Biosensors and Bioelectronics 224 (2023) 115062

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Biosensors and Bioelectronics

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

Development of an Au nanoclusters based activatable nanoprobe for NIR-II

fluorescence imaging of gastric acid
Miao Liang , Qing Hu , Shuxiao Yi , Yajie Chi , Yan Xiao *
Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry-of-Education Key Laboratory for the Synthesis and Application of Organic
Functional Molecules & College of Chemistry and Chemical Engineering, Hubei University, Wuhan, Hubei, 430062, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Gastric acid is an important functional substance secreted by the stomach of the living organisms, reflecting the
Gastric acid gastric physiological condition. The sensing of gastric acid in vivo is of great significance for evaluation of gastric
Activatable nanoprobe function, diagnosis and treatment of gastric diseases and maintenance of organism health but remains chal­
Fluorescence imaging
lenging due to the harsh acid and digestive environment of stomach. This study developed an activatable
Near-infrared II
Au nanoclusters
nanoprobe based on Au nanoclusters (Au NCs) for sensitive and real-time noninvasive near-infrared II (NIR-II)
fluorescence imaging detection of gastric acid in vivo for the first time. The Au NCs were encapsulated by
polydopamine to have enhanced NIR-II luminescence and high stability and combined with methylene blue to
possess the pH responsiveness for gastric acid imaging. The developed nanoprobe could not only monitor gastric
acid secretion in vivo but also imaging the changes of gastric acid caused by feeding, acid-inhibition drugs and
gastric ulcer disease. This study provides a promising avenue for the improvement of the application perfor­
mance of Au NCs and imaging analysis of gastric acid and related gastric diseases.

1. Introduction gastric acid detection is still a formidable challenge but valuable issue.
Therefore, in an effort to tackle this dilemma, opening up new avenues
At present, with the increase in the variety of foods and medicines as for simple, safe and non-invasive sensing of gastric acid in vivo is in
well as the unhealthy eating habits, gastric diseases such as chronic urgent demand.
gastritis, gastric ulcer and gastric carcinoma have developed into a kind Fluorescence (FL) imaging technology has emerged as a forceful
of prevalent diseases, seriously affecting human nutritional intake, strategy to investigate the physiological conditions in vivo in terms of
health and life (Dohi et al. 2019, 2020; Liu et al. 2021b, 2022). The non-invasive, simple operation, high spatial and temporal resolution
real-time monitoring of the gastric acid in vivo can offer major benefits and in situ real-time recognition (Gao et al., 2017; Meng et al., 2021;
in assessing gastric function, timely and accurately diagnosing and Wang et al., 2021). In especial, the emerging near-infrared II (NIR-II,
treating these diseases. As one of substances secreted by the stomach, 1000-1700 nm) fluorescence imaging with reduced tissue
gastric acid is closely related to gastric function and plays vital role in self-absorption and scattering as well as autofluorescence, and deep
the digestion and absorption of nutrients and modulation of the gastric tissue penetration depth is an indispensable technology in field of bio­
environment (Allan Gray and Shiner, 1967; Huang et al., 2019; Ma et al., logical imaging (He et al., 2018; Liu et al., 2020). However, although a
2019). Deviant gastric acid secretion is involved in many gastric diseases series of NIR-II fluorescent materials including quantum dots (QDs),
and can reflect some alterations of physiological and pathological of rare-earth doped nanoparticles (RENPs), organic dyes and so on have
stomach. The traditional detection methods for gastric acid mainly been developed for various biomedical applications in vivo (Ding et al.,
include gastric juice extraction, ingestible electronic capsules and 2021; Huang and Pu, 2020; Zhong and Dai, 2020), real-time in vivo
endoscope (Ghosh et al., 2011; Gursoy et al., 2008; Winder et al., 2019). NIR-II imaging of gastric acid during actual physiological process has yet
However, these methods either suffer from complicated and to be demonstrated. The reasons mainly attribute to that harsh gastric
time-consuming sample collection, serious side effects or are prone to conditions involving the strong acidic and enzymatic bio-contexts may
harm or discomfort to patients, rendering the safe and comfortable lead to instability and fluorescence quenching of most NIR-II emitters,

* Corresponding author.
E-mail address: [email protected] (Y. Xiao).

