![Citation: Zhang, Z. Recent Progress
in Biosensors Based on
Biorecognition Molecules. Biosensors
2023, 13, 842. https://doi.org/
10.3390/bios13090842
Received: 21 August 2023
Accepted: 23 August 2023
Published: 24 August 2023
Copyright: © 2023 by the author.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biosensors
Editorial
Recent Progress in Biosensors Based on Biorecognition
Molecules
Zhen Zhang
School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China;
zhangzhen@ujs.edu.cn
Biosensors are considered a popular technology to rapidly detect targets, and are
generally composed of biorecognition molecules that specifically capture analytes and
signal elements. In terms of methodology, measurements are carried out in a limited time
without sophisticated sample pre-treatments, implying obvious advantages over traditional
instrument methods for rapid analysis [1].
Considering that biosensors are widely applied in environmental pollutant monitoring,
detecting hazardous substances in foods and disease diagnosis [2–4], their advances and
future trends should be highlighted, so we have organized this Special Issue entitled
“Bioassays and Biosensors for Rapid Detection and Analysis”. Among the 10 papers
published, 6 are articles, 3 are communications and 1 is a review.
Huang et al. believe that electrogenerated chemiluminescence (ECL) is a powerful
tool for sensitive and accurate detection of biological analytes and summarize the recent
advances in this field. Moreover, some future trends and challenges of ECL biosensors
are also discussed. They believe that after integration with low-cost photodetectors, ECL
biosensors will have great potential in commercial applications [5]. Meanwhile, several
works focus on contaminant determination in food and environments, like sulfadiazine
(SDZ) [6], deoxynivalenol (DON) [7] and dibutyl phthalate (DBP) [8]. With regard to
the detection systems, antibodies or aptamers were used to recognize the targets, which
contributed to good selectivity. In addition, some novel strategies for signal amplification
were employed to improve their analytical performance [8]. At the same time, sensitive
biosensors were fabricated for the rapid detection of the biomarkers related to some diseases,
which could be achieved easily [9,10].
Most studies focus on the sensitivity improvement of biosensors through introducing
new functional materials or simplifying the measurement procedure, which are definitely
important. However, more attention should be paid to real applications, increasing toler-
ances against various matrices that potentially influence the performance of biosensors.
Interestingly, in this Special Issue, two papers are related to commercial products using
bioassays or biosensors [11,12]. In short, biosensors will be widely used in various fields
after some problems have been overcome, including achieving satisfactory sensitivity and
accuracy and maintaining enzyme activity in varied harsh environments.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
Biosensors 2023, 13, 842. https://doi.org/10.3390/bios13090842 https://www.mdpi.com/journal/biosensors
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![Biosensors 2023, 13, 842
References
1. Zhang, Z.; Zeng, K.; Liu, J. Immunochemical detection of emerging organic contaminants in environmental waters. TrAC Trends
Anal. Chem. 2017, 87, 49–57. [CrossRef]
2. Pilolli, R.; Monaci, L.; Visconti, A. Advances in biosensor development based on integrating nanotechnology and applied to
food-allergen management. TrAC Trends Anal. Chem. 2013, 47, 12–26. [CrossRef]
3. Iwanaga, M.; Tangkawsakul, W. Two-Way Detection of COVID-19 Spike Protein and Antibody Using All-Dielectric Metasurface
Fluorescence Sensors. Biosensors 2022, 12, 981. [CrossRef]
4. Niehues, J.; McElroy, C.; Croon, A.; Pietschmann, J.; Frettlöh, M.; Schröper, F. Bacterial Lighthouses— Real-Time Detection
of Yersinia enterocolitica by Quorum Sensing. Biosensors 2021, 11, 517. [PubMed]
5. Huang, Y.; Yao, Y.; Wang, Y.; Chen, L.; Zeng, Y.; Li, L.; Guo, L. Strategies for Enhancing the Sensitivity of Electrochemiluminescence
Biosensors. Biosensors 2022, 12, 750. [CrossRef] [PubMed]
6. Zheng, X.; Yang, L.; Sun, Q.; Zhang, L.; Le, T. Development and Validation of Aptasensor Based on MnO2 for the Detection of
Sulfadiazine Residues. Biosensors 2023, 13, 613. [CrossRef] [PubMed]
7. Zeng, K.; Yang, J.; Su, H.; Yang, S.; Gu, X.; Zhang, Z.; Zhao, H. Enhanced Competitive Immunomagnetic Beads Assay Assisted
with PAMAM-Gold Nanoparticles Multi-Enzyme Probes for Detection of Deoxynivalenol. Biosensors 2023, 13, 536. [CrossRef]
[PubMed]
8. Meng, H.; Yao, N.; Zeng, K.; Zhu, N.; Wang, Y.; Zhao, B.; Zhang, Z. A Novel Enzyme-Free Ratiometric Fluorescence Immunoassay
Based on Silver Nanoparticles for the Detection of Dibutyl Phthalate from Environmental Waters. Biosensors 2022, 12, 125.
