Analytica Chimica Acta 1007 (2018) 40e49

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Analytica Chimica Acta

journal homepage: www.elsevier.com/locate/aca

Citrate-capped silver nanoparticles as a probe for sensitive and

selective colorimetric and spectrophotometric sensing of creatinine in
human urine
Melisew Tadele Alula a, 1, Leshern Karamchand b, 1, Nicolette R. Hendricks b,
Jonathan M. Blackburn b, *
a
College of Sciences, Department of Chemical and Forensic Sciences, Botswana International University of Science and Technology, Plot 10071, Private Bag
16, Palapye, Botswana
b
Institute of Infectious Disease and Molecular Medicine, Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town 7925, South
Africa

h i g h l i g h t s g r a p h i c a l a b s t r a c t

A colorimetric, LSPR citrate-capped

AgNPs (cc-AgNPs) sensor capable of
selective quantitation of creatinine
was developed.

Creatinine induced concentration-
dependent aggregation of cc-AgNPs
at pH 12.

Inorganic ions and organic molecules
present in urine do not interfere with
cc-AgNPs-assay of creatinine.

The A670/A403 extinction ratio of cc-
AgNPs increased linearly in aqueous
creatinine concentration range of 0
e4.2 mM.

A nanomolar urinary creatinine limit
of detection (66 nM) was achieved
with the cc-AgNPs assay.

a r t i c l e i n f o a b s t r a c t

Article history: Urinary creatinine concentration is a critical physiological parameter that enables reliable assessment of
Received 30 October 2017 patient renal function and diagnosis of a broad spectrum of diseases. In this study, a simple and inex-
Accepted 6 December 2017 pensive sensor comprising monodisperse, citrate-capped silver nanoparticles (cc-AgNPs) was developed,
Available online 28 December 2017
which enabled rapid, sensitive and selective quantitation of creatinine directly in unprocessed urine. The
mechanism of this sensor entails the creatinine-mediated aggregation of the cc-AgNPs (within 1 min)
Keywords:
under alkaline conditions (pH 12). This is attributed to the tautomerization of creatinine to its amino
Citrate-capped silver nanoparticles
anionic species at alkaline pH, which cross-link the cc-AgNPs via hydrogen bond networks with the
Creatinine
Aggregation
negatively charged citrate caps. Creatinine elicited visibly-discernable color changes of the cc-AgNPs
Localized surface plasmon resonance (LSPR) colloids in a concentration-dependent manner up to 10 mM. UV-visible spectroscopic analyses of the
UV-Visible spectrophotometry cc-AgNPs revealed that creatinine elicited a concentration-dependent decrease in intensity of the
localized surface plasmon resonance (LSPR) band centered around 403 nm, with a concomitant increase
in intensity of the red-shifted LSPR band at 670 nm. This observation denotes a creatinine-mediated

* Corresponding author.
E-mail address: jonathan.blackburn@uct.ac.za (J.M. Blackburn).
1
Joint-first authors.