https://doi.org/10.1016/j.bios.2023.115062
Received 18 July 2022; Received in revised form 8 November 2022; Accepted 3 January 2023
Available online 4 January 2023
0956-5663/© 2023 Elsevier B.V. All rights reserved.
M. Liang et al. Biosensors and Bioelectronics 224 (2023) 115062

and some inherent deficiencies of luminescent materials such as inade­ infrared window and opened up a prospective approach for the diag­
quate quantum yield and biocompatibility (Li et al., 2020). Gold nano­ nosis of gastric function and diseases.
clusters (Au NCs), as a new type of NIR-II emitting nanomaterials, with
outstanding chemical stability and photostability, may offer us an 2. Experimental section
excellent opportunity to handle aforementioned limitations (Ma et al.,
2021a). Besides, Au NCs possess numerous superiorities including sim­ 2.1. Synthesis of Au NCs
ple synthesis, ultrasmall size, large Stokes shift, good water-solubility
and biocompatibility, making them promising alternative candidates GSH-protected Au NCs with NIR-II-emitting were synthesized ac­
for bioimaging and biosensing purposes (Qiao et al., 2020; Song et al., cording to a protocol described by Xie et al. with some slight modifi­
2020; Xiao et al., 2021; Yao et al., 2018). Using Au NCs to construct cations (Liu et al., 2019; Yuan et al., 2014). Briefly, freshly prepared
fluorescent nanoprobe for the real-time monitoring of gastric acid in GSH solution (1 mL, 10 mM) and HAuCl4 solution (250 μL, 20 mM) were
vivo may meet the needs of gastric function and disease-related added to 750 μL of ultrapure water under vigorous stirring. Subse­
research. quently, the white Au-(SR) complexes were formed and the solution
Toward this end, this study rational designed a pH-responsive NIR-II became cloudy. After adjusting the pH of solution to 11 by adding 1 M
fluorescent nanoprobe based on Au NCs, as shown in Scheme 1, for real- NaOH, 3 mL of ethanol and 100 μL of NaBH4 (11.4 mM in 0.2 M NaOH)
time imaging of gastric acid in vivo. Glutathione (GSH)-protected NIR-II were successively introduced into the mixture solution under vigorous
emissive Au NCs were first synthesized and then encapsulated with stirring. The solution was kept stirring at 600 rpm for 6 h to form Au
polydopamine (PDA). The encapsulation of PDA endowed Au NCs with NCs. Next, ethanol was added to precipitate Au NCs out of the reaction
enhanced NIR-II fluorescence emission and high stability, which are fluid. The precipitates were collected and washed with ethanol three
imperative in vivo imaging in harsh gastric conditions. In order to en­ times. Thereafter, as-obtained Au NCs were dissolved in ultrapure water
gineer the pH-responsive property of nanoprobe for gastric acid imag­ and purified using an ultrafiltration tube with a molecular weight cutoff
ing, the cationic methylene blue (MB) dye was loaded on the surface of (MWCO) of 10 kDa. Finally, the obtained Au NCs were stored in the
polydopamine-encapsulated Au NCs (Au NCs@PDA) by electrostatic refrigerator (4 ◦ C) for future use.
interaction to regulate NIR-II fluorescent signals of Au NCs. The adsor­
bed MB could quench the NIR-II fluorescence of Au NCs via photoin­ 2.2. Preparation of Au NCs@PDA
duced electron transfer (PET). Under the acidic conditions in gastric
acid, the protonation of PDA surface led to the shedding of cationic MB, 4.74 mg dopamine was dissolved in 49.5 mL PBS solution (10 mM,
and the NIR-II fluorescence of Au NCs@PDA were restored with the pH = 8.5) and then 500 μL of 5 mM Au NCs solution was added under
disappearing of the PET effect between Au NCs and MB. Thus, on the constant stirring. The mixed solution was stirred for 4 h in the air at
basis of this design, an activatable Au NCs@PDA-MB nanoprobe could room temperature to polymerize dopamine. After 4 h of reaction, the pH
be developed as a powerful tool to real-time imaging of gastric acid in of the above solution was adjusted to 6.5 with 1 M HCl to terminate the
vivo with satisfactory sensitivity and response speed. This nanoprobe reaction process. Thus, the polydopamine-encapsulated Au NCs (Au
possesses a series of salient merits: (1) compared with the common used NCs@PDA) were obtained and then dialyzed against ultrapure water
NIR-II fluorescent materials (ICG, Ag2S, NaYF4: Er/Yb), the as-prepared using a dialysis bag (10 kDa) overnight. The as-synthesized nano­
Au NCs materials enjoy outstanding fluorescence stability in harsh materials were stored at 4 ◦ C until use.
gastric conditions; (2) the activatable “off-on” strategy greatly reduces
background signal, resulting in high signal-to-noise ratio in imaging; (3)
the nanoprobe have sensitive and rapid response as well as good 2.3. Preparation of Au NCs@PDA-MB nanoprobe
biocompatibility for the sensing and imaging. With these features, the
as-constructed Au NCs@PDA-MB nanoprobe could successfully respond At room temperature, 360 μM of methylene blue solution was mixed
to gastric acid secretion in mice and monitor the changes of gastric acid with 250 μg/mL Au NCs@PDA solution. The mixture was stirred and
caused by feeding, acid-inhibiting drugs and anti-inflammatory drugs incubated for 5 min to obtain Au NCs@PDA-MB nanoprobes. Then, the
induced gastric ulcer disease. This work validated the possibility of real- mixture solution was centrifuged at 10,000 rpm for 10 min to collect the
time in vivo imaging monitoring of gastric acid in the second near- precipitate of nanoprobes. The precipitates were washed with ultrapure
water for three times. At last, the nanoprobes were redispersed in

Scheme 1. Schematic diagram of the construction and response of Au NCs@PDA-MB nanoprobe.

