j

Journal Pre-proofs
Aggregation-based fluorescence amplification strategy: “turn-on” sensing of
aminoglycosides using near-IR carbocyanine dyes and pre-micellar surfac‐
tants
Sofia A. Zakharenkova, Ekaterina A. Katkova, Irina A. Doroshenko, Anna S.
Kriveleva, Aleksandra N. Lebedeva, Tatyana A. Vidinchuk, Anna V. Shik,
Sergei S. Abramchuk, Tatyana A. Podrugina, Mikhail K. Beklemishev
PII:
DOI:
Reference:
S1386-1425(20)31088-X
https://doi.org/10.1016/j.saa.2020.119109
SAA 119109
To appear in:
Spectrochimica Acta Part A: Molecular and Bio‐
molecular Spectroscopy
Received Date:
Revised Date:
Accepted Date:
2 June 2020
2 October 2020
14 October 2020
Please cite this article as: S.A. Zakharenkova, E.A. Katkova, I.A. Doroshenko, A.S. Kriveleva, A.N. Lebedeva,
T.A. Vidinchuk, A.V. Shik, S.S. Abramchuk, T.A. Podrugina, M.K. Beklemishev, Aggregation-based
fluorescence amplification strategy: “turn-on” sensing of aminoglycosides using near-IR carbocyanine dyes and
pre-micellar surfactants, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2020), doi:
https://doi.org/10.1016/j.saa.2020.119109
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Aggregation-based fluorescence amplification strategy: "turn-on" sensing of
aminoglycosides using near-IR carbocyanine dyes and pre-micellar surfactants
Sofia A. Zakharenkovaa, Ekaterina A. Katkovaa, Irina A. Doroshenkoa, Anna S. Krivelevaa,
Aleksandra N. Lebedevaa, Tatyana A. Vidinchuka, Anna V. Shika, Sergei S. Abramchukb, Tatyana A.
Podruginaa, Mikhail K. Beklemisheva*
aM.V.Lomonosov
Moscow State University, Department of Chemistry, 119991 GSP-1 Moscow,
Russia
bNesmeyanov
Institute of Organoelement Compounds (INEOS), Russian Academy of Sciences,
Vavilova 28, Moscow, 119991, Russia
*e-mail: mkb@analyt.chem.msu.ru
Abstract. This study is aimed at developing sensing schemes without obtaining selective receptors. A
series of simple carbocyanine dyes was synthesized, whose emission was quenched in water with
formation of nanoparticles in the range of 20–100 nm. Fluorescence in near-IR region is “turned on”
in the presence of a drug cation of middle molecular weight (400–700 Da) and sodium dodecyl
sulfate (SDS), as well as anionic drugs and a cationic surfactant (cetyltrimethylammonium bromide,
CTAB). Aggregates (clusters) up to 100–200 nm in size were detected using dynamic light scattering
(DLS) and Rayleigh light scattering (RLS) techniques in the systems: cationic analyte – SDS,
carbocyanine dye – CTAB, and in all brightly fluorescent ternary systems dye – surfactant – analyte.
Small ions (<200 Da) incapable of multi-point binding do not form the aggregates or cause the
emission enhancement. The “turn-on” signal is only observed at the surfactant submicellar
concentrations insufficient to solubilize the dye nanoparticles. Based on these findings, we suggest a
rapid and simple method for the detection of ≥ 4·10–5 mol/L of neomycin in urine. The proposed
strategy paves the way for developing more selective methods.
Keywords: fluorescence spectroscopy, self-assembly, aggregation, ion pair, carbocyanine,
aminoglycoside
Introduction
Fluorescence-based methods for detection of small molecules are inexpensive easy to operate, and
often exhibit low detection limits. Molecular fluorescence sensors are based on different types of
interactions, including supramolecular ones [1, 2]. A challenging problem is sensing of small
molecules without a considerable synthetic effort, which invokes interest to detection strategies based
on aggregation of amphiphilic species [3] and ionic self-assembly [4–6] (several authors prefer to use
term “clusters” instead of “aggregates” [7]). Fluorescent aggregation-based methods, which are
sometimes highly sensitive and selective, are realized using various principles: displacement
processes (AB + С, AC + B) [8], formation of surfactant micelles to encapsulate a hydrophobic
fluorophore [9–11] or joint micelles of the fluorophore and analyte [12], and aggregation of the
fluorophore-containing micelles by a polymeric analyte [13].
