Sodium gluconate capped silver nanoparticles as a highly sensitive and selective colorimetric probe for the naked eye sensing of creatinine in human serum and urine

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Sodium gluconate capped silver nanoparticles as a highly sensitive and selective colorimetric probe for the naked eye sensing of creatinine in human serum and urine Susan Sadeghi , Mohadeseh Hosseinpour PII: DOI: Reference:

S0026-265X(19)32360-4 https://doi.org/10.1016/j.microc.2020.104601 MICROC 104601

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

29 August 2019 31 December 2019 3 January 2020

Please cite this article as: Susan Sadeghi , Mohadeseh Hosseinpour , Sodium gluconate capped silver nanoparticles as a highly sensitive and selective colorimetric probe for the naked eye sensing of creatinine in human serum and urine, Microchemical Journal (2020), doi: https://doi.org/10.1016/j.microc.2020.104601

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Highlights     

A high selective, fast, green and cost effective probe using GA@AgNPs is developed. Induced aggregation of GA@AgNPs by creatinine changes the color from yellow to red. The probe is utilized for visual colorimetric detection of creatinine. The developed method has lower detection limit than that of the clinical standard method. It can be successfully applied in biological fluids with satisfactory results without further pretreatment.

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Sodium gluconate capped silver nanoparticles as a highly sensitive and selective colorimetric probe for the naked eye sensing of creatinine in human serum and urine Susan Sadeghi,a, Mohadeseh Hosseinpoura a

Department of Chemistry, Faculty of Science, University of Birjand, P.O. Box. 97175/615,

Birjand, Iran

Abstract A highly selective and fast analytical assay using sodium gluconate -capped Ag nanoparticles (GA@AgNPs) was developed for the determination of creatinine by UV-Vis spectrophotometric detection. By adding creatinine to the GA@AgNPs solution, the extinction intensity of the surface plasmon resonance (SPR) band of the corresponding UV-Vis spectrum at 393 nm reduced and meanwhile a new absorption peak appeared at 560 nm gradually with increasing creatinine concentratin. Induced aggregation of GA@AgNPs by creatinine, accompanied with color changes from yellow to red can be directly observed by the nacked eye in five minutes. The extinction spectra ratio of A560/A393 was used for the colorimetric determination of creatinine. Effective parameters on the absorption intensity such as the sample pH, the type and buffer concentration, ionic strength and AgNPs concentration were optimized. Under the optimal conditions, a linear relationship between the SPR intensity ratio of A560/A393 values and creatinine concentrations is obtained over the range of 0.3-50 nM with a correlation coefficient of 0.9763 and a limit of detection of 0.2 nM. The relative standard deviation (RSD%) for five measurements of creatinine solution at 1 nM was 4.0%, exhibiting good repeatability. The results indicate that the developed probe can be successfully used for the determination of creatinine in serum and urine samples with satisfactory results. Keywords: Creatinine; Silver nanoparticles; Sodium gluconate; Naked eye sensing; Colorimetric recognition 

Corresponding author. Tel/Fax: +98 5632202009

E-mail address: [email protected] (S. Sadeghi) 2

1. Introduction Peoples have been affected by various types of kidney disease, and sometimes they require to dialysis. Therefore, rapid identification of kidney diseases is important [1]. Creatinine is one of the most common human blood analytes that produces in the human body as the ultimate product of muscle metabolism. In healthy people, creatinine is continuously transmitted to the kidneys using the blood stream and excreted in the urine. Changes in kidney function and creatinine metabolism have an intense effect on serum creatinine concentrations. Thus, creatinine levels in human urine and blood serum indicate kidney function and can be considered as a biomarker [2, 3]. The normal creatinine level in a healthy human serum and urine are in the ranges of 40-150 μM and 2.4-27.0 mM, respectively, but variy more or less depending on gender or disease [4-6]. Up to now, various techniques are available to measure creatinine concentration including luminescence

[7-9],

enzymatic

reaction

[10],

liquid

chromatography [11],

UV-Vis

spectrophotometry [12-14], flow injection [15], tandem mass spectrometry [16, 17], gas chromatography [17], capillary electrophoresis [18], potentiometric sensors [19], colorimetry [20] and electrochemical methods [22]. Although some of these methods offer sensitive detection, but need rigorous sample pretreatment, use expensive equipments or have limited sensitivity and suffer from some interferences in physiological samples. Thus, they do not prefer for routine analysis in a clinical laboratory. Generally, operation colormetric methods have received significant attention due to easy and economic detection and minimize necessary to expensive and sophisticated instruments. Additionally, the presence of some compounds especially amino acids, urea and uric acid contribute to overestimation of urinary creatinine content and influence on the accuracy of the method. Therefore, high selective measurement of creatinine is vital in examination of real samples.

