Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 270 (2022) 120816

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

A copper nanoclusters probe for dual detection of microalbumin and

creatinine
Supitcha Thammajinno a,b,c, Chittanon Buranachai a,b,d, Proespichaya Kanatharana a,b,c,
Panote Thavarungkul a,b,c,d, Chongdee Thammakhet-Buranachai a,b,c,⇑
a
Division of Physical Science, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand
b
Center of Excellence for Trace Analysis and Biosensor, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand
c
Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand
d
Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400, Thailand

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

 Glutathione capped copper

nanoclusters probe was synthesized
with ease.
 The single probe is capable of dual
detection to obtain albumin to
creatinine ratio.
 The probe shows excellent
performance in real-urine analysis.

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

Article history: A fluorescent probe based on glutathione-capped copper nanoclusters (GSH-CuNCs) was developed for
Received 20 September 2021 the detection of dual targets, human serum albumin (HSA) and creatinine, in human urine. The GSH-
Received in revised form 21 December 2021 CuNCs were synthesized by a one-pot green method using ascorbic acid as a reducing agent. The detec-
Accepted 23 December 2021
tion of HSA was in a turn-on mode via electrostatic interaction in a basic condition while the detection of
Available online 29 December 2021
creatinine was in a turn-off mode via non-covalent bonding in an acidic condition. Under optimal condi-
tions, the linear range and detection limit of HSA were 5.0 nM to 150 nM and 1.510 ± 0.041 nM, while
Keywords:
those of creatinine were 30 lM to 1000 lM and 13.0 ± 1.0 lM. This easily fabricated nanocluster probe
Glutathione
Copper nanoclusters
provided a fast response with high sensitivity, and good selectivity. Recoveries from urine samples were
Human serum albumin in the range of 81.44 ± 0.25 to 109.22 ± 0.57% for HSA and 80.57 ± 0.16 to 109.0 ± 0.10% for creatinine. The
Creatinine urinary analytical results from the fluorescent probe were in good agreement (P > 0.05) to those obtained
Albumin to creatinine ratio from immunoturbidimetric and enzymatic methods, signifying the excellent performance of this sensing
Fluorescence detection system.
Ó 2021 Elsevier B.V. All rights reserved.

1. Introduction
A common practice in biosensor and chemical sensor develop-
ment is to focus on detecting one analyte with the highest sensitiv-
ity, good selectivity and the lowest detection limit. However, some
⇑ Corresponding author at: Division of Physical Science, Faculty of Science, Prince
of Songkla University, Hat Yai, Songkhla 90110, Thailand.
E-mail address: chongdee.t@psu.ac.th (C. Thammakhet-Buranachai).
applications involve detecting more than one type of target ana-
lyte, which requires more than one sensor construct to handle. A
well-known example is human serum albumin (HSA) and crea-
tinine detection in urine samples to detect early kidney damage
[1]. Urinary HSA, or microalbumin, is an indicator of kidney dis-
eases [2]. However, the amount of secreted microalbumin also
depends on food uptake [3] and this complicates the diagnosis. A
solution to this problem is to measure creatinine concomitantly
with microalbumin [4]. Creatinine is another biomarker for renal