Aggregation-based technique includes a group of detection methods in which the signal of
analyte does not result from a direct analyte–fluorophore interaction – indirect signalling methods,
which widen the range of possible analytes and expand the opportunities of tuning the sensing
selectivity [14, 15]. A number of indirect signalling-based fluorescent methods were developed using
aggregation [3], including formation of the ternary aggregates of a target analyte with a pre-micellar
surfactant and a fluorescent dye. Such aggregates can be formed when the analyte lowers the critical
micelle concentration (CMC) of the surfactant. The process is visualized upon involvement of a dye,
leading to optical changes [16–18]. In many cases analytes cause emission quenching [17, 18];
however, more practical is signal enhancement that can be achieved by pre-quenching the
fluorophores by a number of ways [15, 19–21], including placement of the dye in water, which results
in its aggregation [22]. However, the concrete form of the dye often remains unknown.
Aminoglycosides are widely used as antibiotics; the instrumental methods for their
determination such as HPLC-MS [23] and other techniques [24, 25] have been developed. Optical
sensing of aminoglycosides is a challenge, since their molecules do not have any chromophoric or
fluorophoric groups. Fluorescent derivatives of aminoglycosides have been obtained by use of
derivatization technique designed for HPLC methods [26]. However, reversed-phase HPLC faces
difficulties in the determination of aminoglycosides because of their poor chromatographic retention
[27]. Several fluorescent methods for aminoglycosides without derivatization were developed [28],
including the methods using aptasensors [29, 30]; an aggregational pattern-based fluorescent method
was used in the discrimination of an artificial mixture of aminoglycosides [12]. Although specific
immune tests for aminoglycosides, which are based on enzyme-linked immunosorbent assay (ELISA)
[31, 32] and requires aminoglycoside conjugation with proteins and animal immunization steps, are
widely used, there is increasing interest in rapid techniques that do not involve the use of enzymes or
antibodies.
2
This proof-of-concept study rests on two fruitful ideas of aggregation-based sensing:
preliminary fluorophore quenching [15, 19, 20] and aggregation of the target compound with a
surfactant [16–18]. Taking into account that the aggregation-based sensing events greatly depend on
hydrophobicity of the fluorophores, we synthesized and studied a series of carbocyanines (I, II, IV–
VI) that are different in their hydrophobicity following the principles described earlier [33]. A
commercially available near-IR dye III was also studied.
The purposes of this study are to reveal the origin of fluorescence enhancement of
carbocyanine dyes observed with model analytes and surfactants and to evaluate the feasibility of
non-covalent sensing of a selected analyte (neomycin). Neomycin is part of medicines for external
use; when applied to damaged skin or granulation tissue, it can be quickly absorbed into the body and
cause kidney problems and hearing difficulty [34]. Neomycin metabolism is negligible; hence, all
absorbed fraction of neomycin is excreted by the kidneys [35]. Also, it can adversely affect the
kidneys [36], which requires its determination in urine.
Materials and Methods
HOOC
N
1
X R
I-III
N
R2
N
I
IV-VI
N
R
I: R1 = R2 = (CH2)10COOH, X = Br–
IV: R = CH3
II: R1 = R2 = (CH2)5CH3, X = I–
V: R = (CH2)5CH3
III (Cy5.5-COOH): R1 = CH3, R2 = (CH2)6COOH, X = Cl–
VI: R = (CH2)10COOH
Scheme 1. Dye structures.
Compounds. Dyes I, II, and IV–VI were synthesized according to [33] as illustrated in
Scheme 2. A more detailed protocol and characterization data are given in the ESI. Carbocyanine III
(Cy5.5-COOH, CAS No 1449612-07-0) was purchased from Lumiprobe (Hannover, Germany). The
structures of compounds used as model analytes are given in Scheme S1 in ESI.
3
NHPh
PhN
RX
N
X
1
Py, Ac2O
N
R1
X
I-II
2b-c
HOOC
N
R1
N
R1
CH3I
HOOC
N
N
I
3
AcONa
4
HOOC
Ac2O
N
I
PhN
NHPh
N
R
X
2a-c
X
N
R
IV-VI
NAcPh
N
R
5a-c
Scheme 2. Synthetic route to dyes I, II (top) and IV–VI (bottom).