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ultrapure water and stored at 4 ◦ C for future use. 2.6. NIR-II fluorescence imaging of gastric ulcer

The gastric ulcer model of mice was constructed according to the

2.4. In vivo NIR-II fluorescence imaging of gastric acid secretion reported literature (Fiorucci et al., 2011; He et al., 2019; Süleyman et al.,
2002). Firstly, the BALB/c mice were fasted for 24 h and further treated
For gastric acid secretion imaging, eight-week-old mice were first for intragastric administration of ibuprofen (500 mg/kg) to induce acute
fasted for 7 h and then intragastrically administered with 200 μL of 200 gastric ulcer. After 4 h, the mice were operated with 100 μL of 200
μg/mL Au NCs@PDA-MB nanoprobe solution. Subsequently, images of μg/mL Au NCs@PDA-MB nanoprobe solution by gavage and used for
the mice were captured at different time points post gavage using the NIR-II fluorescence imaging experiments.
NIR-II fluorescence imaging system. An 808-nm diode laser with power
density of 0.1 W/cm2, which is lower than human skin-sustainable 3. Results and discussion
maximum value (~0.4 W/cm2) permitted by the American National
Standards Institute (Cao et al., 2018; Yuan et al., 2012), was employed to 3.1. Synthesis and characterization of Au NCs
provide the excitation light. The emission was collected with the 1000
nm long-pass (LP 1000) filter. After fluorescence imaging, BALB/c mice The GSH-protected Au NCs with NIR-II emission were synthesized by
were sacrificed and their stomachs were taken for comparison. reducing Au(I)-SG complexes with NaBH4 under alkaline conditions and
The gastric acid imaging experiments of fasting and feeding mice characterized in detail by UV-vis absorption and fluorescence spectros­
were as follows: for gastric acid imaging of fasting mice, BALB/c mice copy, transmission electron microscope (TEM), X-ray photoelectron
were fasted for 7 h, and then intragastrically injected with 100 μL of 200 spectroscopy (XPS) and Fourier transform infrared (FT-IR) spectroscopy.
μg/mL Au NCs@PDA-MB nanoprobe solution; for gastric acid imaging of As shown in Fig. 1A, the UV-Vis absorption spectrum of Au NCs
feeding mice, BALB/c mice were fasted for 7 h, followed by feeding for 2 exhibited two characteristic absorption peaks at 450 nm and 670 nm and
h, and then injected with 100 μL of 200 μg/mL nanoprobe solution. the fluorescence spectrum showed their emission centered at 1050 nm,
Finally, the NIR-II fluorescence signals of the nanoprobe in the stomach which were following with the optical characteristics of Au25 NCs (Liu
of mice were observed by in vivo imaging system. et al., 2019; Yang et al., 2020). The NIR-II fluorescence imaging of Au
NCs at different concentrations confirmed the good dispersibility of Au
NCs in solution (Fig. S1). The luminescence intensity of Au NCs was
2.5. NIR-II fluorescence imaging of gastric acid suppression in mice
linear with the concentration of Au NCs, indicating no obvious
agglomeration-induced quenching phenomenon existed in highly
For the acid suppression model introduction, NaHCO3 was used as
concentrated Au NCs solution. The synthesis of ultrasmall Au nano­
the antiacid drug to regulate the gastric acid in the stomach of BALB/c
materials was verified by TEM images, which displayed that the
mice. Before imaging, BALB/c mice were firstly fasted for 7 h and
as-prepared Au NCs had a good monodispersity with an average size of
randomly divided into three groups. Then, the mice in the two experi­
1.62 ± 0.25 nm and the crystal plane spacing of 0.2 nm (Fig. 1B and C).
mental groups were intragastrically administered with 100 μL of 200 μM
Additionally, in the FT-IR spectrum of Au NCs, the S-H stretching ab­
and 500 μM NaHCO3 solution, respectively. The mice in the control
sorption peak of GSH at 2526 cm− 1 disappeared, indicating that GSH
group were administered with 100 μL water. After 10 min, the mice were
was connected to the surface of Au NCs through the strong combination
all injected with 100 μL of 200 μg/mL Au NCs@PDA-MB nanoprobe
of Au and S (Fig. 1D). The element composition and oxidation state of Au
solution and then imaged at different time points post gavage with the
NCs were determined by XPS analysis. (Fig. S2). The main constituent
imaging system.