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A Few colorimetric methods have been used for the determination of creatinine including enzymatic methods [22], Van Pilsum method [23], Sakaguchi color reaction [24], Folin method [25], Hare method [20], Sullivan and Irreverre methods [26] and Jaffé reaction [27]. Jaffé reaction represents a colorimetric method based on the reaction of creatinine with picric acid in alkaline solution to produce a reddish orange color. The Jaffé method is utilized for routine laboratory creatinine measurement perhaps for its inherent simplicity and cost effectiveness. However, the key limitations of the Jaffé assay are non-specifity and influencing of color intensity upon presence of substances other than creatinine [28]. Additionally, picric acid is corrosive and hazardous that may impair the health of an operator.Therefore, considerable attention has been devoted towards prominent selective, sensitive, fast, and straightforward methods to quantitate creatinine in biological fluids that take less time, require inexpensive investment and safely operation. Currently, a few colorimetric detection methods based on metal nanoparticles including copper, silver, and gold nanoparticles have been reported for improving the determination of creatinine in biological samples [12, 13, 29, 30]. The recent studies showed that the measurement of creatinine could be improved by addition of nanoparticles to the creatinine sample [31-33]. In this work, sodium-D-gluconate capped Ag NPs was used to determination of creatinine. Sodium gluconate is highly soluble in water, nontoxic and biodegradable. The carboxylate group presents in gluconate can adsorbe on the surface of Ag NPs to stabilize them against aggregation as well as to prevent them from oxidation [34]. The hydroxyl groups of gluconate have the tendency to form hydrogen bonding with target analyte. The results indicated that gluconate capped Ag NPs can be used as a naked eye probe for the detection of creatinine. The developed assay dosen’t suffer from common interferences and is highly selective to creatinine. It was utilized for

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determination of creatinine in human plasma and urine without further pretreatments. The observed results are discussed in details. 2. Experimental section 2.1 Materials and apparatus UV–Visible spectra were recorded by UV2501PC-spectrophotometer (Shimadzu, Japan) using 1.0 cm path-length quartz cell. The Benchtop pH meter model BP3001 was utilized to adjust the pH values of the aqueous solutions (Trans Instrument, Singapore). The Transmission electron microscopy (TEM) images of GA@Ag NPs were recorded on a TEM PHILIPS model EM208S, operating at 100 kV. Fourier transform infrared (FT-IR) spectrum of each sample was recorded in the 400-4000 cm-1 wavenumber range with Perkin Elmer Spectrum Two FT-IR spectrometer (Liantrisant, UK). D-Gluconic acid sodium salt was purchased from Sigma-Alderich. (Darmstadt, Germany). All other chemicals used in this study were of analytical grade without further purification and obtained from Merck (Darmstadt, Germany). The nitrate salts of cations and sodium salts of anions were used in interferences study. The water used in all experiments was deionized and provided by an Aqua Max water purification system (Young-Lin, Korea). 2.2 Synthesis of the D-gluconic acid sodium salt stabilized Ag nanoparticles GA@Ag NPs were prepared according to the chemical method described by Pitchumani et al. and Natsuki et al. [35, 36] with a minor modification using NaBH4 as the reducing agent and sodium gluconate as the stabilizer reagent. Briefly, 1 mL AgNO3 (0.25 mM) solution was introduced slowly to a mixture of 20 mL of freshly prepared sodium borohydride solution (2 mM) and 5 mL of sodium gluconate solution (1 mM) with vigours stirring for 2 hours to assemble gluconate on the surface of silver AgNPs. Capping the surfaces of the AgNPs with sodium gluconate, giving a light yellowish solution with a typical absortion band at 393 nm. The 5

