

theoretical calculation indicates that up to 6 units of MeTU can have favorable hydrogen

bonding interaction with one molecule of UA.


14

Figure ESI11

The geometry based on the preliminary DFT calculation showing possible - interaction

of two hydrogen bonded UA units. (Four MeTU molecules per plane are considered).

Top-view (left) and side-view (right).


15

Figure ESI12

Optimized geometry and possible hydrogen bonding of AA with MeTU.

Unlike UA, AA is highly flexible and is not a planar molecule. Although AA can have

hydrogen bonding interaction, it could not effectively aggregate the nanoparticles due to

the fact that TU units are randomly oriented around AA anion.


16

Figure ESI13

UV-vis spectral profile illustrating the selectivity of the colorimetric method. Different

concentrations of interfering analyte (a) glucose and (b) 4-acetamidophenol were

progressively added to the functionalized nanoparticles.

400 600 800

0.2

0.4

0.6

0.8

1.0 (a)

Wavelength(nm)

Ab

sorb

an

ce

100 M

80 M

60 M 40 M 20 M 0 M

480 600 720 840

0.2

0.4

0.6

0.8

1.0(b)

Wavelength(nm)

Ab

sorb

an

ce100 M

80 M 60 M

40 M

20 M 0 M


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Figure ESI14

Absorbance vs time plot at 655 nm in presence of real sample analysis.

0.0 0.2 0.4 0.6 0.8 1.0

0.29

0.30

0.31

0.32

0.33

0.34

Time (min)

Ab

sorb

an

ce a

t 6

50

nm


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Figure ESI15

UV-vis spectra of Au nanoparticles synthesized at different concentration of 2-TU.

The above spectra illustrate the self-aggregation of nanoparticles while increasing the

concentration of 2-TU. The surface plasmon band gradually shifts to higher wavelength

side and the spectral response is very broad, possibly due to the self-aggregation of the

nanoparticles. The colloidal nanoparticle is not stable for long time; the particles

gradually settle down in the sample vial.

360 480 600 720 840

0.2

0.4

0.6

0.8

1.0

90 M

80 M

70 M

50 M

40 M

30 M

10 M

Wavelength(nm)

Ab

sorb

an

ce


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Electronic Supporting Information · 2011-09-07 · 1 Electronic Supporting Information: Enzyme-free colorimetric assay of serum uric acid Raj Kumar Bera, Anakuthil Anoop and C. Retna