Fig. 1. (A) UV absorption (black line) and NIR-II fluorescence (red line) spectra of Au NCs. (B) TEM image of Au NCs (Insert: particle size distribution). (C) High-
resolution TEM image of Au NCs. (D) FT-IR spectra of Au NCs (red line) and GSH (black line). (E) NIR-II fluorescence spectra of Au NCs in the presence of different
concentrations of DA (0-3 mM). (F) NIR-II luminescence images of Au NCs (left) and Au NCs in 3 mM DA solution (right).

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elements of Au NCs are C, N, O, S and Au, which are consistent with in enhanced NIR-II fluorescence of Au NCs, revealing that the presence
synthetic raw materials. The XPS spectrum of Au 4f in Au NCs showed of O2 would quench the NIR-II fluorescence of Au NCs (Fig. S7A). In
two peaks at 84.5 eV and 88.2 eV, which were identified as the binding addition, with the increase of DA concentration, the NIR-II fluorescence
energies of Au 4f7/2 and Au 4f5/2 electrons, respectively. The Au 4f7/2 intensities of Au NCs solution without N2 infusion increased gradually
peak (84.5 eV) located between Au (0) (84.0 eV) and Au(I) (86.0 eV), while that of Au NCs solution with N2 infusion had almost no fluctuation
demonstrating that Au (III) in raw materials was reduced to Au (0) and (Fig. S7B). These results suggested that the DA induced enhancement of
Au (I). Above results confirmed the successful synthesis of the NIR-II luminescence of Au NCs solution was attributed to the con­
ultra-small GSH-protected Au NCs with NIR-II luminescence. sumption of dissolved O2 surrounding Au NCs by DA, which reduced the
quenching effect of O2 on the Au NCs and improved their NIR-II
3.2. DA-induced NIR-II luminescence enhancement of Au NCs fluorescence.