https://doi.org/10.1016/j.aca.2017.12.016
0003-2670/© 2018 Elsevier B.V. All rights reserved.
 M.T. Alula et al. / Analytica Chimica Acta 1007 (2018) 40e49
increase in cc-AgNP particle size via aggregation, as confirmed by transmission electron microscopy
analysis. The cc-AgNP sensor exhibited a linear correlation between the A670/A403 extinction ratio and
creatinine concentration range of 0e4.2 mM in aqueous solutions (R2 ¼ 0.996), and a low detection limit
of 53.4 nM. Hence, the simplicity, short assay time, and high sensitivity and selectivity of our cc-AgNP
sensor affirms its utility as a creatinine monitoring assay for low-resource, point-of-care settings.
1. Introduction
Creatinine is a metabolite of creatine and end-product of ni-
trogen metabolism, which is filtered by the kidneys and excreted
from the human body in urine. The concentration of creatinine in
urine and serum reflects the glomerular filtration rate, which is a
clinically important physiological parameter used to diagnose
kidney disease and monitor renal function [1,2]. Various analytical
methods, such as, chromatography [3e5], electrochemistry [6e8],
capillary electrophoresis [9,10], liquid chromatographyeisotope
dilution mass spectrometry (LCeIDMS) [11], mass spectrometry
[12,13], and surface-enhanced Raman spectroscopy (SERS) [14,15]
have been employed in the quantitation of creatinine in biological
fluids. However, these methods have multiple drawbacks, including
laborious sample preparation steps, complicated and environ-
mentally toxic reagents, the need for expensive, sophisticated in-
struments, as well as highly-trained technicians to operate them.
Hence, conventional laboratory methods are unsuited to, or
impractical for the point-of-care and real-time batch quantitation
of creatinine in human specimens, particularly in low resource
settings. Alternatively, Jaffe's reaction, which is a colorimetric
method based on the complex formation between creatinine and
picric acid in alkaline medium, is well documented [16]. However,
interference from other metabolites and drugs impairs the speci-
ficity of the reagent, which severely limits its clinical application.
Moreover, picric acid is highly corrosive and explosive, and there-
fore unsafe to use in routine clinical analysis. Hence, there is a
pressing need for a fast, simple, inexpensive, highly sensitive and
specific, point-of-care method to detect, and quantitate creatinine
in biological fluids.
Recently, colorimetric sensors based on silver and gold nano-
particles (AgNPs and AuNPs) have received considerable attention
because of their size, shape, composition and distance dependent
optical properties, and high extinction coefficients [17]. Specifically,
the high extinction cross section of AgNPs and AuNPs in the visible
region renders them suitable for colorimetric assays, which are
readily detectable by the unaided human eye.
The color difference between dispersed and aggregated noble
metal nanoparticles is attributed to differences in their localized
surface plasmon resonance (LSPR) properties, which forms the
basis of colorimetric detection. The aggregation of nanoparticles,
which is usually induced by their selective interaction with
specific analytes, results in coupling of the plasmonic nano-
structures. This, in turn, elicits a color change. Hence, the degree of
color change of the colloid is a function of the extent of nanoparticle
aggregation - a process governed by the type and concentration of
the analyte, which affects the electrostatic and/or steric stability of
the plasmonic nanostructures via hydrogen bonding or electro-
static interactions. The color changes of the nanoparticles are
further reflected in the corresponding changes of the absorption
maximum of their UV-visible extinction spectra, which appear as
a red shift, and broadening of the LSPR band. These spectral
changes are readily detectable and quantifiable with modular, USB-
powered UV-visible spectrophotometers, which permit on-site
41
© 2018 Elsevier B.V. All rights reserved.
environmental monitoring, as well as point-of-care biomedical
diagnostics [18,19]. Silver nanoparticles are preferred to gold
nanoparticles of the same size, due to their higher extinction co-
efficients and superior plasmonic properties, lower material costs,
ease and simplicity of synthesis with tunable size and shape, and
practicability [20]. The aforementioned properties of AgNPs are
significant advantages that favor their use as platforms in the
development of point-of-care sensors.
Indeed, several colorimetric sensors based on noble metal
nanostructures have recently been developed for the detection of
analytes, including, metal ions, small molecules, biomolecules, and
microorganisms [21e24]. Similarly, various colorimetric methods
based on AgNPs/AuNPs have been reported for the detection of
creatinine in different biological fluids [25e29]. For example, Yi He
et al. reported the detection of creatinine in spiked human urine
using citrate-capped AuNPs [25]. Jianjun Du et al. reported the
detection of creatinine using uric acid and Hg2þ modified AuNPs
[26]. Sulfonic acid-functionalized silica gel was employed to
selectively extract creatinine from urine samples, which was sub-
sequently detected using AuNPs [27]. Colorimetric sensors based
on picric acid [28] and 2, 20 -thiodiacetic acid (TDA)-capped [29]
AgNPs were developed to detect creatinine in blood, cerebrospinal
fluid and urine. Although these studies achieved significant ad-
vances in developing plasmonic based sensors to detect creatinine,
they still fall short of the requirements of being simple, rapid, and
inexpensive due to: (i) the complicated procedures employed to
functionalize the nanoparticles [26,28,29]; (ii) the laborious pro-
cess required to extract creatinine from the specimen [27]; and (iii)
high cost [25,27]. To address these problems, we report here a
simple, rapid, and inexpensive, colorimetric sensor, based on
citrate-capped AgNPs (cc-AgNPs), to detect and quantitate creati-
nine and demonstrate its highly specific and sensitive detection of
creatinine in aqueous solutions and human urine.
2. Experimental
2.1. Chemicals
All reagents were of analytical grade and used as received
without additional purification. All analyte solutions were prepared
with Millipore ultrapure double deionized water (18 MU). Silver
nitrate was obtained from South Africa Precious Metals (Pty) Ltd.