https://doi.org/10.1016/j.saa.2021.120816
1386-1425/Ó 2021 Elsevier B.V. All rights reserved.
S. Thammajinno, C. Buranachai, P. Kanatharana et al.
dysfunction. It is the final product of creatine metabolism in skele-
tal muscles [5] and is excreted by kidney at a relatively constant
rate [6] in healthy people independent of food uptake [7]; there-
fore, biomarkers in urine are often measured against the concen-
tration of creatinine. Renal dysfunction gives rise to a reduction
in creatinine excretion rate [8]. Accordingly, measuring the con-
centrations of microalbumin and creatinine concomitantly as albu-
min creatinine ratio (ACR) provides a more reliable diagnosis for
kidney malfunction. For example, urine ACR has been widely used
as an indicator to identify kidney disease that can occur as a com-
plication of diabetes [4] or chronic kidney disease [9].
Microalbumin and creatinine can be measured by various con-
ventional analytical methods, such as high-performance liquid
chromatography (HPLC) [10,11] and liquid chromatography (LC)
[12,13]. These methods provide high selectivity but they need high
cost of equipment, require well-trained professionals with involve
several environmentally toxic solvents. In clinical laboratories gen-
erally use immunoassays, among which most are immunoturbidi-
metric assays (ITA) for HSA detection [14] and enzymatic method
applied with creatinine detection [15]. These methods required
the labeling of antibody-based assays and enzymes. Although the
introduction of antibody and enzymes have improved the sensitiv-
ity and selectivity, but it is a high cost and instable of enzymes
[2,16]. Among them, the electrochemical and optical sensors are
considered the two most popular types. Electrochemical sensors
for microalbumin and creatinine detections are highly sensitive
with extra low detection limit and exceptional selectivity [17,18],
however, they are prone to electrical interference and often require
complicate electrode preparation to avoid interference. Optical
sensors offer comparable sensitivity and selectivity but are less
susceptible to external interference and are easier to construct
when compared with the electrochemical type [19,20]. Neverthe-
less, most of the existing sensors is still capable of detecting only
either microalbumin or creatinine but rarely both. In this work
we address the problem by developing a single fluorescence sensor
for a dual detection of microalbumin and creatinine based on cop-
per nanoclusters.
Metallic nanoclusters (NCs) contain a few to several hundred
metal atoms [21]. Therefore, the electrons are well confined and
the clusters become fluorescent [22]. Similar to quantum dots,
metallic NCs have high extinction coefficient and are highly resis-
tant to photobleaching but they are less toxic and easier to synthe-
size [23]. Among various types, copper NC is interesting because it
is relatively inexpensive and easy to synthesize [24,25] and after
stabilized with proper capping agents [21,26,27], the interaction
of CuNCs with certain target analytes can either increase or
decrease their fluorescence intensity [28,29]. For example, a prior
publication reports that xanthine (XA) can bind to the surface of
glutathione-stabilized copper nanoclusters (GSH-CuNCs) via NACu
bond and increase the surface electron density resulting in an
enhancement of fluorescence intensity (turn-on mode) [30], while
rifampicin (RFP), after binding, quenches the fluorescence of
glutathione-stabilized copper nanoclusters (GSH-CuNCs) via the
inner-filter effect (turn-off mode) [31]. Therefore, it is possible to
engineer a CuNCs probe using a proper capping agent, such as
GSH, under suitable sensing conditions to detect more than one
target analyte using different detection modes (turn-on vs turn-
off) without the complications of using separate detection methods
or different sensor development.
In this work, we report the development of a single GSH-CuNCs
fluorescent probe for the detection of dual target analytes, microal-
bumin and creatinine, in order to obtain the ACR values. Our
hypothesis was based on a fact that at a pH in the range of 7.0–
8.0, the GSH is positively charged (pKa of amine group on
GSH = 9.49) [32] while HSA is negatively charged (isoelectric
points (pI) of HSA = 4.7) [33]. Therefore, GSH-CuNCs could interact
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 270 (2022) 120816
with HSA via electrostatic force inducing an aggregation of the
clusters and the enhancement of fluorescence intensity (‘‘turn-
on”) as seen in some prior works [34,35]. The phenomenon of
aggregation-induced emission was occurred under certain condi-
tions such as solvent, ions, pH, and special substances and leads
to aggregation of CuNCs with a strong fluorescence because of
the intramolecular rotation and vibration of NCs are inhibited.
Therefore, the non-radiative pathways were blocked, and mean-
while, the radiative decay was activated in the molecular aggregate
state, resulting by increase in fluorescence intensity of NCs [36]. On
the other hand, at an acidic pH, creatinine is neutral and could
interact with GSH-CuNCs via non-covalent bonding [37]. The com-
plex formation could then quench CuNCs fluorescence (‘‘turn-off”)
because of, e.g. electron transfer between creatinine and nanoclus-
ters [38].
We synthesized the GSH-CuNCs and characterized the interac-
tions between the nanoclusters and the two target analytes (HSA
and creatinine). Afterwards, the affecting parameters of the pro-
posed sensing method were optimized to obtain the highest sensi-
tivity and specificity before validating the sensing performance
and applying for the determination of the two target-analytes in
real urine samples. The results were also compared with those
obtained from conventional methods used by the hospital.
2. Experimental section
2.1. Materials
Chemicals were obtained from manufacturers as follows. Cop-
per (ΙΙ) chloride dehydrate (CuCl2
2H2O) was from Merck (Darm-
stadt, Germany). L-glutathione (GSH) and L-cysteine were from
Sigma-Aldrich (Tokyo, Japan). L-ascorbic acid, dopamine
hydrochloride, creatinine and human serum albumin were from
Sigma-Aldrich (Darmstadt, Germany). Sodium dihydrogen phos-
phate dihydrate (NaH2PO4
2H2O) and sodium phosphate dibasic
dodecahydrate (Na2HPO4
12H2O) were from Ajax Finechem (Auck-
land, New Zealand). Sodium acetate trihydrate (C2H3NaO2
3H2O)
was from AnalaR NORMAPUR (Haasrode, Belgium). Sodium chlo-
ride (NaCl) was from Merck (Copenhagen, Denmark). Uric acid
was from Sigma-Aldrich (Budapest, Hungary) and urea was from
Riedel-de Haën (Bucharest, Romania). All aqueous solutions were
prepared with deionized water (18.2 MX
cm resistivity) from a
water purification system (Milli-Q water Direct, Millipore Corp.,
Billerica, MA, USA).
2.2. Preparation of GSH-CuNCs
The preparation procedure was adapted from the method of
Huang and co-workers [39]. Briefly, 100 mL of 0.20 mM GSH solu-
tion were mixed under vigorous stirring with 312 lL of a 0.050 M
solution of CuCl2 in DI water. The colorless solution immediately
changed to a white emulsion. Then, 2.25 mL of 0.10 mM ascorbic
acid were added dropwise to the white emulsion, which became
a clear solution after 30 min. The clear solution was continuously
stirred for 4 h at 65 °C to obtain a yellow solution of CuNCs, which
was stored in darkness at 4 °C without further purification. To ver-
ify that the synthesis was successful, the UV–vis absorption spectra
of the nanocluster solution was measured by an absorption spec-
trometer (Avaspec 2048, Apeldoorn, The Netherlands).
2.3. Characterization of CuNCs
The morphology and size of CuNCs were studied under trans-
mission electron microscopy (TEM) (JEOL, JEM 2010 system, Tokyo,
Japan) operating at 200 kV. The specimens were prepared by
S. Thammajinno, C. Buranachai, P. Kanatharana et al.
dropping 3.50 lL of the CuNCs solution onto a carbon coated cop-
per grid and then dried at room temperature prior to imaging. In
addition, a portion of the nanocluster solution was lyophilized
and the solid sample was dispersed in KBr discs prior to character-
izing the chemical composition using a Fourier transform infrared
(FT-IR) spectrometer (VERTEX 70, Bruker, Germany). Moreover, the
zeta potential of the GSH-CuNCs in the presence and absence of
HSA and creatinine were evaluated by a particle analyzer (Zeta-
PALS system, Brookhaven Instruments, New York, USA) to verify
the surface charge.
To confirm the success of the synthesis, cyclic voltammetry (CV)
was used to verify that Cu2+ were reduced to Cu+ and Cu0 to pro-
duce CuNCs. The CV was performed using an lAUTOLAB Type III
potentiostat galvanostat (Metrohm Autolab B.V., Utrecht, The
Netherlands) controlled by a Nova 2.1.1 software using a standard
three-electrode cell at room temperature. A glassy carbon elec-
trode (3.0 mm in diameter) as a working electrode was first pol-
ished with 1.0 lm and then 0.30 lm alumina slurries before
being sonicated successively in ethanol and water. A platinum wire
as a counter electrode and Ag/AgCl (in saturated KCl) as a reference
electrode were dipped in 0.10 M NaOH as an electrolyte.
In the part of modification on glassy carbon electrode (GCE),
chitosan cryogel (prepared by freezing and thawing of chitosan
[40]) was used to entrap the CuNCs. Briefly, 6.0 mg of the lyophi-
lized CuNCs was added into 500 lL of chitosan solution (2.0% w/
v) in an amber glass bottle (4.0 mL). The mixture was stirred over-
night at room temperature. Next, 25.0 lL of this mixture was com-
bined with 1.0 lL of 2.5% (v/v) of glutaraldehyde, used as the cross-
linking reagent. Five lL of this mixture was then drop-casted onto
a glassy carbon electrode and stored at 20 °C overnight to achieve
cryogelation and then thawed at 4.0 °C for 30 min to obtain a
CuNCs-CS cryogel/GCE [41,42]. In addition, a chitosan cryogel