Since the luminescence quantum yield (QY) of the nanomaterial 3.3. Preparation and characterization of Au NCs@PDA
plays a vital role in the performance of the constructed nanoprobe, we
first attempted to improve the QY of the as-synthesized Au NCs. In the Inspired by the above interesting findings, polydopamine-
experiments, we found that the NIR-II emission of Au NCs solution could encapsulated Au NCs (Au NCs@PDA) composite materials were pre­
be enhanced by the introduction of dopamine (DA). As presented in pared to achieve the improved NIR-II fluorescence of Au NCs in aqueous
Fig. 1E, the NIR-II fluorescence intensity of Au NCs gradually increased solution. Au NCs@PDA were synthesized by encapsulating Au NCs
with adding of 0–3 mM DA. The NIR-II imaging of Au NCs solution in through self-polymerization of DA under weakly alkaline conditions (Li
centrifugal tube also confirmed this phenomenon (Fig. 1F). In addition, et al., 2016; Nurunnabi et al., 2013). Characterization and performance
the enhancement of luminescence was very rapid. After DA was added, tests of the as-prepared Au NCs@PDA were performed. TEM charac­
the fluorescence of Au NCs reached a maximum within 30 s and was able terization showed Au NCs@PDA were dispersed spherical particles with
to remain in the state (Fig. S3). We tested the change of the QY of Au NCs the size in the range of 80–150 nm and ultra-small Au NCs were packed
upon the introduction of DA. Taking the NIR-II fluorescent dye IR-26 as a in PDA (Fig. 2A and B). Peaks for Au element were found in energy
reference, the QY of Au NCs and Au NCs in 3 mM DA were calculated to dispersive spectroscopy (EDS) of Au NCs@PDA, also supporting the
be 0.34% and 0.65%, respectively (Fig. S4), which are very close to or encapsulation of Au NCs in PDA (Fig. 2C). Additionally, the UV-vis ab­
even higher than the QY of some NIR-II emitters (Liu et al., 2021a; Sun sorption spectrum of Au NCs@PDA showed the superposition of the
et al., 2016). On this basis, whether the luminescence enhancement absorption features of Au NCs and PDA (Fig. 2D). Compared with the Au
induced by DA would be applicable to other metal NCs were then NCs, the Au NCs@PDA exhibited an increased NIR-II fluorescence in­
studied. Some NIR-I emissive NCs and other NIR-II emissive NCs were tensity (Fig. 2E) and negative charge (Fig. 2F). The FT-IR absorption
selected to investigate their fluorescence in the presence of DA. As spectrum of Au NCs@PDA displayed characteristic absorption peaks
shown in Fig. S5, after introducing DA, the fluorescence of NIR-I emis­ consistent with the absorption spectrum of PDA (Fig. S8).
sive metal NCs such as Au22NCs (755 nm), Ag NCs (690 nm) and Prior to the using for practical application, we examined the stability
Au25NCs (800 nm) appeared negligible change. In contrast, the fluo­ of the as-prepared Au NCs@PDA. Firstly, the photostability of Au
rescence intensities of NIR-II emissive Au NCs with different ligands NCs@PDA was studied by irradiating them continuously with laser light
were apparently increased. Therefore, the NIR-II luminescence mecha­ (808 nm, 0.6 W). The NIR-II fluorescence of Au NCs@PDA maintained
nism of metal NCs should be different from their mechanism of NIR-I good photostability under laser irradiation without severe photo­
luminescence (Zhou and Song, 2021). The introduction of DA is a sim­ bleaching (Fig. S9A). Next, the stability of Au NCs@PDA in the high
ple and effective way to improve the NIR-II fluorescence of Au NCs concentration salt solution was explored. The NIR-II emission intensity
solution. of Au NCs@PDA in NaCl aqueous solutions only dropped a little with the
Subsequently, the mechanism of DA-induced NIR-II fluorescence concentrations of NaCl increasing from 0 to 1 M (Fig. S9B). Furthermore,
enhancement of Au NCs was further explored. First, the NIR-II emission the stability of Au NCs@PDA under pH conditions of 1–7 was investi­
intensities of Au NCs upon the addition of different small molecules were gated, which showed only slightly decrease of NIR-II fluorescence in­
investigated (Fig. S6A). The results revealed that small molecules, tensity of Au NCs@PDA as pH decreased (Fig. S9C). To verify the
similar in structure to DA, with multiple phenolic hydroxyl groups and application capability of Au NCs@PDA in fluorescence imaging of
strong reducing ability, such as isoproterenol (ISO) and ascorbic acid gastric acid, the NIR-II fluorescence stability of Au NCs@PDA in simu­
(AA), could enhance the NIR-II fluorescence of Au NCs in solution. lated gastric juice (SGJ, pH = 1.5) was examined. In comparison to
Accordingly, we speculated that the strong reducing ability of DA played commonly used NIR-II fluorescent materials (ICG, Ag2S, NaYF4: Er/Yb),
an essential role in the enhancement of luminescence. Next, UV-vis Au NCs@PDA showed outstanding luminescence stability under the
absorption and XPS analysis were executed to investigate whether the condition of SGJ. The NIR-II fluorescence intensity of ICG, Ag2S and
structure and chemical composition of Au NCs had changed after the NaYF4: Er/Yb decreased more than 80% within 15 min incubation in
addition of DA. As seen in the Fig. S6B, the UV-vis absorption spectrum SGJ, whereas the NIR-II fluorescence intensity of Au NCs@PDA could
of Au NCs did not change after mixing of Au NCs and DA, indicating that maintain almost unchanged (Fig. S10). In addition, there was no sig­
DA did not affect the structure and the HUMO-LUMO energy levels of Au nificant change in position or intensity of the fluorescence peaks of Au
NCs. Likewise, the Au 4f binding energy hardly altered after the addition NCs and Au NCs@PDA in a week (Fig. S11). These experimental studies
of DA, revealing that the state of Au element in Au NCs did not change illustrated the excellent stabilities of Au NCs@PDA, which are essential
(Fig. S6C). Moreover, TEM images stated DA exhibited no significant for practical application.
influence on the morphology and size of Au NCs (Figs. S6D and E). When
removed DA from the Au NCs solution, the fluorescence of Au NCs was 3.4. Preparation of Au NCs@PDA-MB nanoprobe
reduced to its original state (Fig. S6F). These data indicated that DA did
not change the chemical composition, structure and morphology of Au For the construction of gastric acid imaging nanoprobe, methylene
NCs. We thereupon speculated DA may affect the microenvironment blue (MB), an FDA-approved dye, serving as the molecule that regulate
around Au NCs, thereby improving their NIR-II fluorescence. Then, the fluorescent signals, were loaded onto the surface of Au NCs@PDA via
effect of dissolved O2 surrounding Au NCs on their NIR-II fluorescence electrostatic interactions (Fu et al., 2015; Li et al., 2019; Zheng et al.,
was studied. N2 bubbling was utilized to dislodge dissolved O2 from the 2015) (Scheme 1). The loading of MB on Au NCs@PDA was character­
Au NCs solution. Following the infusion of N2, NIR-II fluorescence signal ized by FT-IR spectra and Zeta potential measurements. The absorption
of Au NCs was measured. The removal of dissolved O2 obviously resulted peaks of C– – C(N) vibrations (1610 cm− 1) and asymmetrical bending

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Fig. 2. (A) TEM image of as-prepared Au NCs@PDA. (B) Enlarged TEM image of Au NCs@PDA. (C) EDS analysis of Au NCs@PDA. (D) UV absorption spectra of Au
NCs, PDA, and Au NCs@PDA. (E) NIR-II fluorescence spectra of Au NCs, PDA and Au NCs@PDA. (F) Zeta potentials of Au NCs and Au NCs@PDA.