[CrossRef] [PubMed]
9. Alekhmimi, N.K.; Cialla-May, D.; Ramadan, Q.; Eissa, S.; Popp, J.; Al-Kattan, K.; Zourob, M. Biosensing Platform for the Detection
of Biomarkers for ALI/ARDS in Bronchoalveolar Lavage Fluid of LPS Mice Model. Biosensors 2023, 13, 676. [CrossRef] [PubMed]
10. Khan, A.; Di, K.; Khan, H.; He, N.; Li, Z. Rapid Capturing and Chemiluminescent Sensing of Programmed Death Ligand-1
Expressing Extracellular Vesicles. Biosensors 2022, 12, 281. [CrossRef] [PubMed]
11. Dobrynin, D.; Polischuk, I.; Pokroy, B. A Comparison Study of the Detection Limit of Omicron SARS-CoV-2 Nucleocapsid by
Various Rapid Antigen Tests. Biosensors 2022, 12, 1083. [CrossRef] [PubMed]
12. Johannsen, B.; Baumgartner, D.; Karkossa, L.; Paust, N.; Karpíšek, M.; Bostanci, N.; Zengerle, R.; Mitsakakis, K. ImmunoDisk—A
Fully Automated Bead-Based Immunoassay Cartridge with All Reagents Pre-Stored. Biosensors 2022, 12, 413. [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
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2](https://image.slidesharecdn.com/27345891-250521081118-d54eec0f/85/Bioassays-And-Biosensors-For-Rapid-Detection-And-Analysis-Zhen-Zhang-15-320.jpg)
![Citation: Zheng, X.; Yang, L.; Sun, Q.;
Zhang, L.; Le, T. Development and
Validation of Aptasensor Based on
MnO2 for the Detection of
Sulfadiazine Residues. Biosensors
2023, 13, 613. https://doi.org/
10.3390/bios13060613
Received: 27 April 2023
Revised: 27 May 2023
Accepted: 1 June 2023
Published: 3 June 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biosensors
Communication
Development and Validation of Aptasensor Based on MnO2 for
the Detection of Sulfadiazine Residues
Xiaoling Zheng, Lulan Yang, Qi Sun, Lei Zhang and Tao Le *
College of Life Sciences, Chongqing Normal University, Chongqing 401331, China;
2021110513064@stu.cqnu.edu.cn (X.Z.); 2020110513031@stu.cqnu.edu.cn (L.Y.);
sunqi2017@cqnu.edu.cn (Q.S.); 20132133@cqnu.edu.cn (L.Z.)
* Correspondence: letao@cqnu.edu.cn
Abstract: The monitoring of sulfadiazine (SDZ) is of great significance for food safety, environmental
protection, and human health. In this study, a fluorescent aptasensor based on MnO2 and FAM-
labeled SDZ aptamer (FAM-SDZ30-1) was developed for the sensitive and selective detection of SDZ
in food and environmental samples. MnO2 nanosheets adsorbed rapidly to the aptamer through
its electrostatic interaction with the base, providing the basis for an ultrasensitive SDZ detection.
Molecular dynamics was used to explain the combination of SMZ1S and SMZ. This fluorescent
aptasensor exhibited high sensitivity and selectivity with a limit of detection of 3.25 ng/mL and a
linear range of 5–40 ng/mL. The recoveries ranged from 87.19% to 109.26% and the coefficients of
variation ranged from 3.13% to 13.14%. In addition, the results of the aptasensor showed an excellent
correlation with high-performance liquid chromatography (HPLC). Therefore, this aptasensor based
on MnO2 is a potentially useful methodology for highly sensitive and selective detection of SDZ in
foods and environments.