modified glassy carbon electrode without CuNCs (CS cryogel/
GCE) was also prepared for comparison with the CuNCs-CS cryo-
gel/GCE.
2.4. Steady state and lifetime-resolved fluorescence measurement
All steady state fluorescence measurements were done in a
spectrofluorometer (RF-5301, Shimadzu, Tokyo, Japan). For HSA
detection, the GSH-CuNCs stock solution was diluted and mixed
with HSA standard stock solution at a desired concentration in
phosphate buffer at a basic pH. The dilution was done with a factor
that kept the fluorescence intensity of the blank (i.e. no HSA) low
(70 a.u. out of the maximum intensity limit of the instrument
at 1000 a.u.), because HSA detection was done in a turn-on mode;
the fluorescence intensity increases with increasing the HSA con-
centration. For creatinine detection, the GSH-CuNCs stock solution
was diluted with acetate buffer and mixed with creatinine stan-
dard stock solution at a desired concentration at an acidic pH with
a different factor from that used in HSA detection to ensure that
the initial fluorescence intensity from blank solution (i.e. no crea-
tinine) was high (165 a.u.). This is because creatinine detection
was done in a turn-off mode. Then, the mixture was transferred
into a quartz microcuvette for fluorescence measurement (Starna
Scientific Ltd., Essex, U.K.) and its fluorescence spectra was
recorded with the excitation wavelength at 360 nm and the emis-
sion wavelengths ranging from 380 to 700 nm and slid widths of
5 nm for excitation and 10 nm for emission. Finally, the integrated
fluorescence intensity of GSH-CuNCs in the presence (F) and
absence (F0) of the target analytes (either HSA or creatinine) were
calculated. F/F0 was used as the signal for HSA detection, whereas
F0/F was used as the signal for creatinine detection.
To clarify the quenching mechanism, the fluorescence lifetimes
was employed to explore the mechanism of the fluorescence
response of GSH-CuNCs towards creatinine. The GSH-CuNCs solu-
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 270 (2022) 120816
tions in the absence and presence of creatinine were measured
under a time-resolved fluorometer using a 370 nm pulsed laser
as the excitation light source (FLS 980, Edinburgh Instruments, Liv-
ingston, United Kingdom).
2.5. Optimization of operational conditions
Optimization was performed by changing a single parameter at
a time while other parameters were kept constant. Standard solu-
tions of HSA (5.0–150 nM) and creatinine (30–1000 lM) were pre-
pared and analyzed (three replicates for each tested condition). The
F/F0 and F0/F of HSA and creatinine standards were plotted against
their respective concentrations. The optimal value was the one that
provided the steepest slope (sensitivity) of the calibration plot, the
shortest analysis time and the lowest limit of detection (LOD) and
limit of quantification (LOQ).
2.6. Analysis of real samples
Urine samples were kindly provided by Songklanagarind Hospi-
tal (Hat Yai, Songkhla, Thailand). The samples collecting and han-
dling protocol met the criteria of the Exemption Determination
as was acknowledged by Human Research Ethics Committee of
Faculty of Medicine, Prince of Songkla University (REC.63-510-
19-2). They were diluted with an appropriate dilution factor for
HSA and creatinine detection using 0.15 M phosphate buffer at
pH 7.4 and 0.10 M acetate buffer at pH 5.0, respectively, then ana-
lyzed under the optimal conditions. The obtained concentrations of
HSA and creatinine were statistically compared by the Wilcoxon
signed rank test to those obtained from the immunoturbidimetric
and enzymatic standard methods used by the hospital.
3. Results and discussion
3.1. Characterization of GSH-CuNCs
The UV–vis absorption spectrum of GSH in DI water (Fig. 1b)
showed no absorption peak while the spectrum of GSH-CuNCs in
DI water showed a sharp peak at 292 nm that indicated the suc-
cessful synthesis of GSH-CuNCs. The absence of a plasmon band
of Cu nanoparticles at 507 nm in this spectrum indicated that the
synthesized GSH-CuNCs were of a high purity [25,39]. Under UV
light at 365 nm, the as-prepared GSH-CuNCs solution emitted an
intense blue fluorescence (Fig. 1b cuvette I) which was compared
with the non-fluorescence of DI water (cuvette II). When the exci-
tation wavelength was varied from 300 to 370 nm at 10 nm incre-
ments, the successive peaks of fluorescence emission occurred at
the same wavelength of 428 nm. The maximum intensity of fluo-
rescence emissions occurred at the excitation wavelength of
360 nm (Fig. 1c) and this was used as the excitation wavelength
for the remaining experiments.
The molecular structures of GSH and GSH-CuNCs were charac-
terized by FT-IR spectroscopy. In the FTIR spectrum of GSH
(Fig. 1d), absorption peaks of the ANH2 group were observed at
3348 and 3250 cm 1 for NAH, and 3026 cm 1 for CAH stretching
vibration bands. Peaks around 3000 cm 1 were attributed to the
absorption bands of OAH stretching. Characteristic peaks were also
presented at 2525 cm 1 for the SAH stretching vibration of the
ASH group while the peak at 1715 cm 1 was attributed to the
C@O stretching vibration of the ACOOH group. The sharp NAH
stretching bands, present in the spectrum of GSH at 3348 and
3250 cm 1, were not presented in the spectrum of GSH-CuNCs.
Instead, a wide band presented at about 3406 cm 1, which should
be attributed to the absorption of the associated hydroxyl (AOH)
and amino (ANH2) groups. The SAH stretching peak of GSH at
S. Thammajinno, C. Buranachai, P. Kanatharana et al.
Fig. 1. (a) UV–vis absorption spectra of GSH and GSH-CuNCs were produced at wavelengths from 200 to 700 nm: (b) Photographs show the fluorescence of (I) CuNCs in
solution compared to (II) DI water under UV light at 365 nm: (c) Fluorescence emission spectra of CuNCs were produced at excitation wavelengths from 300 to 370 nm:
(d) FT-IR spectra of GSH and GSH-capped CuNCs.
2525 cm 1 was also absent in the spectrum of GSH-CuNCs. These
results indicated the attachment of GSH on the surface of CuNCs
via the amine and thiol groups [43].
To further characterize CuNCs, cyclic voltammetry was carried
out to study their electrochemical behavior. Owing to the small
size of CuNCs, chitosan cryogel (CS-cryogel), which can be very
easily made by freezing and thawing of chitosan was employed
to entrap these nanomaterials [40,44].
The electrochemical tests were carried out in 0.10 M NaOH
aqueous solution at a scanning rate of 10 mV s 1. The forward scan
range was from the potential of 1.20 to + 0.80 V, followed by the
reverse scan back to the onset potential ( 1.20 V). The CV of
CuNCs-CS cryogel/GCE exhibited characteristic anodic current
peaks at 0.36 and 0.048 V, corresponding to Cu (0)/Cu (I) and
Cu (I)/Cu (II), respectively (Supplementary information, Fig. S1).
The cathodic peaks for Cu (II)/Cu (I) and Cu (I)/Cu (0) is at 0.37
and 0.74 V, respectively. These peaks were not observed with
the CS cryogel/GCE. These CVs further demonstrated the successful
preparation of CuNCs, and confirmed that the synthesized CuNCs
had the same properties as previous works [44,45].
3.2. Sensing mechanism for HSA and creatinine detection
The possible detection mechanism of HSA and creatinine by the
GSH-CuNCs is proposed in Fig. 2. In the presence of HSA, the iso-
electric point (pI) of HSA is 4.7 [33]. At pH 7.4, that is higher than
the pI, HSA is negatively charged. Since the amine group of GSH-
CuNCs is positively charged [32], HSA induces the GSH-CuNCs to
come closer and interact via electrostatic attraction, which affects
the aggregate of nanoclusters. The aggregation increases fluores-
cence intensity and the GSH-CuNCs are working in the turn-on
mode (Fig. 2a).
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 270 (2022) 120816
To confirm this possible mechanism, TEM image and zeta
potential were obtained. TEM images showed spherical GSH-
CuNCs with an average diameter of 1.70 ± 0.66 nm (n = 100) (Sup-
plementary information, Fig. S2a). Aggregation of GSH-CuNCs in
the presence of HSA had an average diameter of 62 ± 37 nm
(n = 55) (Supplementary information, Fig. S2b). The size of GSH-
CuNCs aggregations in the presence of HSA was similar to those
reported in earlier research [35], confirming that HSA induces the
nanoclusters to form aggregations, resulting in a change in the size
of the nanoclusters.
Besides TEM images, zeta potential was used further to confirm
the interaction between GSH-CuNCs and HSA. The zeta potentials
of GSH-CuNCs were measured before and after the addition of
HSA. The GSH-CuNCs provided a zeta potential value of
25.1 mV, indicated that the negative charge was exposed on the
surfaces of the CuNCs. The addition of 50, 100 and 150 nM HSA
changed the value to 23.78 mV, 15.36 mV and 9.81 mV,
respectively. The decrease of the negatively charge value with
higher concentration of HSA indicating that the negatively charge
surface of GSH-CuNCs was neutralized and the interaction between
HSA and GSH-CuNCs occurred most likely via electrostatic force,
which induce the nanoclusters to come closer together.
In an acidic condition at pH 5.0, creatinine is electrostatically
neutral and could interact with GSH-CuNCs probe via non-
covalent bonding to form non-fluorescent complexes, resulting in
electron transfer from creatinine to the nanoclusters, causing a
reduction in fluorescence emission as a ‘‘turn-off” mode (Fig. 2b)
[37,38]. Fluorescence lifetime time measurement was employed
to explore the possible mechanism of the GSH-CuNCs probe for
the detection of creatinine. From fitting the fluorescence decay
curves, the average fluorescence lifetimes of the GSH-CuNCs in
the absence and presence of creatinine were found to be
S. Thammajinno, C. Buranachai, P. Kanatharana et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 270 (2022) 120816