vibrations of CH3 (1397 cm− 1) of MB existed in the FT-IR spectrum of Au calculated to be − 0.66 eV and 0.58 eV, respectively. The HOMO energy
NCs@PDA-MB nanoprobe (Fig. S12A). Besides, the loading of MB of MB (− 0.455 eV) is less negative than that of the HOMO energy of Au
induced the Zeta potential elevation from − 26 mV to − 18 mV of Au NCs, but is more negative than their LUMO energy (Yang et al., 2011).
NCs@PDA, indicating that positively charged MB was adsorbed on Au Consequently, the electron transfer from excited Au NCs to MB is
NCs@PDA (Fig. S12B). After combined with Au NCs@PDA, MB could feasible (Fig. 3A). The experimental results also confirmed this phe­
quench the NIR-II fluorescence of Au NCs via photo-induced electron nomenon. The NIR-II fluorescence intensity of Au NCs@PDA decreased
transfer (PET). We determined the highest occupied molecular orbital gradually with the loading of increasing concentrations of MB (Fig. 3B).
(HOMO) and lowest unoccupied molecular orbital (LUMO) of Au NCs by The decrease in fluorescence intensity showed a good linear relationship
cyclic voltammetry measurements. From the results displayed in with the concentration of MB (Fig. 3C). The NIR-II imaging of Au
Fig. S13, the HOMO and LUMO energy levels of Au NCs can be NCs@PDA before and after loading MB dramatically showed the

Fig. 3. (A) Schematic diagram of the PET process between Au NCs and MB. (B) NIR-II fluorescence spectra of Au NCs@PDA in different concentrations of MB (0-360
μM). (C) The relationship between the NIR-II fluorescence intensity of Au NCs@PDA and MB concentration (Insert: NIR-II luminescence images of Au NCs@PDA
before and after the addition of 360 μM MB). (D) NIR-II fluorescence spectra of Au NCs@PDA-MB nanoprobe under different pH conditions. (E) Plot of the fluo­
rescence intensity of Au NCs@PDA-MB nanoprobe as a function of pH value. (F) NIR-II luminescence images of Au NCs@PDA-MB nanoprobe at different
pH conditions.