Glucose, sodium chloride, sodium hydroxide, sodium citrate
tribasic dehydrate, ascorbic acid, glycine, and urea were obtained
from Sigma-Aldrich. Uric acid, creatinine (98%), Fe2(SO4)3.xH2O,
FeSO4.7H2O, and Zn(NO3)2.6H2O were purchased from Alfa Aesar.
Potassium chloride, and CaCl2.2H2O were obtained from MERCK.
Glutamic acid was purchased from Fluka.
2.2. Instrumentation
UV-visible spectra were recorded using a Cary 50 UV-visible
spectrophotometer. The cc-AgNPs were deposited on silicon
nitride mesh substrates, and transmission electron microscopy
42 M.T. Alula et al. / Analytica Chimica Acta 1007 (2018) 40e49
(TEM) images were obtained using a field emission transmission
electron microscope (FE-TEM, JSM-6500F, JEOL Ltd., Tokyo, Japan),
operating at an accelerating voltage of 5 kV.
2.3. Synthesis of citrate-capped silver nanoparticles
Citrate-capped AgNPs were prepared based on the previously
reported method using ascorbic acid as the reducing agent and
citrate as the stabilizer [30]. Briefly, 8.0 mL of aqueous solution
containing a mixture of ascorbic acid (final concentration of
0.6 mM) and trisodium citrate (final concentration of 3.0 mM) was
prepared, and the pH adjusted to 10.5 with 0.1 M NaOH. To this
solution, 80 mL of 0.1 M AgNO3 aqueous solution was added slowly
with gentle, continuous stirring at 30  C for 15 min. The color of the
solution changed from colorless to yellow immediately upon
addition of AgNO3. The resulting yellow-colored, cc-AgNP colloids
were used to detect and quantitate creatinine following adjustment
of the pH of the colloids to 12 with 600 mM NaOH solution (final
NaOH concentration: 12 mM).
2.4. General procedure for detection and quantitation of creatinine
Detection and quantitation of creatinine was performed at room
temperature. Briefly, 10 mL of 600 mM NaOH solution was added
into 480 mL of undiluted cc-AgNPs to adjust the pH of the colloids to
12. Subsequently, 10 mL of various concentrations of creatinine were
added to the alkaline cc-AgNPs colloids. After incubating for 1 min,
50 mL aliquots of the various creatinine/cc-AgNP colloid mixtures
were diluted with deionized water to a final volume of 1 mL and
analyzed by UV-visible spectrophotometry.
2.5. Urine analysis
The detection and quantitation of creatinine in human urine was
performed using a similar procedure to that employed for the
detection of creatinine in aqueous solutions. Briefly, 15 mL of urine
was sampled from a healthy male volunteer and diluted with 15 mL
deionized water. Thereafter, 10 mL of the diluted urine specimen
was mixed with 490 mL of alkaline cc-AgNP colloids. The urine/cc-
AgNP colloid mixture was incubated for 1 min at room tempera-
ture and analyzed by UV-visible spectrophotometry.
3. Results and discussion
3.1. Properties of cc-AgNPs
The initial yellow-colored cc-AgNPs colloid exhibited a narrow
LSPR band centered around 403 nm. This is indicative of the for-
mation of homogeneously dispersed cc-AgNPs with relatively
smaller diameters. TEM analysis revealed that the cc-AgNPs
exhibited quasi-spherical morphology and a size distribution of
27.4 ± 5.4 nm (Fig. 2A). The LSPR and size distribution characteris-
tics of the cc-AgNPs synthesized in this study were similar to those
reported by Qin et al. [30]. The relatively small sizes of the nano-
particles can be explained by the redox potential of the precursor
solutions, with specific reference to silver, ascorbic acid and the pH
of the solution. As the pH increases, the redox potential of ascorbic
acid decreases, whereas that of silver remains constant. The
increasing difference between the redox potentials of ascorbic acid
and silver, in turn, makes the reaction proceed faster. Thus, at
higher pH, more silver nuclei are formed, albeit with smaller sizes
[30,31]. The synthesized cc-AgNPs colloids remained stable for
more than one month when stored in the dark at 4  C.
3.2. Optimization of the creatinine detection assay
The interaction of creatinine with AgNPs is highly dependent on
the pH of the reaction mixture. In the presence of sodium hy-
droxide, AgNPs interact preferentially with creatinine as opposed
to other chemical species such as uric acid [14], which is likely due
to the pH-dependent tautomerization of creatinine. Here, the effect
of pH on the properties of cc-AgNP colloidal solutions, with appli-
cation to the detection of creatinine, was investigated systemati-
cally. Initially, three distinct mixtures of cc-AgNP colloids
containing (a) NaOH only (12 mM NaOH final concentration, pH
12); (b) 4.5 mM creatinine only; or (c) NaOH (12 mM NaOH final
concentration, pH 12) plus 4.5 mM creatinine were prepared sepa-
rately, and changes in color and LSPR bands of each were assessed
after 1 min of incubation at room temperature (Fig. 1). In mixtures
(a) and (b), neither the color, nor the LSPR bands of the colloids
changed relative to the pure cc-AgNPs colloid, which confirmed
that neither NaOH (12 mM, pH 12), nor creatinine alone (at pH 6.6),
respectively, could induce aggregation of the cc-AgNPs, in agree-
ment with the study by Mohammadi et al. [29]. The absence of
aggregation of the cc-AgNPs in the presence of NaOH (12 mM, pH
12) alone can be explained by the negatively-charged state of cit-
rate resulting from complete deprotonation of all three carboxylic
groups at alkaline pH (pKa1: 3.13, pKa2: 4.76, pKa3: 6.40 for citrate),
thus ensuring strong electrostatic repulsion between the individual
cc-AgNPs. Moreover, El Badawy et al. specifically demonstrated that
the zeta potential of cc-AgNPs decreases from 40 mV to ~-60 mV
as the pH increases from 2 to 10, at ~10 mM NaCl ionic strength,
again without aggregation of the cc-AgNPs [32]. These observations
confirm that Naþ and OH ions themselves do not induce aggre-
gation of the cc-AgNPs at a concentration of 12 mM.
In the third mixture (c), however, a significant color change of
the cc-AgNP colloids from yellow to dark blue was observed (Fig. 1,
inset). In addition, the decrease in the LSPR band centered around
403 nm, with a concomitant red shift in the LSPR band to 670 nm
were observed (Fig. 1, spectra). The decrease in the LSPR band
centered around 403 nm reflects a reduction in concentration of
individually-dispersed cc-AgNPs in suspension, while the red-shift
in the LSPR band to 670 nm reflects an increase in cc-AgNP particle
0.6
0.5
(a) (b) (c)
0.4
Extinction (arb. unit)
(a)AgNPs+NaOH
0.3 (b)AgNPs+creatinine