Fig. 2. Sensing mechanism in the presence of (a) human serum albumin (HSA) at pH 7.4 induces the electrostatic interaction between HSA and GSH-CuNCs following the
aggregation-induced emission enhancement (turn-on mode). Inset: a fluorescence spectra of GSH-CuNCs with concentration of HSA increase the FL intensity also increase
and (b) the detection of creatinine at pH 5.0 was interacted via non-covalent bonding with GSH-CuNCs based on static quenching via formation of ground state complex
(turn-off mode). Inset: a fluorescence spectra of GSH-CuNCs with concentration of creatinine increase the FL intensity also decrease.

1.858 ± 0.059 ns and 1.833 ± 0.063 ns, respectively (Supplemen-
tary information, Fig. S3). The unchanged average fluorescence life-
time after the addition of creatinine indicates that creatinine may
quench the fluorescence of the GSH-CuNCs via a static (non-
fluorescent complex formation) quenching mechanism. The slight
increase in the average diameter of GSH-CuNCs from
1.70 ± 0.66 nm to 2.22 ± 0.70 nm (n = 85) in the presence of crea-
tinine observed via the TEM images (Supplementary data,
Fig. S2c) was likely due to the formation of ground state complex
between creatinine molecules and copper atoms [46].
Interestingly, Fig. 3B shows a downward deviation from a pure
linear Stern-Volmer plot. This is unlikely due to the presence of
both static and dynamic quenching because, in that case, the devi-
ation would have been upward [47]. Instead, this downward
behavior was reported in the case of buried fluorophores inacces-
sible to the quencher, such as tryptophan’s fluorescence quenching
by trifluoroacetamide [48]. We then hypothesize that some of our
CuNCs are easily accessed and statically quenched by creatinine
whereas the others are not due to heterogenous surface function-
alization. This gave rise to the initial decrease in the nanocluster’s