Keywords: aptamer; MnO2; aptasensor; sulfadiazine
1. Introduction
Sulfadiazine (SDZ) is an antibiotic of the sulfonamide family, which is widely applied
to prevent and treat bacterial infections in livestock [1,2]. However, SDZ can lead to residues
in animal-derived foods when overused. These residues can not only accumulate in humans
through the food chain and cause serious health problems, but can also pollute soil or
water by the pathway of animal excretion, leading to ecotoxicological contamination [3,4].
After discovering its harmful effects, the maximum residue level of SDZ has been set at
100 μg/kg in the EU, the USA, and China. Therefore, monitoring the residues of SDZ
in food and the environment is of great significance [5,6]. In previous studies, SDZ was
detected by HPLC, capillary electrophoresis, enzyme-linked immunosorbent assay, and
time-resolved immunoassay [7–11]. Although these approaches are highly sensitive, the
necessity of specialized operators, high costs, weak stability of antibodies, and the potential
of cross-reactivity limit their application. Therefore, a simple, effective, and economical
method is required to measure SDZ in food and environmental risk monitoring.
Aptamer development as a new type of recognition probe provides new ideas for the
development of its detection. Aptamers are single-stranded DNA (ssDNA) or RNA that
specifically bind to target molecules. Aptamers have superior stability, excellent affinity,
strong specificity, and easy in vitro screening compared to antibodies [12–14]. With these
advantages, various aptamer-based sensors have been widely developed for environmental
monitoring and food safety inspection (e.g., fluorescent, electrochemical, surface-enhanced
Raman scattering, and photoelectrochemical sensing signals) [15–17]. In comparison, flu-
orescent aptasensors have been widely used in the field of detection for their ability of
sensitivity, low cost, and selectivity, which make them particularly useful in monitoring
Biosensors 2023, 13, 613. https://doi.org/10.3390/bios13060613 https://www.mdpi.com/journal/biosensors
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food and environmental samples [18,19], and fluorescent aptamer sensors based on nano-
materials (quantum dots, graphene oxide, nanosilicon, etc.) have attracted considerable
attraction [20–22]. In particular, manganese dioxide (MnO2) has attracted much attention
for its low cost, strong molar extinction coefficient, environmental tolerance, and non-
toxicity, which has been widely used in biomedical and biosensing fields [22,23]. As a
constantly developing two-dimensional nanomaterial, MnO2 nanosheets are easy to pre-
pare on a large scale and possess broad absorption peaks that can overlap with the emission
spectra of many fluorescent materials, making them considered to be quencher candidates.
In addition, because MnO2 has a large specific surface area and good biocompatibility, it
can greatly improve the detection of targets. Xu et al. reported that MnO2 nanosheets
adsorb ssDNA through van der Waals forces interacting with bases, thus exhibiting strong
absorption and efficiency of fluorescence burst [24]. Consequently, Qin et al. established
an upconversion MnO2-based biosensor for the detection of carbendazim pesticides in
food [25]. Li et al. constructed an open fluorescence sensor based on carbon dot-labeled
oligodeoxyribonucleotide and MnO2 nanosheets for the detection of mercury(II) [26]. How-
ever, MnO2-based fluorescent aptasensors for the detection of SDZ residues have not been
reported so far.
In this research, we constructed an aptasensor for selective detection of SDZ using
FAM-labeled aptamer (FAM-SDZ30-1) and MnO2 nanosheets as donor-acceptor pair for
the first time. Molecular dynamics simulations were performed to study the reason for the
high specificity for SDZ by SDZ30-1. The performance of the established aptasensor was
tested in food and environmental samples by adding different concentrations of SDZ to
five samples, including soil, lake water, river water, egg, and beef. Finally, we compared
the results of this method with HPLC and explored their correlation, demonstrating that
the aptasensor can detect SDZ in a variety of samples.