(c)AgNPs+creatinine+NaOH
0.2
0.1
0.0
350 400 450 500 550 600 650 700 750 800
Wavelength (nm)
Fig. 1. Extinction spectra of cc-AgNP colloids mixed with (a) NaOH only (12 mM final
concentration, pH 12) (b) 4.5 mM creatinine only, or (c) 4.5 mM creatinine and NaOH
(12 mM final concentration, pH 12). The inset picture depicts the cc-AgNP colloids from
which the extinction spectra were recorded under the above-mentioned conditions.
 M.T. Alula et al. / Analytica Chimica Acta 1007 (2018) 40e49
Fig. 2. TEM images of quasi-spherical cc-AgNPs (diameter: 27.4 ± 5.4 nm). (A) cc-AgNPs mixed with creatinine alone remained dispersed, whereas, (B) cc-AgNPs mixed with
creatinine and sodium hydroxide rapidly aggregated. The color inset images depict the corresponding colorimetric appearances of the bulk cc-AgNP colloids. (For interpretation of
the references to color in this figure legend, the reader is referred to the Web version of this article.)
size via aggregation, since the resonant wavelength of two coupled
cc-AgNPs significantly red-shifts relative to that of the individual,
homogeneously-dispersed nanoparticles [33]. Importantly, the
degree of color change, and the decrease in LSPR intensity (around
403 nm) of the colloids were highly dependent on the concentra-
tion of creatinine, as depicted in Fig. 4. It is known that nanoparticle
size increases proportionally with the degree of aggregation.
Therefore, the LSPR wavelength red-shifts with increasing particle
size due to the near-field coupling in the resonant wavelength peak
of the interacting nanoparticles. Collectively, these observations
suggest that, at alkaline pH, creatinine strongly interacts with, and
drives the aggregation of cc-AgNPs.
The ability of creatinine to induce aggregation of cc-AgNPs was
studied further by transmission electron microscopy (TEM). In the
presence of creatinine alone, the cc-AgNPs remained well dispersed
(Fig. 2A), which corresponded with the narrow LSPR band centered
around 403 nm (Fig. 1b). However, intense aggregation of the cc-
AgNPs at alkaline pH occurred only upon addition of creatinine,
as denoted by a rapid color change of the colloid (Fig. 2B), which
corresponds with the red-shift of the LSPR band to 670 nm (Fig. 1c).
These observations clearly demonstrate that trace amounts of
creatinine can induce the aggregation of cc-AgNPs, but, only at
alkaline pH. Therefore, this approach obviates the need for the
laborious surface modification of the AgNPs with molecules such as
picric acid [28] and 2, 20 -thiodiacetic acid [29] to facilitate the
interaction between creatinine and bare AgNPs.
3.3. Effect of reaction time on creatinine-mediated cc-AgNP
aggregation
The effect of reaction time on the interaction between the cc-
AgNPs and creatinine was investigated. Extinction ratios (A670/
A403) were acquired for the cc-AgNPs incubated with different
creatinine concentrations (0.5e10 mM) in alkaline medium (pH 12,
12 mM NaOH final concentration) in 0.5 or 1 min time intervals, up
to 10 min. Regardless of the concentration of creatinine, the ag-
gregation of cc-AgNPs plateaued, albeit to different degrees, within
the first min of reaction i.e. after 1 min, no significant characteristic
changes in either spectral or colorimetric features were observed
for the tested creatinine concentrations. As shown in Fig. 3A, this
effect is most pronounced at both 5 and 10 mM creatinine, whereby
the A670/A403 extinction ratios increased sharply and linearly to
different, concentration-dependent intensities within the first 30 s
43
of reaction, and then remained almost constant over the remaining
duration of observation. A similar trend was also reported by with
TDA-capped AgNPs in response to creatinine [29]. The sudden
decrease in the LSPR band within the first 30 s of reaction, at higher
concentrations of creatinine, depicts the rapid association of the
homogenously dispersed cc-AgNPs into larger nanoparticle aggre-
gates. Fig. 3B clearly illustrates this phenomenon with the extinc-
tion spectra acquired from the mixture of cc-AgNPs and 10 mM
creatinine at various time intervals up to 10 min. Since the aggre-
gation rate was fast, the difference in color changes at the different
time intervals beyond 1 min were difficult to identify colorimetri-
cally (Fig. 3A). Additionally, extended reaction time may result in
precipitation of the cc-AgNPs at higher creatinine concentrations
that, in turn, adversely affects the analytical linear range. Therefore,
1 min was selected as the standard reaction time for subsequent
assay of creatinine both in aqueous solutions and human urine. The
rapid aggregation of the cc-AgNPs at alkaline pH, which occurred
only upon addition of creatinine, implies an alkaline-dependent,
chemical conformation change of creatinine that specifically
drives the aggregation of the cc-AgNPs. Importantly, Qin et al. [30]
demonstrated that the cc-AgNPs remained homogeneously
dispersed and exhibited progressive blue-shifting of the LSPR band
when incubated in increasingly alkaline pH at 100  C for 2 h, which
the authors attributed to an increase in sphericity of the cc-AgNPs
due to Ostwald ripening. Furthermore, the method employed here
to synthesize the cc-AgNPs results in complete consumption of Agþ
ions after 2 min of reaction at pH 10.5 [30]. Taking these factors into
consideration with our experimental conditions (10 min duration
at room temperature and pH 12), our observations are, therefore,
highly unlikely to be artefacts of nanoparticle instability issues, or
the reduction of unreacted Agþ ions to Ag0. Interestingly,
Mohammadi et al. observed that their TDA-capped AgNPs exhibited
maximum absorbance in response to creatinine at pH 10, which
decreased upon addition of either HCl or NaOH [29], whereas pH 12
was optimal for the detection of creatinine in our study. A plausible
reason for the discrepancy between our observations and those of
Mohammadi et al. is that citrate is a triprotic acid, whereas 2, 20 -
thiodiacetic acid is a diprotic acid, and therefore more sensitive to
screening by Naþ ions or protonation by H3Oþ ions, which affects its
ability to hydrogen bond to creatinine.
It is also important to consider the electrical double layer (EDL)
theory, which states that the diffuse double layer of metal nano-
particles decreases in thickness with increasing ionic strength,
44 M.T. Alula et al. / Analytica Chimica Acta 1007 (2018) 40e49