5
S. Thammajinno, C. Buranachai, P. Kanatharana et al.
fluorescence intensity with increasing creatinine concentration. At
the concentration higher than 1000 lM, however, all nanoclusters
that are easily accessed are all quenched while the rest are still flu-
orescent even when the concentration of the quencher is raised
further. Despite this heterogeneity, we are confident in the repro-
ducibility of the synthesis and as far as creatinine sensing is con-
cerned, we are using only the linear portion of the plot.”
On the other hand, zeta potential of GSH-CuNCs in acetate buf-
fer pH 5.0 exhibited a value of 15.05 mV, suggested that there is
an electrostatic repulsion among the GSH-CuNCs helping to stabi-
lize CuNCs from aggregations. Upon the addition of 30, 50 and
300 lM creatinine, the apparent zeta potential of the CuNCs
decreased from 15.05 to 12.51 mV, 12.51 to 10.50 mV and
10.50 to 8.77 mV, respectively. The reduction in the zeta poten-
tial value in the presence of creatinine suggested that surface
charge neutralization involved between GSH-CuNCs and creatinine
via non-covalent bonding and this led to the formation of non-
fluorescent complexes between the creatinine and GSH-CuNCs
probe [38]. It is unlikely that creatinine causes a destruction of
the nanocluster that results in the decrease in fluorescence inten-
sity because the extinction peaks of the nanocluster do not signif-
icantly change in the presence of various concentrations of
creatinine (Supplementary information, Fig. S4). Therefore, an effi-
cient electron transfer between creatinine and nanoclusters was
occurred in acetate buffer pH 5.0 which quenches the fluorescence
emission.
3.3. Optimization of conditions for HSA and creatinine detection
3.3.1. pH and concentration of buffer
pH was the most important parameter in this study because
it directly affected the binding between GSH-CuNCs and both
target analytes. For the proposed sensing mechanism to work,
the moieties in the reactions required the appropriate basic
and acidic buffer ranges. Since there was no single buffer with
Fig. 3. Fluorescent spectra and calibration plots were produced from (a) HSA and (b) creatinine detection under the optimum 15 min reaction time. Calibration plots mapped
F/F0 for HSA (turn-on mode), and F0/F for creatinine (turn-off mode) against concentration at an excitation wavelength of 360 nm. Insets show zooms of the fluorescence
spectra peaks of GSH-CuNCs in the presence of different concentrations of the analytes.
6
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 270 (2022) 120816
a good buffer capacity that could cover the range of pH from
4.0 to 8.0 [49], two common buffers were applied together:
phosphate buffer in a pH range from 5.80 to 8.0 for HSA detec-
tion and acetate buffer in a pH range from 3.40 to 5.90 for
creatinine detection.
The effect of basic pH was investigated for HSA at 7.0, 7.2, 7.4,
7.6 and 8.0. The sensitivity of the response was not much different
at each pH (Supplementary information, Fig. S5a). This was possi-
bly because the isoelectric point (pI) of HSA is 4.7 [33] and within
the applied pH range, HSA remained negatively charged and could
always interact with GSH via electrostatic bonding. Since the sen-
sitivity at pH 7.4 was slightly higher than at other pH values, this
pH was used for further studies.
Creatinine detection was tested in acetate buffer at pH 4.0, 4.5
and 5.0, and in phosphate buffer at pH 6.0 and 7.0 (Supplementary
information, Fig. S5b). Detection sensitivity increased from pH 4.0
to 5.0 and decreased at higher pH. This was likely because at pH
5.0, creatinine (pKa  5.02) [50] remains in a neutral form, and
GSH which contains amide, carboxylic acid and amino groups
[32] can induce creatinine towards the CuNCs via hydrogen bond-
ing, enhancing the likelihood of interaction between creatinine and
copper atoms on the CuNCs surface. At pH higher than 5.0, crea-
tinine is in a negatively charged form, and repulsion will occur
between GSH and creatinine, lowering the possibility of the inter-
action. So, acetate buffer at pH 5.0 was used in the remaining
studies.
The concentration of phosphate buffer at pH 7.4 was optimized
at 0.050, 0.10, 0.15, 0.20 and 0.25 M while the acetate buffer at pH
5.0 was optimized at 0.010, 0.050, 0.10 and 0.15 M. Sensitivity
toward HSA increased in the phosphate buffer from 0.050 to
0.15 M (Supplementary information, Fig. S6a) and sensitivity
toward creatinine increased in the acetate buffer from 0.010 to
0.10 M (Supplementary information, Fig. S6b). In both cases, sensi-
tivity decreased at higher concentrations. It is likely that at too
high a concentration, ions might block access to the GSH-CuNCs
S. Thammajinno, C. Buranachai, P. Kanatharana et al.
surface, making difficult paths for the targets to interact [51].
Therefore, 0.15 M phosphate buffer at pH 7.4 and 0.10 M acetate
buffer at pH 5.0 were the optimal buffer conditions for HSA and
creatinine detection.
3.3.2. Reaction time
The effect of reaction time on HSA and creatinine detection was
evaluated at 10, 15, 20 and 25 min. For both targets, detection sen-
sitivities increased from 10 to 15 min and became relatively con-
stant thereafter (Supplementary information, Fig. S7a and S7b).
Sensitivity was higher at 15 min, so this was the time chosen as
the optimal value.
3.4. Analytical performances
3.4.1. Linear dynamic range, limit of detection and limit of
quantification
Under optimal conditions, the system was tested in the turn-on
mode, detecting HSA concentrations from 1.0 to 300 nM and in the
turn-off mode, detecting creatinine concentrations from 10 to
3000 lM. The response to HSA concentration was linear in the
range of 5.0 nM to 150 nM (Fig. 3a) and to creatinine concentration
in the range of 30 lM to 1000 lM (Fig. 3b). The limits of detection
(LODs) and limits of quantification (LOQs) were calculated based
on LOD = 3Sa/b and LOQ = 10Sa/b, where Sa is the standard devia-
tion of the intercept and b is the slope of the calibration curve.
The LODs were 1.510 ± 0.041 nM for HSA and 13.0 ± 1.0 lM for cre-
atinine while the LOQs were 5.04 ± 0.14 nM for HSA and
44.0 ± 3.0 lM for creatinine.
It is reasonable to argue that the fluorescence enhancement
and quenching are not very obvious. So, another commercial
spectrofluorometer (LS-55, PerkinElmer, Waltham, MA, USA)
was used to test and confirm the fluorescence enhancement
and quenching of the reaction between GSH-CuNCs probe and
the two targets. The fluorescence emission spectra were recorded
from 380 nm to 700 nm with the excitation wavelength of
360 nm, as has been used with the first spectrofluorometer (RF-
5301, Shimadzu, Tokyo, Japan). As expected, under the same opti-
mal condition for HSA detection, the probe’s fluorescence signal
increases with increasing concentration of HSA; likewise, under
the optimal condition for creatinine detection, the probe’s fluores-
cence signal decreases with increasing creatinine concentration
(Fig. S8 and Table S1 in supplementary information). This agrees
with those observed under the first fluorometer. There are slight
differences between the two data sets taken from the two fluo-
rometers, such as sensitivities of creatinine detection, but we
believe that those values are instrument dependent that have to
be considered when characterizing a sensor. Therefore, we can
conclude that the increase and decrease of the probe’s fluores-
cence signal really come from the interaction between GSH-
CuNCs probe and HSA and creatinine, respectively, regardless of
the fluorometers being used, providing that the detections are
done under the optimal conditions.
3.4.2. Reproducibility
The reproducibility of the synthesized GSH-CuNCs as a fluores-
cent probe for the determination of HSA and creatinine was evalu-
ated from the performances of six different preparations of the
sensor. Up to seven concentrations of HSA and creatinine in their
respective linear ranges (each concentration n = 3) were detected
and the sensitivities of the six preparations varied from
(196.0 ± 1.4)  10 6 to (198.0 ± 2.0)  10 6 nM 1 (RSD = 0.41%)
for HSA and from (96.0 ± 1.0)  10 6 to (98.0 ± 3.0)  10 6 lM 1
(RSD = 0.65%) for creatinine. Thus, the synthesis and the detection
processes exhibited good reproducibility (P > 0.05).
7