aqueous solution of as-synthesized Ag nanoparticles nominated GA@Ag NPs was stored in the dark before use. The GA@Ag NPs remained stable for two weeks at 4 ºC . 2.3 Assay for creatinine A 100 μL aliquot of different creatinine standard solutions was added to 2 mL of GA@Ag NPs solution (9.6 µM) and diluted with phosphate buffer (pH 7, 0.05 M) to 5 mL. The absorption spectra of the GA@Ag NPs in the presence of creatinine were recorded after 5 min. The gradual decrease in intensity of maximum absorbance of GA@Ag NPs at 393 nm and subsequent increasing the absorbance at 560 nm as a result of interaction with creatinine was occurred. The extinction spectra ratio of A560/A393 was used to determine creatinine. 2.4 Analysis of physiological sample The serum and urine samples were obtained from healthy people. Physiological fluids were filtered (0.25 µm) and diluted 200 times by deionized water. 100 μL of each diluted sample was spiked with 25–75 μL of 1µM creatinine solution. Then, 2 mL of GA@Ag NPs (9.6 µM) was added to the mixture and total volume was made up to 5 mL by adding phosphate buffer, so that final concentrations were within the linear range of the developed assay. The creatinine concentration was analyzed with standard addition method. The Jeffé assay used in clinical laboratoies was also conducted for comparative purpose. 3. Results and discussion 3.1 Characterization of the GA@Ag NPs

The synthesized silver nanoparticles was characterized by UV-Visible (UV-Vis.) spectroscopy, Fourier Transform Infrared (FTIR) spectroscopy and transmission electron microscopy (TEM). The UV-Visible spectrum of GA@Ag NPs solution was recorded in the wavelength range of 300-800 nm at room temperature. The spectrum of yellow color GA@Ag NPs solution exhibits 6

the plasmon resonance characteristic band of Ag NPs at 393 nm. By addition of creatinine, a new peak at 560 nm appears along with decreasing the intensity peak at 393 nm. This peak at higher wavelength may be as a result of aggregation of Ag NPs induced by creatinine [36]. Meanwhile, the color of solution changes from yellow to red (Fig. 1A). Thus, the GA@Ag NPs can be used for visual detection of creatinine. Possible capping mechanism of Ag NPs by sodim gluconate as dispersing agent demonstrated using FTIR spectroscopy. The FT-IR spectra of the GA and GA@Ag NPs are illustrated in Fig. 1B. The absorption peaks located at 3000-3500 cm-1 range in GA spectrum can be assigned to the stetching vibration of hydroxyl groups. The absorption peak at 1635 cm-1 corresponding to stretching vibration of carboxyl group in GA shifted to 1629 cm-1 in GA@Ag NPs and became weaker. The broad absorption band in the range of 3000-3500 cm-1 in the spectrum of GA@Ag NPs is attributed to the hydroxyl groups of gluconate. The absorption bands at 2978 cm-1 and 2896 cm-1 observed in GA and GA@Ag NPs spectra are assigned to C-H stretching vibration frequencies. The absorption peaks observed at 713 and 1061 cm-1 for GA@Ag NPs FTIR spectrum is related to Ag-O stretching vibration mode. On the basis of these observations, it is confirmed that the Ag NPs are capped by gluconate. The morphological structure and average distribution of GA@Ag NPs was investigated by TEM. The uniform distribution and spherical shape of the particles with size distribution of 17 2 nm observed from TEM images (Fig. 1C-a). Upon addition of creatinine to the GA@Ag NPs, aggregation of nanoparticles started to occur (Fig. 1C-b). Fig. 1 3.2 Suggested mechanism

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The principle of colorimetric method for determination of creatinine based on GA-Ag NPs is presented in Scheme 1. FTIR results indicated that sodium gluconate interacts with Ag NPs surface through the carboxylate group to stabilize and well disperse Ag NPs in aqueous solution. Sodium gluconate is in anionic form at pH values close to 7. The negatively charged gluconate forms an anionic layer around Ag NPs, kept the nanoparticles stable in aqueous solution [35]. Creatinine has and