0.6 B 0 min
1.0 A 0.5 min
1 min
0.5 2 min
0.8

Extinction (arb. unit)

3 min
0.4 4 min
5 min
0.6
A670/A403

7 min
0.3 10 min
0.4
0.2

0.2 0.1

0.0 0.0
0 1 2 3 4 5 6 7 8 9 10 11 300 350 400 450 500 550 600 650 700 750 800
Incubation time (min) Wavelength (nm)
Fig. 3. (A) Effect of incubation time on A670/A403 extinction ratio of the cc-AgNP colloids incubated with creatinine concentrations of 0.5 (-) 1.0 ( ), 2.5 ( ), 5.0 ( ) and 10 mM ( ) in
alkaline medium (pH 12, 12 mM NaOH). (B) Extinction spectra of cc-AgNP colloids acquired after mixing and incubating with 10 mM creatinine in alkaline medium (pH 12, 12 mM
NaOH) over a time course of 0e10 min.

Fig. 4. (A) Image of cc-AgNP colloids mixed with different concentrations of creatinine at pH 12 (12 mM NaOH) depicting the colorimetric changes visible up to 10 mM creatinine, (B)
Changes in extinction spectra of cc-AgNP colloids in response to increasing concentrations of creatinine, up to 12 mM at pH 12 (12 mM NaOH), (C) Plot depicting the sigmoidal
relationship between the A670/A403 extinction ratio of the cc-AgNPs colloids and creatinine concentration at pH 12 (12 mM NaOH).

which, in turn, permits a greater degree of particle-particle inter-
action resulting in aggregation of the nanoparticles. However, El
Badawy et al. demonstrated that cc-AgNPs, with similar physico-
chemical properties to the nanoparticles prepared in this study, do
not aggregate at strong alkaline pH, due to a reduction in the zeta
potential of the cc-AgNPs, even in the presence of monovalent ions,
such as, Naþ, Cl and NO 3 , at 10 mM ionic strength [32]. Addi-
tionally, Huyhn and Chen demonstrated that divalent electrolytes
such as, CaCl2 and MgCl2 were more efficient at inducing aggre-
gation of cc-AgNPs at significantly lower concentrations (2.1 mM
and 2.7 mM, respectively) than NaCl (47.6 mM) [34]. In the present
experimental setup, divalent metal ions were absent, while the
ionic strength conferred solely by Naþ ions, upon addition of NaOH
to the cc-AgNPs, was only 12 mM at most, and therefore insufficient
alone to drive the aggregation of the cc-AgNPs.
3.4. Assay sensitivity and detection limit
The sensitivity of cc-AgNPs toward creatinine was determined
by both colorimetry and UV-visible spectrophotometry using a
reaction time of 1 min (Fig. 4AeC). Fig. 4A clearly illustrates the
discernable colorimetric changes of the cc-AgNP colloids in a
creatinine concentration range of 0.5e10 mM. Fig. 4B depicts the
spectral changes of the cc-AgNP colloids in response to increasing
concentrations of creatinine after 1 min of incubation, where a
gradual decrease in extinction intensity at 403 nm with a
 M.T. Alula et al. / Analytica Chimica Acta 1007 (2018) 40e49 45

concomitant increase in extinction intensity at 670 nm was

observed. Fig. 4C depicts the sigmoidal relationship between the 1.0
creatinine concentration and A670/A403 extinction ratio of the cc-
AgNP colloids. Briefly, the A670/A403 extinction ratio increased
gradually and followed a linear correlation with the creatinine 0.8
concentration range of 0e4.2 mM, then increased sharply from 4.2
to 5.5 mM, and subsequently plateaued above 8 mM creatinine. The
0.6

A670/A403
plateau in the A670/A403 extinction ratio above 8 mM creatinine
denotes saturation of the reaction due to the rapid and complete DF, ×5.33
aggregation of all homogeneously dispersed cc-AgNPs in suspen- 0.4
sion. A linear correlation was obtained between the A670/A403 DF, ×2.53
extinction ratio and creatinine concentration in the range of DF, ×1.66
0e4.2 mM, with a regression coefficient (R2) of 0.996 and the 0.2
DF, ×1.23
following equation;
DF, ×1.00
4 4 0.0
y ¼ 0.01655(±2.3347910 )x þ 0.05286(±2.9501710 ),

where y is the A670/A403 extinction ratio, and x is the concentration 0 2 4 6 8 10

of creatinine. Hence, the highest sensitivity was achieved at 4.2 mM,
and the lower limit of detection for creatinine was determined to be
53.4 nM, based on the standard deviation of the response, and the
slope of the calibration curve obtained from the linear correlation
range for 1 min of incubation. This detection limit is far below the
typical creatinine concentration range in human urine of
1.77e26.52 mM (20e300 mg/dL) [35]. Hence, our cc-AgNPs sensor
meets the requirement of a lower detection limit for creatinine in
the sub-millimolar range. Importantly, this colorimetric study
confirmed that minute variations in the concentration of creatinine,
within the linear range, elicit discernable colorimetric and UV-
visible spectral changes, specifically, at alkaline pH.
We also investigated the effect of the cc-AgNPs colloid con-
Concentration of creatinine (μM)
Fig. 5. Effect of the cc-AgNPs colloid concentration on the degree of nanoparticle
aggregation induced by creatinine. The efficacy of quantitation of creatinine decreased
as the cc-AgNPs colloid became increasingly dilute. Different concentrations of cc-
AgNP colloids were prepared by diluting the synthesized colloids with ultrapure
deionized water to various dilution factors (DF): 1, 1.23, 1.66, 2.53, and 5.33.
cc-AgNP colloids either remained unchanged, or increased only
marginally relative to the blank (untreated) cc-AgNPs (Fig. 6). It is
relevant to note that the inorganic cations were tested as salts e
Ca2þ, Kþ, and Naþ cations as chloride (Cl) salts, Fe2þ and Fe3þ
cations as sulfate (SO2
4 ) salts, and the Zn
2þ
cation as a nitrate (NO3)