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 270 (2022) 120816
3.4.3. Precision
The precision of the proposed sensor was evaluated by spiking
standard solutions of HSA and creatinine in urine samples obtained
from Songklanagarind Hospital. The final concentrations of HSA in
the spiked urine samples were 5.0, 10.0, 50.0, 100.0 and 150.0 nM,
whereas those of creatinine were 30.0, 70.0, 300.0, 500.0 and
1000.0 lM. Six replications of each spiked sample were analyzed
under the optimal conditions. The relative standard deviations (%
RSD) varied between 0.10 and 0.60 for the determination of HSA
and between 0.12 and 0.50 for the determination of creatinine.
These values were better than the AOAC guideline RSDs, which
are 7.3% at the concentration of 10 mg L 1 and 5.3% at the concen-
tration of 100 mg L 1 for HSA and creatinine, respectively [52].
Therefore, the analytical precision achieved with the developed
sensor was good for HSA and creatinine detection.
3.4.4. Stability
The stability of the GSH-CuNCs was tested to determine how
long the fluorescent sensing probe remained usable after it was
synthesized. Calibration curves were plotted using freshly pre-
pared GSH-CuNCs to determine HSA and creatinine in their linear
ranges (up to 7 concentrations, n = 3) and the sensitivities obtained
were set as 100% at week zero. The GSH-CuNCs solution was kept
in darkness at 4 °C and used to detect HSA and creatinine in the
same concentration ranges on a weekly basis. During the first
14 weeks, relative sensitivity remained stable at an average of
99.64 ± 0.67% for HSA and 99.84 ± 0.50% for creatinine. Sensitivity
then slowly declined and dropped below 90% after 20 weeks (Sup-
plementary information, Fig. S9a and 9b). The probes were there-
fore considered stable for up to 20 weeks [53].
3.4.5. Selectivity of the sensor
Human urine may present many interferences to the detection
of the target analytes. Selectivity was therefore tested toward rel-
evant biological molecules and salts at the highest level normally
found in human urine. The molecules tested and their levels were
uric acid at 4.46 mM [54], ascorbic acid at 3.40 mM [55], cysteine
at 2.50 mM [56], urea at 500 mM [57], Na+ at 260 mM [58] and
Cl at 170 mM [59]. Dopamine was tested at a higher than normal
level, at 2.50 mM, because its level in human urine may depend on
age [60]. Also, HSA was tested at a higher than normal level, at
0.0040 mM, [61] as an interference to creatinine detection and cre-
atinine was tested as an interference to HSA detection at 15.0 and
30.0 mM, its middle and maximum levels in human urine [62]. The
concentrations to be determined in this study were from the mid-
dle of the linear range of the calibration plots, at 0.10 lM for HSA
and 0.50 mM for creatinine.
Taking account, the level of the analytes of interest in real sam-
ples, detection should be carried out in a 6-time dilution of HSA
and 40-time dilution of creatinine so that their concentrations
would fall within the calibration ranges. Therefore, the interfer-
ences were diluted with buffer accordingly.
During HSA detection (Fig. 4a), negligible increases in the fluo-
rescence intensity of the biological molecules were observed:
dopamine 0.31%, Na+ 0.41%, Cl 0.32%, ascorbic acid 0.45%, uric acid
0.68%, 15 mM creatinine 0.66% and 30.0 mM creatinine 0.98%.
These were well below the acceptable 5% change of signal [63].
During creatinine detection (Fig. 4b), no interference effects were
observed. All interferences produced the same signals as the blank
solution (GSH-CuNCs in buffer), indicating that the relevant mole-
cules and salts had negligible effects on the detection of HSA and
creatinine.
Each of the studied interferences was also mixed with HSA at
0.10 lM or creatinine at 0.50 mM. The fluorescence emission
intensities of all mixtures showed no significant change and were,
therefore, within the acceptable limit of signal change of less than
S. Thammajinno, C. Buranachai, P. Kanatharana et al.
Fig. 4. The charts show the selectivity of the proposed sensor toward (a) HSA and (b) creatinine in the presence of other biological molecules and salts found in human urine
compared with a blank (GSH-CuNCs). Selectivity was also tested using solutions of (c) HSA at 0.10 lM and (d) creatinine at 0.50 mM mixed with interfering biological
molecules and salts.
5% (Fig. 4c and 4d). Thus, detection of HSA and creatinine with this
method was highly selective. Even though HSA enhances while
creatinine quenches the probe’s fluorescence, HSA does not inter-
fere with creatinine detection because HSA does not increase the
probe’s fluorescence under the optimum condition of creatinine
detection (i.e., acidic solution). Likewise, creatinine does not inter-
fere with HSA detection because creatinine does not quench the
probe’s fluorescence under the optimum condition of HSA detec-
tion (i.e., slightly basic solution).
To detect these two compounds, the same sample was divided
into two portions; each of which was pipetted into a different tube
containing different buffer solution: 0.15 M phosphate buffer at pH
7.4 for HSA and 0.10 M acetate buffer at pH 5.0 for creatinine. Then
the fluorescence signal from each reaction was measured sepa-
rately before analyzing the concentration of the two analytes. Since
the probe binds selectively to HSA or creatinine, depending on the
conditions of the solutions, as shown in Fig. 4A-D, we are able to
detect both HSA and creatinine from the same samples with the
same probe.
3.5. Determination of HSA and creatinine in human urine samples
Urine samples were diluted into the appropriate linear ranges
before detection. Samples were diluted 6 or 20 times (depending
on their original fluorescence intensity) with 0.15 M phosphate
buffer at pH 7.4 for HSA determination and 40 times with 0.10 M
acetate buffer at pH 5.0 for creatinine determination.
The matrix effect of urine samples was first studied by spiking
with HSA and creatinine standard solutions to obtain final concen-
trations after dilution in the range of 5.0 nM to 150 nM and 30 to
1000 lM, respectively. The different buffer solutions used for the
target analytes were also spiked in the same way. The spiked urine
8
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 270 (2022) 120816
samples and spiked buffer solutions were analyzed and the
obtained integrated fluorescence intensities were plotted against
their respective concentrations. The slopes of the calibration plots
obtained from the spiked samples and spiked buffer solutions were
compared by two-way ANOVA. The two slopes were significantly
different at the confidence level of 95% (P < 0.05), indicating the
presence of a matrix effect. Therefore, the standard addition
method was used to quantify the concentration of both HSA and
creatinine in urine samples.
Fifteen samples were analyzed and the results were compared
to the results obtained from the immunoturbidimetric and enzy-
matic methods used by the hospital. The results obtained from
all urine samples with the developed fluorescence detection
method were in good agreement with the concentrations of target
analytes obtained from the conventional methods used by the hos-
pital (P > 0.05) (Fig. 5a, 5b and 5c).
To further validate the developed fluorescence sensor, the accu-
racy of the method was also evaluated in terms of recoveries,
which were in the range of 81.44 ± 0.25–109.22 ± 0.57% for HSA
detection and 80.57 ± 0.16–109.0 ± 0.10% for creatinine detection
(Table S2). These values were within the acceptable limits between
80 and 110% (AOAC, 2011). Therefore, the GSH-CuNCs provided
good recovery, good precision, good accuracy, and could detect
HSA and creatinine in urine samples.
The analytical performances of the GSH-CuNCs fluorescent
probe were compared with performances reported for other sen-
sors for HSA and creatinine detection (Table 1). For HSA detection,
the linear range, reaction time and LOD of the proposed method
were either comparable or better than the results of the reported
fluorescence and colorimetric methods [19,64]. Importantly, the
LOD value (1.5 nM) was more than sufficient for the determina-
tion of urinary HSA (33.0–400 nM) [61]. In the case of creatinine
S. Thammajinno, C. Buranachai, P. Kanatharana et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 270 (2022) 120816