values of 4.9 and 9.2, respectively. At pH value 7, creatinine deprotonates and

converts to anionic tautomers and an equilibrium of the amino and the amine tautomers in creatinine occurs [37]. Thus, aggregation of creatinine on the surface of the modified nanoparticles via hydrogen bond formation between NH and carboxyl groups of creatinine and hydroxyl groups of gluconate occurs [38]. Therefore, crosslinking Ag NPs through hydrogen bonding with creatinine induce aggregation of nanoparticle . This event changes the color of Ag NPs solution from yellow to red, so that it provides a simple and inexpensive probe to the determination of creatinine. 3.3 Optimization of GA@Ag NPs synthesis In order to increase the sensitivity of the creatinine determination, important parameters in the measured absorption intensity should be optimized. 3.3.1 Effect of silver ion concentration To prepare GA@Ag NPs, 1 mL silver nitrate solutions with different concentrations (0.25, 0.5, 1 mM) were added slowly to the mixtures of 20 mL freshly prepared sodium borohydride solutions (2 mM) and 5 mL sodium gluconate solution (5 μM). In these experiments, the molar concentration ratio of sodium borohydride to silver ion was varied as 160, 80 and 40, respectively.The most absorbance for GA@Ag NPs was found for 0.25 mM silver nitrate solution, so that this concentration of silver ion was used for further experiments (Fig. S1A). 8

3.3.2 Effect of sodium gluconate concenteraction Stabilizing AgNPs via capping agent during the course of AgNPs synthesis in aqueous solution is critical. The influence of the capping agent concentration on AgNPs formation was studied by using different concentrations of sodium gluconate (0.1-10 mM). The GA@Ag NPs using 1 mM sodium gluconate showed high relative absorbance intesity. This may be due to the fact that the refractive index of the environment around the nanoparticles changes which in turn changes the intensity of the SPR peak. Increasing the concentration of sodium gluconate to 1 mM probably increases the thickness of the dielectric layer of capping and increases the SPR peak intensity [39, 40]. More increasing of sodium gluconate concentration reduced the interaction of surface plasmons of Ag NPs with light, thereby the absorption intensity of GA@Ag NPs decreased (Fig. S1B). At lower concentration of gluconate, capping of AgNPs can not be completely formed which lead to poor dispersing Ag NPs in an aqueous solution. 3.4 Optimization of significant variables affecting creatinine measurement 3.4.1 Effect of pH Absorption intensity of GA@AgNPs was affected by pH changes. Therfore, the effect of pH was evaluated in the range of 3-10. The sample pH was adjusted by using 1M sodium hydroxide or hydrochloric acid solutions. The trend observed in the absorption spectra with variation in pH values represented an increase of absorption at 393 nm with increase in pH value up to 7 and afterwards it decreases (Fig. 2A). This is might be due to the fact that the gluconate ions physically adsorbed onto the surface of the Ag NPs without forming any chemical bonding. At low pH values, the hydronium ions protonates the negative charges of the carboxylate in gluconate (pKa of gluconic acid = 3.86) on the surface of nanoparticles, leading to start agglomeration of Ag NPs and subsequently diminishes hydrogen bonding with creatinine [13]. Whereas, replacement of gluconate ions with hydroxide ions may be a reason of decreasing 9

sensitivity of the probe to creatinine at high pH values. Apart from that, the gradual variations of pH-dependent absorption spectra on two sides of pH= 7 upon addition of creatinine is observed and the A560/A393 ratio is the highest at this pH. As aforementioned, creatinine exists in tautomeric forms at pH=7 that can interact with GA@Ag NPs and the extinction ratio A560/A393 increases (inset of Fig. 2A).Therefore, pH 7 was selected as the optimum pH for further studies. 3.4.2 Effect of type and concentration of buffer Type and concentration of buffer affect absorption spectrum of nanoparticles. In order to investigate the effect of different buffers on the determination of creatinine, three types of buffer (citrate, phosphate and Mcilvaine) at pH 7 were selected. The results indicate that phosphate buffer provides the best response for determining of creatinine (Fig. S2A). Furthermore, different concentrations of phosphate buffer at pH 7 in the range of 0.01-0.1M were investigated. As Fig. S2B shows, the extintion ratio of A560/A393 for determination of creatinine in the presence of 0.05 M phosphate buffer is the maximum and selected for subsequent studies. 3.4.3 Effect of GA@AgNPs concentration Carboxylate and hydroxyl groups of gluconate on the surface of AgNPs provide hydrogen binding sites for creatinine. The higher interaction between carboxylate and hydroxyl groups of gluconate with –NH and –CO groups of creatinine, the more hydrogen bonding forms and higher sensitivity of the measurement is obtained. So, the concentration of the GA@AgNPs plays a crucial role in the determination of creatinine. Fig. 2B indicates that the best response is obtained using 2 mL of GA@AgNPs solution (9.6 µM). Higher volumes more than 2 mL nanoparticles had no significant effect on the response. Fig. 2 3.4.4 Effect of ionic strength