centration on the degree of nanoparticle aggregation induced by salt e none of which elicited changes in the A670/A403 extinction
creatinine and, in turn, its effect on the efficacy of quantitation of ratio relative to the blank (untreated) control. Therefore, it can also
creatinine. Different concentrations of cc-AgNP colloids were pre- be deduced that none of the inorganic anions, Cl, NO 2
3 , and SO4 ,

pared by diluting the synthesized colloids with ultrapure deionized act as interferents to induce cc-AgNPs aggregation at the tested
water to dilution factors (DF) of 1, 1.23, 1.66, 2.53, and 5.33. The concentrations. Only creatinine elicited a significant, ~24-fold in-
A670/A403 extinction ratios for each mixture comprising cc-AgNP crease in the A670/A403 extinction ratio, as well as a yellow-to-blue
colloids (at different dilution factors), and a series of creatinine
standard solutions were studied systematically. The changes in the
A670/A403 extinction ratios were used to assess the degree of cc-
AgNPs aggregation. We observed that the rate of creatinine-
1.0
mediated cc-AgNP aggregation was inversely proportional to the
concentration of the cc-AgNP colloids. Specifically, as the concen-
tration of the cc-AgNP colloids decreased, aggregation occurred 0.8
faster such that all homogeneously dispersed cc-AgNPs aggregated
completely, resulting in a very narrow analytical linear correlation
0.6
A670/A403

range. In contrast, a relatively large linear correlation between the

A670/A403 extinction ratio and creatinine concentration was
observed with the concentrated (undiluted) cc-AgNPs colloid
(Fig. 5). Therefore, undiluted cc-AgNP colloids were used 0.4
throughout this study.

3.5. Assay selectivity

0.2
The selectivity of our method was examined against potentially
interfering inorganic ions and organic molecules, including Ca2þ,
Kþ, Fe2þ, Fe3þ, Naþ, Zn2þ, ascorbic acid, glucose, glutamic acid, urea, 0.0
Urea