Fig. 5. The charts compare results from (a) HSA detection and (b) creatinine detection with GSH-CuNCs and standard methods and the chary (c) shows relative ACRs.

detection, the developed method provided a good linear range
with a comparable reaction time. Although, the LOD for creatinine
in this work was higher than in other works [20,38,64,65], it was
good enough for the determination of creatinine in human urine
(3.6–27.0 mM in males and 3.30–22.50 mM in females) [62]. The
great advantage of the developed sensor is probably the storage
stability of 20 weeks. In addition, the preparation reproducibility
of the GSH-CuNCs showed a good % RSD (n = 3). Values of %
recovery in urine samples for HSA detection and creatinine were
also acceptable and comparable with the recoveries of other
methods.
4. Conclusions
A fluorescent probe utilizing glutathione-capped copper nan-
oclusters was successfully developed for the determination of
the dual targets, human serum albumin and creatinine in
human urine. The detection principle was based on electrostatic
interaction between the nanoclusters and human serum albu-
min to turn on fluorescence and non-covalent bonding between
the nanoclusters and creatinine to quench, or turn off, fluores-
cence. Under optimal conditions, the developed method pro-
vided good linear ranges and low limits of detection for both
targets. Furthermore, when applied to the analysis of human
serum albumin and creatinine in human urine samples, the
developed method produced highly accurate and precise analyt-
ical results which were consistent with those obtained from the
immunoturbidimetric and enzymatic methods used by the col-
laborating hospital. In addition, the fluorescence probe was also
highly selective toward human serum albumin and creatinine in
the presence of biological molecules and salts typically found in
urine.