RESULTS AND DISCUSSION
1. Fluorescent Properties of Carbocyanine Dyes
Organic solution. In organic solvents (ethanol, THF) dyes I–VI show absorption maxima at
~350 nm and 650–680 nm and fluorescence bands at 400–500 nm and 650–750 nm (Fig. 1a, b),
which correspond to their molecular forms. The quantum yields of the NIR emission do not exceed
~2% (Table S1 in ESI).
Aqueous solutions without surfactants. The dilution of the ethanolic (or THF) solution of the
new dyes I or II with ≥10-fold excess of water or an aqueous buffer produces a visually transparent
solution, which exhibits weak red absorption (Fig. S2) and almost no NIR emission, while the
fluorescence at 400—500 nm remains prominent (Fig. 1c). In this study we focused on NIR emission
as it is more responsive to model analytes.
4
Fig. 1. Absorbance (a) and fluorescence (b) spectra of ethanolic solutions of dyes I – VI; fluorescence
spectra of dyes I – III: (c) in an aqueous solution without surfactant (pH 7.4, 7 mM phosphate buffer)
and (d) with submicellar (0.7 mM) or micellar (24 mM) SDS in the same buffer.
Transmission electron microscopy (TEM) evidences (Fig. 2a) that in water the dye forms ballshaped nanoparticles of size 20–100 nm (Fig. 2a). No regular nanoparticles were found by TEM in
the sample of the dried ethanolic solution of dye I (Fig. S3). As mentioned above, the nanoparticles of
dye I demonstrate very low fluorescence intensity in the region higher than 600 nm (Fig. 1d). A
similar aggregational form was observed in TEM for another fluorophore [37].
5
Fig. 2. TEM images of the solutions of dye I: a – in water (a THF solution (1 g/L) was mixed with
water (1 + 40, v/v) and dried on the copper grid); b – in water containing submicellar SDS (0.7 mM).
Aqueous solutions with surfactants. Surfactants at submicellar concentrations (0.1–0.03
critical micelle concentration (CMC) value) do not considerably increase the dyes emission compared
with the buffer solution (Fig. 1d). Presumably, in submicellar solutions the dyes also exist in the form
of nanoparticles, as found for dye I in TEM (Fig. 2b). At micellar concentration of surfactants (3
CMC values) the dyes are strongly emitting (Fig. 1d), most probably, due to distribution of the dye
molecules in the micelles. This process is supposed to be accompanied by de-aggregation and
inhibition of radiationless decay, which in turn can be explained by the rigidization of the dye in the
micelle environment [38].
2. Fluorescence in the dye – analyte – surfactant systems
The effect of model analytes on the emission intensity of dyes was studied at physiological pH
7.4. In the absence of surfactant, no effect of analytes was noticed with respect to the blank
experiments.
Analytes in submicellar surfactants. In the premicellar solutions of SDS or CTAB, the
emission of dyes I–III is quenched (similar to pure water, section 1). The feasibility of fluorescence
enhancement was studied by adding differently charged medicinal drugs. In the 500-nm emission
range, no regular effect of the model analyte on the emission was found (data not shown). By
contrast, the NIR fluorescence was enhanced for the combinations of an analyte with the oppositely
charged surfactant: cationic species (aminoglycosides) with SDS (Fig. 3,a) and anionic species
6
(cephalosporins, penicillins) with CTAB (Fig. 3,b). Similar results were obtained with the other dyes
(Fig. S4 in ESI). The signals could be also acquired by photographing 96-well plates (Fig. 3 insets)
with a NIR photo camera (the setup is shown in Fig. S1).
Studies of a wider range of compounds in the system dye I – analyte – submicellar surfactant
revealed that the species of molecular weight less than 200 Da, even those charged oppositely to the
surfactant, induced smaller signals compared to those of the drugs (Fig. 4a, b).
Let us construct a conceivable scheme of the observed fluorescence enhancement for dye I
and aminoglycosides that yield the highest signals. At the first stage, the cationic drugs (charge
+3...+5 at pH 7.4, https://www.drugbank.ca/) form aggregates with the oppositely charged SDS. The
high charge of these cations could therefore be considered as the main driving force for binding with
SDS. However, anionic drugs (cephalosporins and penicillins) also yield a signal with an oppositely
charged surfactant (CTAB, Fig. 4,b), though their physiological charge does not exceed –1 (the lower
charge may be the reason for lower selectivity for the anionic species (Fig. 4,b) compared to
aminoglycosides (Fig. 4,a). Therefore, it is probably important that the analyte could form multi-point
bonds in order to be bound in the aggregates. The studied drugs do have several centers of binding
(their structures are shown in Scheme S1 in ESI). The other party in this binding (Scheme S3) could
be micelle-like structures of the surfactant formed upon aggregation (the micelles per se are absent in
the initial submicellar solution).