Glycine
Uric acid
Ascorbic acid

Creatinine
Glutamic acid
Glucose

Blank

Ca2+ Fe2+ Fe3+ K+ Na+ Zn2+

glycine, and uric acid. Solutions of each chemical analyte were
prepared in ultrapure deionized water to a final concentration of
10 mM, then incubated individually with cc-AgNP colloids at pH 12
for 1 min, and analyzed for their ability to induce cc-AgNP aggre-
gation. Under these conditions, none of the tested chemical ana-
Fig. 6. Selectivity of the cc-AgNPs assay toward creatinine illustrated by comparison of
lytes elicited discernable colorimetric changes of the colloids in the the changes in A670/A403 extinction ratios of the cc-AgNP colloids after incubation with
absence of creatinine. Additionally, the A670/A403 extinction ratio various inorganic and organic species (10 mM), and creatinine (10 mM) under alkaline
for each inorganic/organic analyte incubated individually with the conditions (pH 12).
46 M.T. Alula et al. / Analytica Chimica Acta 1007 (2018) 40e49
colorimetric change of the cc-AgNPs colloid relative to the blank
control (Fig. 6). It is also important to note that only high purity
creatinine (98% purity; Alfa Aesar) was used in this study, which
contained no contaminating ions that could potentially induce cc-
AgNPs aggregation. These observations are consistent with those
of Parmar et al., who tested the same set of inorganic and organic
analytes investigated in this study, each at 10 mM, against picric
acid-capped AgNPs, but also observed selectivity of their sensor
only toward creatinine [28]. Importantly, uric acid did not elicit any
change in the A670/A403 ratio compared to the blank control, due to
both uric acid and the cc-AgNPs being negatively-charged at alka-
line pH, which ensures electrostatic charge repulsion between the
two. This is a significant improvement on the study by Alula and
Yang [14] in which the authors developed Ag@ZnO/Fe3O4 nano-
particle composites to detect creatinine in urine by SERS, but also
observed significant interference from uric acid in the samples.
In summary, these observations confirm that the organic and
inorganic analytes tested here do not interfere with our assay, due
to their inability to induce cc-AgNP aggregation at equimolar con-
centrations, further validating the selectivity of the cc-AgNPs to-
ward creatinine under alkaline conditions.
3.6. Detection of creatinine in human urine
To demonstrate the practicality of our method, the sensor was
further validated by detecting creatinine directly in a human urine
sample. The urine sample was initially tested without further
pretreatment to remove the urinary matrix, which reportedly in-
hibits aggregation of the nanoparticles [27]. In this assay, however,
the undiluted urine triggered intense aggregation of the cc-AgNPs,
such that rapid color change was observed, thereby impeding
quantitative analytical measurements. Hence, dilution of the urine
sample was necessary to reduce the concentration of creatinine and
other matrix components present in the urine sample such that
changes in the A670/A403 extinction ratio and colorimetric appear-
ance of the cc-AgNPs fell within the linear regression range of the
assay.
Different concentrations of creatinine were spiked into the
diluted urine sample, based on the standard addition method, and
subsequently analyzed via the same protocol applied to the quan-
titation of creatinine in pure aqueous solutions, as described earlier.
A diluted urine sample, without spiked creatinine, served as a
control to confirm that the additional changes in the colorimetric
A 0 μM
0.6
Extinction (arb. unit)
0.5
0.4
0.3
0.2
0.1
0.0
400 500 600
Wavelength (nm)
Fig. 7. (A) Extinction spectra of cc-AgNP colloids mixed with urine samples that were spiked with different concentrations of creatinine. The black spectrum corresponds to the
diluted urine sample without spiked pure creatinine (0 mM). (B) Calibration curve for the quantitation of creatinine in urine samples generated by the standard addition method.
appearance and A670/A403 extinction ratio of the cc-AgNP colloids
were actually attributable to the spiked creatinine, over and above
the signal changes elicited by the endogenous creatinine of the
urine sample. The duration of reaction, the changes in colorimetric
appearance as well as the A670/A403 extinction ratio observed for
the cc-AgNP colloids mixed with creatinine-spiked, diluted urine
samples, and diluted urine samples without spiked creatinine were
all similar to that of aqueous creatinine samples. Specifically, the
A670/A403 extinction ratio of the cc-AgNPs increased linearly with
the concentration of spiked creatinine in the diluted urine samples,
which was observed similarly for the pure aqueous creatinine so-
lutions (Fig. 7A). Therefore, these observations suggest that the
components of the urinary matrix had been diluted sufficiently
such that they did not interfere with the interaction between
creatinine and the cc-AgNPs under alkaline conditions.
An 8-point standard addition method was used to quantify
endogenous creatinine in the urine sample (Fig. 7B). A linear cor-
relation was obtained between the A670/A403 extinction ratio and
the spiked creatinine concentration, with a regression coefficient
(R2) of 0.9803 and the following equation;
y ¼ 0.06841(±3.49103) x þ 0.06603(±3.48103),
where y is the A670/A403 extinction ratio, and x is the concentration
of spiked creatinine.
Extrapolation of the linear correlation curve in Fig. 7B to the x-
intercept (y ¼ 0) yields a value of 0.965 ± 0.002 mM creatinine and
therefore an actual urinary creatinine concentration of 965 ± 2 mM
(0.965 ± 0.002 mM) in the tested sample (based on a 1000-fold
dilution of the sample), which is close to the lower end of the
typical reported urine creatinine concentration range
(1.77e26.52 mM) [35]. Thus, this result demonstrates the practical
application of our cc-AgNP sensor to the detection and quantitation
of creatinine in clinical urine samples with the necessary sensitivity
and specificity. The advantages and low limit of detection of our
assay are highlighted in the context of the previously reported
methods for the detection of creatinine in Table 1.
3.7. Proposed mechanism of creatinine-mediated cc-AgNP
aggregation at alkaline pH
Before describing the plausible mechanism by which creatinine
mediates aggregation of the cc-AgNPs at alkaline pH, it is necessary
0.3 μM
0.5 μM 0.25 B
0.75 μM
1.0 μM
1.5 μM 0.20
A670/A403
2.0 μM
2.5 μM 0.15
3.0 μM
3.5 μM
4.0 μM 0.10
0.05
700 800 0.0 0.5 1.0 1.5 2.0 2.5
Concentration of spiked creatinine (μM)
 M.T. Alula et al. / Analytica Chimica Acta 1007 (2018) 40e49
Table 1
Comparison of the present method with some previously reported methods employed in the detection of creatinine in urine.
No. Technique Limit of
Detection
(mmol L1)
1 High performance liquid chromatography 1.8
2 Amperometric sensor 0.6
3 Capillary electrophoresis method 4.4
4 Gold nanoparticles-based colorimetric 80
assay
5 Gold nanoparticles-based detection after 121.2
solid phase extraction
6 Simple, rapid, sensitive, and selective 0.066
sensor based on citrate-capped silver
nanoparticles
to briefly review the nature of the surface interaction between
citrate molecules and the AgNPs. Frost et al. demonstrated, by
attenuated total reflectance Fourier transform infrared (ATR-FTIR)