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In order to evaluate the effect of ionic strength, determination of creatinine in different concentration of NaCl solutions in the range of 0-0.5 mM was examined. It was found that the absorption intensity ratio of A560/A393 for GA@AgNPs in the presence of creatinine did not change with increasing NaCl concentration. Thus, interactions of the creatinine with GA@AgNPs are not affected by ionic strength that favors this probe for determination of creatinine in biological fluids. 3.4.5 The effect of reaction time The extintion ratio A560 /A393 in interaction of creatinine with GA@AgNPs was investigated as a function of time in the range of 1 to 10 min with 1 min time interval. The aggregation occurs readily and the absorption ratio increased up to 5 min and then no further change is observed with increasing the reaction time, meanining that the reaction is completed in about 5 min. Therefore, 5 min after the addition of creatinine used for absorbance measurement. 3.5 Analytical performace of the assay To evaluated the assay for the determination of creatinine, analytical parameters such as linear range, detection limit and precision were determined. As aforementioned, the degree of color changes increases with increase of creatinine concentration (Fig. 3A). Under optimum conditions, the relative change in absorption ratio (A560

nm/A393 nm)

increased

linearly with increasing creatinine concentration in the range of 0.3-50 nM with a correlation coefficient of 0.9763 and a limit of detection 0.2 nM (Fig. 3B).The precision of the method based on five replicate measurments of creatinine solution at 1 nM was 4.0%. The detection limit of this assay is much lower than that of the Jaffé method. Therefore, this assay can be utilized as a highly sensitive method for the determination of creatinine. Fig. 3 3.6 Selectivity of the assay 11

Urine and blood sample have complicated matrices and some possible speices that commonly found in these biological samples may affect the creatinine content. To study selectivity of the developed method, binary solutions containing creatinine (1 nM) and possible interferents were treated by the described assay in which the interferent concentration was varied until changing  10% in the response of the probe obtained. The results are summarized in the Table 1. As it can be seen, most existing ions and compounds including uric acid, urea, glucose, and ascorbic acid with the tolerance limit of 1000 and co-existing species such as aspartic acid, cysteine, methionine and isoleucine with tolerance limit of 750 have no significant interferences on the measurement of creatinine. This investigation suggesting that the assay is high specific to creatinine and can apply in human plasma and urine samples for fast and accurate determination of creatinine. Table 1 3.7 Application The concentrations of creatinine in human serum and urine 24-h is considered as a biomarker of kidney function. To demonstrate the applicability of the assay in real samples, human blood serum and 24-h urine samples were analyzed for quantitation of creatinine via the same procedure described in section 2.4. Diluted both serum and urine samples were used as control samples. The results are summarized in Table 2. The obtained recoveries were in the range of 95.1% -109.3 % and 92.2% to 103.5 % with RSD% values of 2.9% - 5.9 % and 1.2 % - 8.0 % for human serum and 24-h urine samples, respectively. These observations indicate that the biological fluids have been diluted suffiently and demonstrate reliability of the assay for creatinine detection. Additionally, the serum and urine samples were analysed by the Jaffé

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method using picric acid (Table 2). It is clear the recoveries are in good agreement with those obtained with the Jaffé method. Table 2 3.8 Comparison of the proposed method with other colorimetric methods using metal nanoparticles for determination of creatinine Several colorimetric sensors based on noble metal nanoparticles have been developed recently for the detection of creatinine [12, 13, 19, 37, 38, 41, 42]. As indicated in Table 3, the linear range of the developed method is wider than that most of other colorimetric reported methods and the detection limit is lower than that of the compared methods. Additionally, the developed GA@AgNPs based nanoprobe has advantages such as bening, cost effective, easy, fast response, and highly sensitive to creatinine. A feature of the present assay is that it can be used without further pretreatment to clean the matrices of human plasma and urine samples. Importantly, uric acid and urea didn’t interfere in creatinine determination in this assay. These observations are consisted with the results obtained by using citrate capped Ag NPs [37] and shows better selectivity than that obtained with L-cystein capped Cu NPs [42]. Hence, this probe dosen’t suffer from background interferences in urine and is highly selective for the determination of creatinine in biological fluids. Table 3 4. Conclusions The developed probe based on GA@AgNPs is highly selective, fast, and cost effective for colorimetric recognition and determination of creatinine. The developed assay has wide linear range and low detection limit so that it is very sensitive for both qualitative and quantitative creatinine analysis. The assay can be successfully utilized in biological fluids with satisfactory results without need to further pretreatment to removing the matrices. The developed method has 13