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fluorescence quenching (Fig. 3C, insert), suggesting that MB had a good gastric acid imaging in vivo. The BALB/c mice were intragastrically
quenching effect on the NIR-II fluorescence of Au NCs. administrated with Au NCs@PDA-MB nanoprobes and then imaged by
NIR-II fluorescence imaging system with 808 nm laser excitation
3.5. The pH and gastric acid response of Au NCs@PDA-MB nanoprobe (Fig. S19). Accompanied by gastric acid secretion, the NIR-II fluores­
cence signal of the nanoprobe could be clearly detected at the mouse
Under acidic conditions, the protonation of the phenolic hydroxyl stomach without dissection (Fig. 4A). Benefiting from reduced scat­
group on the surface of PDA decreased their surface negative charge and tering and negligible autofluorescence of NIR-II fluorescence imaging,
weakened their adsorption capacity for MB (Zheng et al., 2015). no obvious background fluorescence signal was observed beyond the
Thereby, MB would desorb from the Au NCs@PDA and their quenching target region in mice. With the increase of gastric acid secretion, the
effect on the luminescence of Au NCs would disappear, resulting in the NIR-II fluorescence intensity in the stomach of the mouse became strong,
restoration of the NIR-II luminescence of Au NCs in acidic conditions. To which could clearly delineate the outline of the stomach. The diameter
confirm the ability of the nanoprobe to respond to acidic pH, the NIR-II of the stomach determined from the full widths at half-maximum
fluorescence of Au NCs@PDA-MB nanoprobe upon different pH condi­ (FWHM) of imaging data was comparable to the actual measured
tions were investigated. The results showed the quenched NIR-II fluo­ stomach diameter after dissection of the mice, demonstrating accurate
rescence of Au NCs@PDA-MB nanoprobe recovered with the decreasing visualization capabilities of the nanoprobe for stomach (Fig. 4B and C).
of the pH of solution, which could be visualized by naked eyes (Fig. 3D, To further verify the response ability of Au NCs@PDA-MB nanoprobe
E and F). The nanoprobe possessed a fast response speed which would be to gastric acid in vivo, the performance of the nanoprobe to distinguish
beneficial for in vivo imaging. Under the condition of pH 2, the lumi­ between feeding and fasting mice was estimated. It has been reported
nescence of the Au NCs recovered in 1 min (Fig. S14A). After confirming that fasting mice have no food to stimulate gastric acid secretion,
the acidic pH responsiveness of the nanoprobe, the response of the resulting in less gastric acid in gastric juice, while feeding mice with
nanoprobe to gastric acid was further examined. The NIR-II fluorescence food stimulation generated a large amount of gastric acid (Feldman and
of the nanoprobe in water, simulated urine, simulated saliva and SGJ Barnett, 1991; Mahar et al., 2012). As shown in Fig. S20, after the
was measured, respectively. Only in SGJ, the NIR-II fluorescence of gavage of the nanoprobe for 5 min, the NIR-II fluorescence intensity of
nanoprobe was specifically activated and the luminescence could the stomach in feeding mice was significantly stronger than that of
remain stable for a long time (Figs. S14B and C). Based on above results, fasting mice, illustrating that the diet stimulated gastric acid secretion.
the Au NCs@PDA-MB nanoprobe is a promising tool for imaging of This phenomenon was further proved that the constructed Au
gastric acid in vivo. NCs@PDA-MB nanoprobe could respond to the change of gastric acid in
stomach.
3.6. Biotoxicity and in vivo metabolism of Au NCs@PDA-MB nanoprobes
3.8. NIR-II fluorescence imaging of the gastric acid suppression in mice
The low biotoxicity of nanomaterials is a prerequisite for their in vivo
application. To evaluate biotoxicity of the as-constructed Au NCs@PDA- Gastric acid suppression is very essential for treatment of gastric acid
MB nanoprobe, a series of experiments including the cytotoxicity, disorder-related diseases including gastroesophageal reflux, peptic ulcer
interference on mouse body weight and H&E staining analysis were and so on (Metz et al., 2000; Schubert and Peura, 2008). The research on
carried out. Firstly, the cytotoxicity of the nanoprobe towards HeLa cells gastric acid suppression has driven much of the development of
was assessed by methyl thiazolyl tetrazolium method. As presented in acid-suppression therapy. We thus sought to study the utilization of Au
Fig. S15, after the incubation of HeLa cells with different concentrations NCs@PDA-MB nanoprobe to imaging of gastric acid suppression in vivo.
of Au NCs@PDA-MB nanoprobes for 12 h, the cell viability maintained Sodium bicarbonate (NaHCO3), as an antacid, is often used to inhibit the
above 80%, which presented a low cytotoxicity of the nanoprobe.
Additionally, the flow cytometric analysis of calcein AM and propidium
iodide double staining displayed that the proportion of viable cells in the
concentration range of 0–120 μg/mL nanoprobes were all over 90%,
manifesting that the Au NCs@PDA-MB nanoprobe has no obvious
cytotoxicity in these concentration conditions (Fig. S16). Furthermore,
the weight change of BAlB/c mice within a week after intragastric
administration of the nanoprobes was measured. Consistent with the
weight changes in control group, the weight of the mice in the experi­
mental group did not decrease significantly (Fig. S17). On basis of this,
the feces of mice after the gavage of nanoprobe were collected for the
analysis of metabolism of the nanoprobe. The collected feces were
digested by aqua regia and subjected to inductively coupled plasma
atomic emission spectrometry (ICP-AES) analysis for the quantification
of the amount of Au element. The content of Au in feces accounted for
84.8% of the initial gavage content, indicating that most of the admin­
istered Au NCs@PDA-MB nanoprobes were excreted in the feces
(Table S1). Besides, the above mice were sacrificed and dissected 7 day-
post nanoprobe injection, and their hearts, livers, spleens, lungs and
kidneys were harvested for hematoxylin and eosin (H&E) staining
analysis, and no pathological change was observed (Fig. S18). These
results suggested that Au NCs@PDA-MB nanoprobes did not cause
apparent toxicological response and could be applicable for in vivo
imaging application.
Fig. 4. (A) NIR-II fluorescence imaging of the stomach of BALB/c mice at
3.7. NIR-II fluorescence imaging of gastric acid secretion in vivo different time points after Au NCs@PDA-MB nanoprobes gavage. (B) Intensity
profiles of stomach at cross section in figure A. (C) The photograph of the
We next attempted to employ the Au NCs@PDA-MB nanoprobe for stomachs dissected from BALB/c mice.

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M. Liang et al. Biosensors and Bioelectronics 224 (2023) 115062