At the second stage, the SDS – analyte aggregates formed could solubilize the dye
nanoparticles to enhance the fluorescent signal (the situation with CTAB slightly differs). This stage
is considered in section 3 below.
Fig. 3. Fluorescence spectra (and NIR images of the plates) with dye I: (a) in submicellar SDS:
without added analytes (1) and with analytes: cationic (blue colors: 2–5) or anionic (red colors: 6, 7);
(b) in submicellar CTAB: without analytes (1) and with cationic (blue colors: 2, 3) or anionic (red
7
colors: 6–9) analytes. Analytes: 2 – amikacin, 3 – neomycin, 4 – gentamicin, 5 – streptomycin, 6 –
ceftriaxone, 7 – cefazoline, 8 – ceftazidime, 9 – cefotaxime. Conditions: pH 7.4 (7 mM phosphate
buffer); 1.5 mM analyte, 5 M dye I, 0.7 mM SDS or 0.1 mM CTAB (here and below the
concentrations are given for the reaction mixture).
Fig. 4. NIR fluorescence intensity in the system dye I – submicellar SDS (0.7 mM, left) and dye I –
submicellar CTAB (0.1 mM, right) in the presence of 1.5 mM of various species at pH 7.4. The molar
weight of the cation or anion of the species, whichever is heavier, is shown beside its name. The
charge at pH 7.4 (according to https://www.drugbank.ca/) is given in parentheses.
* As sulfates. **As sodium salts.
Analytes in micellar surfactants. In micellar solutions of surfactants the dyes exhibit high NIR
emission (Fig. 1d); this is the maximum signal developed with the dyes under such conditions, for
which reason it is not increased by the analytes (Fig. S5).
8
3. Aggregation in the systems dye – analyte – surfactant
The aggregational phenomena were studied to understand the reasons behind the emission
enhancement we observed (Figs. 3, 4).
Analyte – surfactant aggregation. According to the DLS data, the submicellar (0.03–
0.1×CMC) SDS solutions with cationic drugs contained particles with an average size of 60 nm (Fig.
5). For the micellar concentrations of the surfactant (3×CMC), no similar aggregates were observed,
probably, due to ability of the surfactant micelles to solubilize the surfactant – analyte AGs [39].
Fig. 5. Particle sizes according to DLS technique in micellar (3×CMC) and submicellar (0.03×CMC)
solutions of SDS with and without streptomycin. Concentrations: 0.24 or 24 mM SDS, 1.5 mM
analyte, pH 7.4.
Dye – analyte – surfactant aggregation: cationic analytes with SDS. In studying aggregation
in the presence of fluorophores, the DLS technique faces problems caused by the strong dye
fluorescence under the laser light. Characterization of the dye – analyte – surfactant systems was
performed using Rayleigh light scattering (RLS) spectra that qualitatively reflect the particle sizes.
The analyte – SDS aggregates showed intense RLS spectra, not significantly changing upon
adding a dye (Fig. 6a,b and Fig. S6 in ESI). This means that the particle size is not appreciably
increased, which allows us to hypothesize that the dye enters already formed aggregates. As a result,
the dye – analyte – SDS ternary aggregates are formed.
However, not every large cationic species will give rise to emission enhancement. For
example, amikacin does not form an aggregate with SDS, as evidenced by the RLS spectra, and
consequently does not form a ternary aggregate in the presence of the dye. No considerable emission
is observed with this drug (Fig. 6c; for detailed spectra and other examples, see Fig. S6 in ESI).
9
Thus, the RLS and fluorescence sudies demonstrate that emission is turned on only it there are
aggregates of the (cationic) analyte and (anionic) surfactant. If the dye has an affinity for these binary
aggregates, it is solubilized in them, transforming from an aggregated conditon (nanoparticles) to
molecular state, thereby increasing its quantum yield.