detection limit much lower than that of clinical standard method and can be used to analysis of creatinine in point of care applications. Acknowledgement The authours thank the Doctoral Scientific Fund (1396-D-12158) for financial support.

Author statement Contributions of authors to this work are as follows, Mohadese Hosseinpour: Formal analysis, Data curation, Conceptualization, Methodology, Writing - Original draft preparation. Susan Sadeghi: Supervision, Conceptualization, Methodology, Resources, Visualization, Data curation, Writing- reviewing and Editing

Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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org/10.1016/ j.aca.2017.12.016 [38]. Y. He, X. Zhang, H. Yu, Gold nanoparticles-based colorimetric and visual creatinine assay, Microchem. Acta, 182(2015) 2037-2043, https://doi. org/10.1007/s00604-0151546-0. [39] K.-E. Fong, L.-Y. L.Yung, Localized surface plasmon resonance: a unique property of plasmonic nanoparticles for nucleic acid detection, Nanoscale,,5 (2013)12043-12071 https://doi.org/: 10.1039/c3nr02257a [40]. I. O. Osorio-Román, V. Ortega-Vá Squez, C. Victor Vargas, R. F. Aroca, Surfaceenhanced spectra on D-Gluconic acid coated silver nanoparticles, Appl. Spect., 65(2011) 838-843, https://doi.org/10.1366/11-06279. [41]. H. Du, R. Chen, J. Du, J. Fan and X. Peng, Gold nanoparticle-based colorimetric recognition of creatinine with good selectivity and sensitivity, Ind. Eng. Chem.Res. 55(2016) 12334-12340, http://dx.doi.org/10.1021/acs.iecr.6b03433. [42]. U. Sivasankaran, T. C. Jos, K. G. Kumar, Selective recognition of creatinine development of a colorimetric sensor, Anal. Biochem. 544(2018)1-6, https://doi.org/ 10.1016/j.ab.2017.12.017.

17

Figure captions Scheme 1 Schematic representation of creatinine induced aggregation GA@Ag NPs. Fig. 1 (A) UV-Vis extinction spectrum of GA@Ag NPs (a) and GA@Ag NPs in the presence of creatinine (10 nM) (b), (B) FTIR spectrum of GA@Ag NPs, (C) TEM images of GA@Ag NPs in the absence (a) and presence (b) of creatinine. Fig. 2 (A) Effect of pH on the absorption intensity of GA@Ag NPs solution (inset shows the pH effect on the A560/A393 absorption ratio in the presence of 10 nM creatinine) and (B) Effect of volume of GA@Ag NPs on the A560/A393 absorption ratio in the presence of 10 nM creatinine. Fig 3. (A) The UV-Vis. absorption spectra of GA@Ag NPs in the presence of different concentration of creatinine (0.1 to 100 nM ); (B) Corresponding calibration plot of A560/A393 vs. creatinine concentration (inset shows the linear concentration range); (C) Photograph of color change of GA@Ag NPs with various concentration of creatinine

18

Scheme 1

H O N

H3C

NH

N H

O

N

H3C

N

N

N H

O N

N

N H

H

N H

O CH3

H

N H

H3C N

O

H3 C N H

N

H3C

O

N

NH

H

AgNP

O

N H

H

NH N

H

HN H3C N

AgNP

O

H3 C N

CH3 N

N

H H

H3C N N

CH3

N

O N

H

N

HN

HN

AgNP

O

N

O

O H N

H N N

O

AgNP

H3C

N

N

NH N

CH3

H HN H

N

CH3

O

Ag Ag Ag

Ag

Ag Ag

Ag

CH3 Ag

N N H

N H

O

19

Ag

O

OH OH

OH

= OH OH

OH

Fig. 1A

0.7

A

0.6

Absorbance (a.u)

0.5 0.4

a b

0.3 0.2 0.1 0 300

400

500 Wavelength (nm)

20

600

700

800

Fig. 1B

Transmittance (%)