spectroscopy, that two carboxylate groups as well as the hydroxyl
group of citrate bind to the surface of AgNPs (Fig. 8) [36]. Since two
of the three carboxylate groups of each surface-interacting citrate
molecule are bound to the surface of the AgNP, only the remaining
carboxylate group is available to contribute to the negative surface
charge of the AgNP. At mildly acidic conditions (pH 6.6 in this
study), all carboxylate groups of citrate are fully deprotonated
(pKa1: 3.13, pKa2: 4.76, pKa3: 6.40), and remain fully deprotonated at
alkaline conditions (pH 12 in this study) (Fig. 8), which sustains the
electrostatic repulsion between the individual cc-AgNPs, thus
maintaining their stabilization in a homogeneously dispersed state
[32].
Creatinine has a pKa1 of 4.9 and pKa2 of 9.2 [37]. At pH values
close to its pKa1, creatinine rapidly interconverts between its two
neutral tautomers i.e. 2-amino-1-methyl-imidazoline-4-one
(amino tautomer) and 2-imino-1-methyl-imidazolidine-4-one
(imino tautomer) [38], with both tautomers capable of inter-
converting with the creatininium cation as well, as observed by
Raman spectroscopy [39] (Fig. 8). Furthermore, Gao et al. demon-
strated that creatinine also exists in equilibrium between its amino,
imino, and creatininium cation tautomers at pH 6 [39]. In the
present study, we observed that creatinine failed to induce aggre-
gation of the cc-AgNPs at pH 6.6, where the amino, imino, and
creatininium cation tautomers are still likely at equilibrium with
each other. Although the amino, imino and creatininium cation
tautomers can form hydrogen bonds with the single exposed
carboxylate group of each surface-adsorbed citrate molecule, these
intermolecular hydrogen bonds are likely to be only transient due
to the rapid interconversion between these tautomeric forms of
creatinine (Fig. 8).
Above neutral pH, however, Antoniou et al. observed by mass
spectrometry that the anionic species of creatinine became
increasingly prevalent as the pH increased, which reached a
maximum ion count (~3290) at pH 11.1, followed by a sharp
decrease in ion count at higher pH (~500 at pH 12.5) [37]. Fig. 8
(adapted from Antoniou et al.) depicts the formation of the carb-
anion and oxoanion species exclusively from the amino tautomer of
creatinine at alkaline pH [37]. At pH 12 e at which we observed
creatinine-mediated aggregation of the cc-AgNPs e the carbanion
and oxoanion species of the amino tautomer would still be present,
albeit at ~46% of the maximum level at pH 11.1, according to the
47
Comments Ref
Good linear response achieved with an acceptable [3]
concentration range at physiologically-relevant levels.
Based on the formation of copper-creatinine complex on a [6]
copper electrode surface. Uric acid was found to interfere
with the assay.
Laborious deproteination and centrifugation procedures [10]
were employed.
The detection limit is high and procedure is costly. [25]
Laborious solid phase extraction was employed to [27]
overcome urinary matrix interferences.
The method requires no additional laborious surface Present work
modifications of silver nanoparticles or conjugation with
other linker molecules, and yielded a wide linear correlation
between the cc-AgNPs extinction ratio and concentration of
creatinine.
findings by Antoniou et al. The amino tautomer, which is the most
thermodynamically stable form of creatinine [40], is therefore
favored at alkaline pH via formation of the carbanion and oxoanion
species, with the negative charge localized over the C5 or O8 atoms,
respectively (Fig. 8). Thus, stable hydrogen bonds likely form be-
tween the carbanion/oxoanion amino tautomers and the single,
exposed negatively-charged carboxylate group of the citrate caps.
Furthermore, Naþ ions can electrostatically interact with the
carbanion/oxoanion species of the surface bound creatinine tau-
tomers, which further screen the negatively-charged citrate caps of
the cc-AgNPs from each other. As a result, an intermolecular
hydrogen bonding network would form between surface-bound
creatinine molecules that effectively ‘cross-link’ the interacting
cc-AgNPs, thus leading to aggregation. It should also be mentioned
that Gangopadhyay et al. recently demonstrated, by SERS-based
reaction monitoring, the decyclization of creatinine to creatine at
pH 12; however, this was observed only after 30 min of reaction
[41]. Given the reaction time of 1 min at pH 12 in our study,
creatinine decyclization is therefore not a likely cause of the
creatinine-mediated aggregation of the cc-AgNPs.
4. Conclusions
The detection and quantitation of creatinine in urine specimens
is an important test that enables clinicians to diagnose renal
dysfunction occurring in a broad spectrum of diseases. The ability
to rapidly and reliably quantitate creatinine with high sensitivity
and specificity in a complex specimen matrix such as urine, without
sophisticated analytical equipment, is especially useful in low-
resource settings. To address this need, we developed a simple
and inexpensive, colorimetric citrate-capped AgNP sensor, which
demonstrated sensitive and selective detection of creatinine in
aqueous solutions and in human urine within one minute. The
detection mechanism relies on the creatinine-mediated aggrega-
tion of the cc-AgNPs at pH 12, which elicits a visibly-detectable
color change. The ccAgNP sensor exhibited a linear correlation
between the A670/A403 extinction ratio and creatinine concentra-
tion range of 0e4.2 mM (R2 ¼ 0.996), and a low detection limit of
53.4 nM. Importantly, this approach requires no additional surface
modification of the AgNPs, or conjugation with other linker mole-
cules that would render synthesis of the AgNPs laborious and
detection complicated. Furthermore, neither sample pretreatment,
nor selective extraction of creatinine are required to facilitate its
detection in urine with our method. The advantages of simplicity,
48 M.T. Alula et al. / Analytica Chimica Acta 1007 (2018) 40e49

Fig. 8. Schematic diagram depicting the pH-dependent tautomerism of creatinine, and its bonding interactions with citrate-capped AgNPs under mild acidic, and alkaline con-
ditions. Aggregation of the cc-AgNPs, at pH 12, is mediated by formation of a hydrogen bonding network between the amine group of the carbanion/oxoanion amino tautomers of
creatinine and the negatively-charged, carboxylate group of the surface-adsorbed citrate molecules. Additionally, electrostatic interactions between Naþ ions and the carbanion/
oxoanion amino tautomers of creatinine further screen the negatively-charged citrate groups between individual cc-AgNPs in close proximity.

short assay duration, high sensitivity and selectivity demonstrate Partnership. LK thanks the NRF (Grant No. 99517) for a postdoctoral
the utility of our cc-AgNP sensor as a creatinine monitoring assay innovation fellowship; MTA and NRH were supported by post-
for low-resource, point-of-care settings. doctoral fellowships from the SAMRC.

Acknowledgements References

JMB thanks the National Research Foundation (NRF) for a South
African Research Chair grant (Grant No. 64760). This research was
supported by a grant to JMB from the South African Medical
Research Council's (SAMRC) Strategic Health Innovation
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