Dye – analyte – surfactant aggregation: anionic analytes with CTAB. A different pattern has
been observed for the cationic surfactants. Judging by the RLS intensity (Fig. 6,b and Fig. S6) we
observe (1) no formation of large particles in the system analyte – CTAB, but (2) presence of a large
aggregate dye – CTAB. The dye – CTAB aggregates (observed for dyes I and II) may be formed due
to CTAB cation interaction with the  system of the dye (Scheme S3 in ESI), as it is typical of
quaternary ammonium salts to give complexes with the -systems of aromatic compounds [1, 40].
Fluorescence in the ternary systems dye – CTAB – analyte is only observed for some anionic drugs
(ceftriaxone, ceftazidime and cefotaxime) that form large ternary aggregates (Fig. 6b,c). There is no
fluorescence with CTAB and other species giving large anions (cefazoline or penicillin); at the same
time, these drugs do not form large aggregates dye – CTAB – analyte (judging by the RLS intensity
that is far lower that for dye I – CTAB, Fig. S6 in ESI).
Based on this data, we can suppose that fluorescence of anionic analytes appears only in the
ternary aggregates dye – CTAB – analyte. It should be analyzed how the ternary aggregates can be
formed. In case of cationic drugs and SDS (previous subsection), the SDS ensembles dissolved the
dye nanoparticles. In case of anionic drugs, we can imagine two scenarios of ternary aggregates
formation: (1) the dye – anionic drug aggregate reacts with the cationic surfactant, which entails
solubilization of the dye in the surfactant micelle-like substructure of the ternary aggregate thus
formed; (2) the ternary aggregate is formed during simultaneous interaction of the three components:
dye nanoparticles, ions of the surfactant and the drug. The existing techniques do not allow for
distinguishing between these two scenarios; however, both of them result in conversion of the dye
into its molecular (fluorescent) form (Scheme 3).
Not every analyte of the proper charge will cause fluorescent enhancement. The RLS spectra
show that small species that are unable to give multiple non-covalent bonds (such as arginine or
diethylamine in the presence of SDS, Fig. 4 or S7) do not form the aggregates with the oppositely
charged submicellar surfactants. This is in agreement with the fact that only large ions (having
multiple binding centers) can form the aggregates and selectively enhance fluorescence.
Overall, fluorescence is turned on in the ternary aggregates dye – surfactant – analyte,
independently of the the surfactant and analyte charges. (Scheme 3). This mechanism can be
implemented if the analyte species is large and multifunctional (to allow for multi-point binding) and
the dye fluorescence without the analyte is pre-quenched in a submicellar surfactant.
10
Fig. 6. Rayleigh light scattering and fluorescence (in the inserts) spectra of the systems: neomycin
(Neo) – SDS – dye I (a) and ceftriaxone (Ceftr) – CTAB – dye I (b); correlation diagrams between the
NIR fluorescence and RLS intensities of the ternary aggregates (analyte – surfactant – dye I): with
SDS (c) and CTAB (d). The RLS and emission spectra for the other analytes are given in Fig. S6 in
ESI. In the correlation diagrams (c, d) the RLS signals were calculated as the intergal intensities of
the ternary aggregates spectra within 360–650 nm excluding the intensities of the binary aggregates
(surfactant – dye I).
11
Scheme 3. Suggested mechanism for fluorescence enhancement: dye fluorescence quenching in water
(process I); for cationic analytes: aggregation with SDS (process IIa) and solubilization of the dye
nanoparticles in the formed aggregate (process IIb); for anionic analytes: aggregation of dye
nanoparticles with CTAB (process IIIa) and inclusion of the anionic analyte into the aggregate with
solubilization of the dye nanoparticles (process IIIb).
12
4. Effects of the nature of the dye and surfactant on fluorescence enhancement
Above we considered the emission enhancement scheme for dye I and two surfactants (SDS
and CTAB). Below we provide the data for some other surfactants and dyes.
Nature of the surfactant. Studies of a number of surfactants with dyes I–III and four selected
analytes (Fig. S8) showed that the most efficient surfactants for sensing cationic and anionic analytes
are SDS and CTAB, respectively. All other surfactants used (including non-ionic ones) yield either a
high blank signal or (more frequently) almost no signal with respect to the blank.