9
S. Thammajinno, C. Buranachai, P. Kanatharana et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 270 (2022) 120816

Table 1
Comparison of the performances of the GSH-CuNCs fluorescence sensor with the sensor performances of other fluorescence and colorimetric methods for HSA and creatinine
detection.

Sensing probe Method Linear range LOD Reaction Reproducibility Stability Sample Recovery Ref.
time (%)
HSA detection
(nM) (nM)
poly(thymin)-CuNPs Fluorescence 150–2500 82 15 min – – Serum 97.0–101.0 [19]
Ex 340 nm
Em 610 nm
CIZS QDs Fluorescence (0.075– 45 – – – Serum 88.14–94.39 [64]
Ex 380 nm 100)  103
Em 570 nm
citrate-AuNPs Colorimetric 20–100 1.4 15 min – – Artificial 96.7–103.8 [67]
A650/A520 urine
GSH-CuNCs Fluorescence 5.0–150 1.5 15 min RSD 0.41% 20 weeks Urine 81.44 ± 0.25– This
Ex 360 nm (n = 3) 109.22 ± 0.57 work
Em 428 nm
Creatinine detection
(lM) (lM)
AuQC@gluten Fluorescence 20–520 0.0020 15 min – – Blood 98.16–99.5 [65]
Ex 380 nm
Em 680 nm
BSA-CuNCs Fluorescence 5.0–60 0.050 12 min – >18 days Urine 97.63–102.43 [38]
Ex 525 nm
Em 643 nm
6
CC-AuNCs Fluorescence 0.00010–0.10 7.54  10 30 min RSD 4.2% >1 month Serum and 96.00–107.0 [20]
Ex 365 nm (n = 5) urine
Em 481 nm
TDA-AgNPs Colorimetric 0.010–1.0 0.0030 5 min – – Serum and 89.4–102.1 [66]
A560/A390 urine
GSH-CuNCs Fluorescence 30–1000 13.0 15 min RSD 0.65% 20 weeks Urine 80.57 ± 0.16– This
Ex 360 nm (n = 3) 109.0 ± 0.10 work
Em 428 nm

CRediT authorship contribution statement Compliance with ethical standards

Supitcha Thammajinno: Methodology, Investigation, Formal
analysis, Writing – original draft, Visualization. Chittanon Bur-
anachai: Writing – review & editing. Proespichaya Kanatha-
rana: Resources, Validation, Writing – review & editing.
Panote Thavarungkul: Conceptualization, Methodology, Valida-
tion, Formal analysis, Resources, Writing – review & editing.
Chongdee Thammakhet-Buranachai: Conceptualization,
Methodology, Validation, Formal analysis, Resources, Writing –
review & editing.
Urine samples in experiments were performed with the
approval of the Exemption Determination as was acknowledged
by Human Research Ethics Committee of Faculty of Medicine,
Prince of Songkla University (REC.63-510-19-2).
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

Declaration of Competing Interest

Appendix A. Supplementary material
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared Supplementary data to this article can be found online at
to influence the work reported in this paper. https://doi.org/10.1016/j.saa.2021.120816.

Acknowledgements References

This work was financially supported by the Thailand Center of
Excellence in Physics (ThEP), Prince of Songkla University and
Faculty of Science, Prince of Songkla University (grant no. ThEP-
61-PHM-PSU2), the Research Assistantship, Faculty of Science,
Prince of Songkla University (contract no. 1-2559-02-010) and
the Center of Excellence for Innovation in Chemistry (PERCH-
CIC). Partial support is also gratefully acknowledged from the
Graduate School Dissertation Funding for Thesis, the Center of
Excellence for Trace Analysis and Biosensor (TAB-CoE), Division
of Physical Science, Department of Chemistry, Department of Phy-
sics, Faculty of Science, Prince of Songkla University, Hat Yai,
Songkhla, Thailand.
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