gastric acid in gastric disease therapy (Ma et al., 2021b; Quade et al., could find obvious inflammation and hyperemia, gastric mucosal
2020). The BALB/c mice were pre-treated with different amounts of swelling and thinning. Besides, the H&E staining analysis of gastric
NaHCO3 by gavage and then intragastrically administered with the Au tissue showed the erosion of the surface epithelial cells, the decrease of
NCs@PDA-MB nanoprobes to observe the inhibition of gastric acid. In mucosal thickness and the edema and inflammatory cell infiltration in
Fig. 5A, after the administration of nanoprobe, the NIR-II fluorescence the submucosa, proving that ibuprofen successfully induced GU in mice
signal in the stomach of control mice appeared at the initial stage and (Fig. 6). Taken together, the Au NCs@PDA-MB nanoprobe might offer an
became obvious after 2 min, while the signal in acid-suppressed mice attractive strategy for imaging of GU in vivo.
emerged later and with weaker intensity. The fluorescence signal in­
tensity detected in the stomach of mice was decreased with the 4. Conclusion
increasing amount of pretreated NaHCO3 (Fig. 5B). These experimental
results declared that the nanoprobe was able to imaging of gastric acid In summary, this study synthesized GSH-protected Au NCs with NIR-
inhibition in vivo. The developed Au NCs@PDA-MB nanoprobe has a II-emitting and found that the introduction of DA could improve the
certain potential application value in drug development or gastric dis­ NIR-II fluorescence of Au NCs in solution. Enlightened by this interesting
ease therapy. finding, we encapsulated Au NCs with polydopamine, thus obtaining Au
NCs with enhanced NIR-II luminescence and high stability. On this basis,
a pH-responsive Au NCs@PDA-MB nanoprobe were developed by
3.9. Imaging analysis of gastric ulcer in vivo
loading MB on the surface of PDA-encapsulated Au NCs for the first real-
time NIR-II fluorescence imaging monitoring of gastric acid in vivo. On
Ibuprofen, as a kind of non-steroidal anti-inflammatory (NSAIDs)
account of the high sensitivity, rapid response and good biocompati­
drug, is often used to treat inflammatory diseases such as rheumatoid
bility, the developed nanoprobe could successfully be used to monitor
arthritis and relieve headaches or fever caused by colds (Süleyman et al.,
gastric acid secretion in vivo and imaging the different feeding states,
2002). However, excessive use of NSAIDs can cause gastric ulcer (GU), a
acid-suppressive drugs and gastric ulcer disease induced changes of
common gastrointestinal disease that can induce some complications,
gastric acid. The nanoprobe offers a potential tool for the gastric acid
such as gastrointestinal bleeding, ulcer perforation, and even gastric
imaging and the research on gastric related diseases in clinical medicine.
cancer (Fiorucci et al., 2011; He et al., 2019). The occurrence of GU can
The results furnish new insights toward the improvement of the optical
lead to the weakening of gastric function and its clinical manifestation is
properties of Au NCs and the NIR-II imaging of gastric acid in vivo.
the decrease of gastric acid secretion. In view of the good optical
properties and response ability of the Au NCs@PDA-MB nanoprobe, we
CRediT authorship contribution statement
proceeded to discuss the feasibility of using this nanoprobe for imaging
of GU in vivo. The BAlB/c mice were fed with ibuprofen suspension to
Miao Liang: Methodology, Investigation, Data curation, Writing –
induce acute GU. The results pertinent to the imaging displayed the
original draft. Qing Hu: Methodology, Data curation. Shuxiao Yi:
gastric NIR-II fluorescence signal intensity of control group was signif­
Investigation, Software. Yajie Chi: Validation. Yan Xiao: Supervision,
icantly higher than that of the GU group, confirming that the gastric acid
Writing – review & editing, Project administration, Funding acquisition.
secretion of the control mice was more than that of the mice with GU
(Fig. 6). This result demonstrated the Au NCs@PDA-MB nanoprobe
could differentiate drug-induced ulcerated stomach from normal stom­ Declaration of competing interest
ach by responding to gastric acid. To verify the successful construction
of GU model, we dissected the control and GU mice, and found that the The authors declare that they have no known competing financial
gastric diameter of normal mice was 6.5 mm while that of GU mice was interests or personal relationships that could have appeared to influence
swollen up to 8.5 mm (Fig. 6). In addition, the gastric tissue of GU mice the work reported in this paper.

Fig. 5. (A) Schematic illustration of the NIR-II fluorescence imaging of the gastric acid suppression in mice. (B) NIR-II fluorescence imaging of control and acid-
suppressed mice at different time points after the injection of Au NCs@PDA-MB nanoprobes. (C) Comparison of the NIR-II fluorescence signals from the stomach
of mice with different amounts of NaHCO3 treatment at 10 min post-injection of nanoprobes. (**: P < 0.01, ****: P < 0.001).

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M. Liang et al. Biosensors and Bioelectronics 224 (2023) 115062

Fig. 6. (A) NIR-II fluorescence imaging, photographs of stomach, and H&E staining of gastric tissue (left to right) of normal mice and gastric ulcer mice. (B)
Comparison of the fluorescence signals from the stomach of the normal and gastric ulcer mice. (***: P < 0.001).

Data availability Huang, J., Pu, K., 2020. Activatable molecular probes for second near-infrared
fluorescence, chemiluminescence, and photoacoustic imaging. Angew. Chem. 132
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Data will be made available on request. Huang, W., Chen, R., Peng, Y., Duan, F., Nie, L., 2019. In vivo quantitative photoacoustic
diagnosis of gastric and intestinal dysfunctions with a broad pH-responsive sensor.
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Natural Science Foundation of China (Grants 21605043) and the Natural J. Mater. Chem. B 4 (23), 4216–4226.
Li, R., Wang, W., Kong, Y., Jiang, J., Han, Z., 2020. Engineering corona structure on gold
Science Foundation of Hubei Province (Grants 2016CFB125). nanoclusters for gastrointestinal imaging by red-shifted emissions in the second
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