To explore role of surfactant, two systems were examined in TEM (Fig. 7): dye I – ceftriaxone
– CTAB (fluorescent) and dye I – cetriaxone – benzyldimethyltetradecylammonium chloride
(BDTAC) (non-fluorescent). When CTAB is used, no dye nanoparticles were found over the whole
TEM sample, which implies that the dye was completely solubilized in the aggregates, as can be
clearly seen in the images (Fig. 7a, b). On the contrary, in the case of BDTAC (Fig. 7b, c), numerous
dye nanoparticles, which are apparently unable to solubilize in this surfactant – analyte pair and thus
remain non-fluorescent, were observed. These results imply that the dye nanoparticles can be
converted into molecular form when fluorescent aggregates are formed; on the other hand, no
dissolution is observed in the system without fluorescence. We believe these data are a strong
argument in favor of the suggested mechanism for fluorescence enhancement (Scheme 3).
The results (Fig. 7) confirm that not all surfactants of the proper charge will form ternary
aggregates and cause emission enhancement: there should be certain affinity of the surfactant to the
dye nanoparticles.
Fig. 7. TEM images of dye I – ceftriaxone mixture with two surfactants: a, b – CTAB (NIR
fluorescence), c, d – benzyldimethyltetradecylammonium chloride (no NIR fluorescence).
Conditions: 1.5 mM analyte, 5 M dye I, 0.1 CMC of each surfactant, no buffer. The NIR images of
the vials with the said solutions are also shown.
13
Effect of dye hydrophobicity. All dyes provide signals with anionic and cationic analytes
(ceftriaxone and neomycin, respectively, are shown as examples in Fig. 8, see also Fig. S4). However,
sensing is only feasible on a low background level (signal without analytes). The low backround in
submicellar CTAB or SDS is observed for the most hydrophobic dyes, I and II. Emission of the less
hydrophobic dyes is insufficiently quenched in water, and these dyes demonstrate a higher
background signal with SDS (dye VI), CTAB (dyes III, V) or both surfactants (dye IV); the most
hydrophilic dye IV is not useful for sensing at all under these conditions. The hydrophobic property
of a carbocyanine dye is important for the suggested sensing scheme because the dye should (1) form
quenched nanoparticles in aqueous solution (ensuring low background signal) and (2) be efficiently
included in the ternary aggregates along with the analyte and the counter-ion (ensuring the high signal
of interest). From this viewpoint, dye III (logP = 6.9) is inferior to dyes I and II (logP = 9.7 and 9.0)
as it yields a higher blank signal in the submicellar CTAB (Fig. 8). Future development should be
directed to synthesizing carbocyanine dyes with logP of 9 and higher.
Fig. 8. NIR fluorescence image of the plate wells with surfactant – analyte mixtures with dyes I–VI
of different lipophilicity (measured as logP*). The surfactants are added at concentrations of onetenth of their CMC (0.1 mM CTAB, 0.7 mM SDS), pH 7.4 (7 mM phosphate buffer), 1.5 mM
analyte, 5 M dye.
* Distribution coefficient of the molecular form in n-octanol–water system calculated using
https://www.molinspiration.com/cgi-bin/properties
14
5. Sensing of neomycin with dye I and SDS
The selective signal induced by the aminoglycoside antibiotics allowed us to develop a NIR
test for neomycin using dye I and submicellar SDS. Testing was performed in a 96-well plate, the
signal was recorded by using a NIR photo camera (Fig. S1) and digitized using ImageJ software.
Selection of conditons. Dependence of the signal on the reactant concentrations is shown in
Fig. 9. Excessive amount of dye results in a lower signal, probably, due to exceeding the
solubilization capacity of the aggregates. The working pH range is 4–8, and the SDS concentration in
the reaction solution should not exceed 2 mM (note that the CMC of SDS in pure water is 8 mM).
The signal can be measured directly after mixing the solutions and does not change for hours
thereafter. The chosen conditions are given in caption to Fig. 9.
Fig. 9. NIR fluorescent signal of dye I in the presence of neomycin and without it for different
concentrations: of the dye (a), SDS (b), and pH (c). General conditions: pH 7.4 (7 mM phosphate
buffer), 0.7 mM SDS, 2.5 M dye I, 0.4 mM neomycin. In varying pH, the following buffers were
used in 7 mM concentrations (by anion): borate (pH 9–10), phosphate (pH 5.5–8.1), acetate (pH 3.5–
5.0), and HCl (pH 1.8–2.8).
Selectivity of neomycin sensing was characterized by cross-reactivity and interferences. To
evaluate cross-reactivity, the system response to various organic species and inorganic salts was
studied without neomycin. As it can be seen from Fig. 10a, signals were obtained from neomycin
itself and other aminoglycosides (amikacin, gentamycin and streptomycin, wells No 3–5), while the
compounds of other classes yielded virtually no emission. To study the interferences of the same
species with neomycin detection, the signals of their mixtures with neomycin were measured (Fig.