B

GA@Ag NPs

3900

3400

2900

2400

GA

1900

Wavenumber (cm-1)

21

1400

900

400

Fig. 1C

a

22

Fig. 1C

b

Fig. 2

23

1 0.3 A560/A393

A 0.8

0.2 0.1 0

0.6

A 393

2

4

6 8 pH

10

0.4

0.2

0 2

4

6

8 pH

10

12

14

0.3

B

0.25

A560/A393

0.2 0.15 0.1 0.05 0 0

1

2 3 Volume of GA@Ag NPs (mL) 24

4

5

Fig. 3 0.7

A

0.6

Absorbance

0.5

0.4 0.3 0.2 0.1 0 300

400

500 600 Wavelength(nm)

700

800

1.2 1

B

A560/A393

A560/A393

0.8 0.6 0.4 0.2

0 0.00

1.2 1 0.8 0.6 0.4 0.2 0 0.00

y = 19.87x + 0.059 R² = 0.976

0.02

0.04

0.06

Conc. of creatinine (µM)

0.02

0.04 0.06 0.08 Concentration of Creatinine (M)

25

0.10

0.12

26

Table 1 Effect of various inorganic and organic substances on the determination of creatinine (1 nM). Conditions: Phosphate buffer 0.05 M (pH= 7); CAgNPs= 9.6 µM, 2 mL; time reaction= 5 min. Tolerance limit

Interferents

(mol ratio)

Recovery (%)

Fe3+

1000

102.4

Zn2+, Cu2+

1000

93.6-99.8

K+, Na+

1000

96.6-101.4

Cl-, I-

1000

101.9-104.9

1000

91.1-104.1

Al3+

750

91.1

Ca2+

750

104.1

Cysteine, Metheonine, Aspartic acid, Isoleucine

750

90.7-98.8

Uric acid, Urea, Glucose, Ascorbic acid, Histidine, Tryptophan, Valine, Arginine, Succinic acid, Phenylalanine

27

Table 2 Comparison of real samples analysis by the developed method with standard method

Founded by Samples

Founded by

Added

this method

RSD (%)

Recovery

Jaffe assay

(nM)

(nM)

(n=3)

(%)

(µM)

0

0.06

-

-

ND

Blood

5

5.5  0.3

5.9

109.3

5. 2  0.1

1.9

104.0

Serum

10

9.5  0.6

6.0

95.1

10.1  0.4

3.9

99.4

15

15.5  0.4

2.9

103.1

15.0  0.3

2.0

100.0

0

0.3

-

95.7

ND

5

5.0  0.4

8.0

100.0

4.2  0.4

9.5

84.0

10

9.2  0.1

1.0

92.2

10.4  0.4

3.8

104.2

15

15.5  0.2

1.2

103.5

14.5  0.3

2.1

96.6

24-h Urine

ND= Not detected

28

RSD (%) (n=3)

Recovery (%)

Table 3. Comparison of the present method with other colorimetric methods for detection of creatinine using metal nanoparticles

Colorimetric probe

Label-free gold nanoparticles (AuNPs) after solid phase extraction Thiodiacetic acidstabilized Ag nanoparticles(AgNPs)

Linear range (µM)

LOD (nM)

RSD (%)

Response time (min)

Real sample

Ref.

133-353

121103

9.7

3

Urine

12

0.01-1.0

3.0

-

5

Serum plasma and urine

13

19

0.010-1.0

8.4

-

4

Human blood and cerebrospinal fluid sample

0-4.2

53.4

-

1

Urine

37

100-20000

80103

4.3-6.4

24

Urine

38

0.2-1.4

12.7

-

5

L-Cystein-stabilized copper nanoparticles (L-Cys-CuNPs)

0.333-5.33

45.4

3.6

7

Artificial serum and urine

42

Gluconic acid-stabilized silver nanoparticles (AgNPs)

0.0003-0.05

0.02

4.0

5

Plasma and urine

This work

Picric acid- stabilized silver nanoparticles

Citrate-stabilized silver nanoparticles (AgNPs)

Citrate-stabilized gold nanoparticles (AuNPs) as a colorimetric probe

Citrate-stabilized gold nanoparticles (AuNPs), adenosine and Ag+

29

Bovine serum and urine mimic

41