10b; Fig. S9 in ESI contains the quantitative data for the images of Fig. 10). Neomycin response was
only reduced by Cu2+ and Fe3+ salts due to their quenching effect; smaller amounts of these ions did
not interfere (0.05 mM Cu2+ and 1.5 mM Fe3+ in the final solution). We can conclude that the
15
detection of neomycin is selective with respect to organic species, including large anionic molecules
(cephalosporins, penicillins), as well as non-charged species, amino acids and inorganic salts.
Fig. 10. Images of the plates for the system dye I – SDS without neomycin (a) and with it (b) in the
presence of equal molar amounts of diverse species. Numbers of wells: 1a — blank, 2a, 2b – only
neomycin, 3 — amikacin*, 4 — gentamicin, 5 — streptomycin*, 6 — arginine, 7 — diethylamine, 8
— cefazolin**, 9 — ceftazidime**, 10 — cefotaxime**, 11 — ceftriaxone**, 12 — ampicillin**, 13
— aspartic acid, 14 — trisodium citrate, 15 — pefloxacin, 16 — phenylalanine, 17 — glycine, 18 —
proline, 19 — histidine, 20 — cysteine, 21 — glutamine, 22 — serine, 23 — aniline, 24 — glucose,
25 — sulfathiazole, 26 — urea, 27 — K4P2O7, 28 — chloride**, 29 — nitrate**, 30 — sulfate**, 31
— acetate**, 32 — KCl, 33 — CaCl2, 34 — KI, 35 — MgCl2, 36 — (NH4)2SO4, 37 — FeCl3, 38 —
CuSO4. Conditions: 0.7 mM SDS, pH 7.4 (7 mM phosphate buffer), 4 M dye I, 1.5 mM neomycin.
Note. The NIR light, as not perceived by eye, should be imaged in black and white, but in
Figs. 10, 11 we are keeping the red hue yielded by the digital camera as more convenient for
perception.
* As sulfates **As sodium salts
Determination of neomycin in urine. Urine samples collected from healthy volunteers of
Moscow University were spiked with neomycin solution at different concentrations. Determination
was performed in a 96-well plate with photographic recording of the NIR signal. The amount of urine
was 1/7 (v:v) of the resulting solution, as higher amounts increased the background signal. The
16
calibration plots for the determination of neomycin in aqueous buffer practically coincided with those
in urine, which confirmed the high selectivity of the method (Fig. 11). The fluorescent signal is
proportional to the log concentration of neomycin within 0.05 – 2 mM (initial concentration in the
aqueous solution or sample) with an average RSD of 0.07 (in water) and 0.09 (with urine) for the said
concentration range and a limit of detection of 4×10–5 M (by 3s criterion).
Fig. 11. Calibration plot for sensing neomycin in distilled water and in urine. Insert: images of the
plates with the corresponding concentrations of neomycin in urine and water. The following solutions
were added to each well: 30 L of 8 mM SDS, 30 L of 67 mM phosphate buffer, 30 L of 25 M
dye I in water, 50 L of urine sample spiked with neomycin or an aqueous neomycin solution, and
220 L of water. Four parallel runs were performed for each concentration of neomycin. The
exposure time was 0.2 s. Confidence intervals for the error bars were calculated as
, where t is
Student's coefficient (3.18), s is standard deviation, and n is number of runs (4).
Detection of other aminoglycosides under the conditions of neomycin detection. Other
aminoglycosides can also be detected in urine. The logarithmically linear relationships between
fluorescence intensity and concentration in urine were observed for gentamycin and streptomycin
(Table S2) with LODs for of 50 and 100 М, respectively. The interferences were similar to those
found for neomycin (Fig. 10).
17
Conclusions
This study reveals a number of aggregational effects that deserve interest. First is the
formation of hydrophobic dye nanoparticles in water. Quenching in water is usually associated with
the aggregation of the dye, but the information on the concrete structure of those aggregates is very
scarce.
Second is the ability of the dye nanoparticles to solubilize in the analyte – surfactant
aggregates and the ability of anionic analytes to enter the dye – surfactant aggregates; both processes
involve transfer of the dye into its fluorescent (probably, molecularly dispersed) form within the
aggregates, resulting in emission enhancement. Lack of investigation of such processes possibly
resulted in gap in the literature. The formation of the aggregates is governed (1) by the analyte charge
(the aggregates only can be formed from species oppositely charged to the used surfactant), (2) the
ability of analyte to form multiple bonds (small ions do not induce aggregation). Such requirements
are necessary but insufficient, as the third component must have an affinity to the binary aggregate as
follows: dye to the cationic analyte – surfactant, or anionic analyte to the dye – surfactant. These
limitations will allow for tuning the selectivity by varying the nature of the surfactant and
fluorophore.
The third phenomenon to be emphasized is an unusual role of surfactant as not a micelleforming but an aggregate-forming agent. A cationic surfactant can aggregate the dye nanoparticles,
presumably due to cation- interactions; an anionic surfactant aggregates with the highly charged
aminoglycosides. However, it is likely that within those aggregates the surfactants exist in the form of
micelle-like structures bound together (with the analyte ions or the dye nanoparticles) by cooperative
bonding. It is essential, that these aggregational events only take place in submicellar solutions, as
surfactant micelles solubilize all types of aggregates. The micellar solutions are not of interest from
the sensing viewpoint, as the dyes are brightly fluorescent in the micelles without the analytes.
Fourthly, the selective signal of aminoglycosides over other species is not caused by selective
interaction with the dye (because the analytes have no direct contacts with the dye) or even with the
surfactant (though aminoglycosides do form the aggregates with SDS). Rather, the selective signal is
due to formation of the aggregate, which is cross-linked by multiple non-covalent bonds to yield a
nanoparticle that, in turn, is capable of solubilizing the dye and converting it into the fluorescent
form.
From the practical viewpoint, the sensing scheme provides an approach for the detection of
large species, multiple charged ions or molecules capable of multi-point binding. In comparison to
18
other instrumental techniques, the method is more rapid and simple; the signal can be registered by a
modified digital camera without using a stationary instrument. As compared to immune-based tests,
the suggested method does not require any enzymes or antibodies. In contrast to direct fluorescent
methods, the described approach does not require a sophisticated synthesis of receptors; the synthesis
of fluorophores is performed using standard methods, and even commercially available dyes can be
used. The "turn-on" signal in the NIR region minimizes the interferences from a biological matrix.
Such schemes may be used as an alternative to fluorophore–spacer–receptor (FSR) sensors.
The selectivity and sensitivity of the fluorescent methods based on the suggested scheme can
be further improved as described below. We suggest that a selective sensing method can be developed
for a target analyte provided a complementary counter-ion (not a surfactant) could be found to form a
stable ion pair with the analyte. If both particles can form multi-point bonds, the resulting ion pairs
will be able to produce large aggregates (nanoparticles). A requirement to these nanoparticles is
formation of hydrophobic domains which are necessary to solubilize a hydrophobic dye with
emission enhancement. This strategy has exciting prospects as it paves the way for obtaining
fluorescent signals for a wide range of ionizable species of middle molecular weight such as
medicinal drugs or other physiologically active compounds that are capable of multi-point binding.
Successful development of such methods will widen the range of analytes of interest to be determined
by fluorescent techniques.
Acknowledgements
The authors thank Russian Foundation for Basic Research for the financial support (grant No 20-0300334a). We are indebted to Dr. N.S.Melik-Nubarov for an exceptionally fruitful discussion. We
thank Aleksey Dobrotvorsky (photodrom.com) for equipping the photo camera with the NIR light
filter and Dr. N.A.Paramonov for language advice.
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Credit author statement
Sofia A. Zakharenkova: investigation, writing – original draft, visualization
Ekaterina A. Katkova: investigation,
Irina A. Doroshenko: investigation,
Anna S. Kriveleva: investigation,
Aleksandra N. Lebedeva: investigation,
Tatyana A. Vidinchuk: investigation,
Anna V. Shik: investigation,
Sergei S. Abramchuk: investigation,
Tatyana A. Podrugina: methodology, investigation,
Mikhail K. Beklemishev: conceptualization, supervision, funding acquisition, writing – review and editing
22
23

Formation of quenched carbocyanine nanoparticles in water

Submicellar surfactants as aggregate-forming agents

Fluorescence enhancement in dye–analyte–surfactant aggregates

Selectivity to large organic ions capable of cooperative binding

Rapid detection of neomycin in urine without using a spectrofluorimeter
24