2. Materials and Methods
2.1. Chemicals and Materials
The SDZ 30-1 (5-AACCCAATGGGAT-3, Kd = 65.72 nM) was selected in our labo-
ratory, which was purified using HPLC and synthesized by Sangon Biotech, Shanghai,
China [24]. All antibiotics, e.g., SDZ, tetracycline (TC), furaltadone (FTD), and norfloxacin
(OFL) were bought from Sigma-Aldrich, St. Louis, MO, USA. KMnO4, hexadecyl trimethyl
ammonium bromide (CTAB), buffer, and other reagents were purchased from Aladdin Co.,
Ltd., Ontario, CA, USA. The accuracy of the method was verified by HPLC on LC-20A
(Shimadzu, Kyoto, Japan). Fluorescence intensity measurements were performed at the
excitation wavelength of 492 nm and emission wavelength of 518 nm using Varioskan LUX.
2.2. Synthesis of MnO2
The MnO2 nanosheets were prepared as previously described [23]. Specifically, 0.5
g KMnO4 was dissolved in 450 mL of water and stirred for 30 min, then 1.5 g of CATB
was added to form a stable emulsion. Next, 50 mL of 0.1 M MES (pH 6.0) was added to
the above mixture and stirred for 12 h until a black color was formed. The product was
centrifuged at 8000 rpm for 10 min and then washed thrice alternately with water and
alcohol. Finally, the collected product was dried under a vacuum at 60 ◦C for 12 h. MnO2
was characterized by transmission electron microscope images (TEM), the UV-Vis, EDS
elemental mapping and Fourier transform infrared spectrometer (FTIR).
2.3. Molecular Dynamics Simulation
The interactions between aptamers and different targets were compared through
molecular dynamics simulations [27,28]. Firstly, the structures and parameters of the
aptamer and target molecules were obtained from SwissParam. Subsequently, implicit
solvation simulations were performed for the aptamer to target under constant temperature,
volume, pressure, etc., while the obtained structures were subjected to an explicit solvent
model of molecular dynamics simulations. Finally, the stability was measured using the
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![Biosensors 2023, 13, 613
root mean squared deviation (RMSD) produced by the molecular dynamics simulations
trajectory. When the RMSD curve showed a slope upward tendency, the conformation of
the system might undergo significant movement, and the smooth oscillation of the RMSD
curve around a certain height indicated that the system had reached equilibrium. Other
than that, a lower RMSD value means that the structural deviation of the binomial has
small and higher stability.
2.4. Optimization of Aptasensor Conditions
We optimized the detection conditions to obtain high sensitivity and selectivity with
the established aptasensor. First, to determine the optimal concentration of FAM-SDZ30-1
with MnO2, different concentrations of MnO2 (0, 25, 50, 75, 100, 125, 150, 175, 200 μg/mL)
were incubated with 50 nM aptamer. After centrifugation, the fluorescence intensity of
the supernatant was measured, and the optimal concentration of MnO2 was determined.
Subsequently, at the optimal MnO2 concentration, different concentrations of aptamer (50,
100, 200, 400, 600, 800 nM) were incubated with 100 ng/mL SDZ, followed by the addition
of MnO2 to the mixture, incubation, and centrifugation to determine the optimal aptamer
concentration based on fluorescence intensity. To identify the optimal reaction time of this
sensor, MnO2 was added to the FAM-SDZ30-1 and incubated for 0, 2, 4, 6, 8, 10, 12, and
14 min, and the optimal time of the aptamer with MnO2 was determined by the value of
fluorescence. To find the effects of the reaction time of SDZ with the FAM-aptamer on
the fluorescence intensity, the aptamer was then incubated with SDZ for 10, 20, 30, 40, 50,
and 60 min. The fluorescence intensity of the supernatant was measured, and the best
incubation time of the aptamer with the target was determined. Finally, to explore the
effects of the binding buffer on the aptasensor, 400 nM of FAM-SDZ30-1 was incubated with
100 ng/mL of SDZ with four different buffers (HEPES, TE, PBS, Tris-HCl), followed by the
addition of 150 μg/mL of MnO2, incubated and centrifuged, resulting in the determination
of the optimized buffer according to the fluorescence in the supernatant. Similarly, the
aptasensor was kept at different pH values (6.4, 6.9, 7.4, 7.9, 8.4) and operated as described
above to determine the optimal pH based on the fluorescence intensity.
2.5. Standard Curves of the Aptasensor
Briefly, 40 μL of FAM-labeled aptamer (2 μM) was mixed with SDZ at various con-
centrations (5, 10, 20, 40, 80, 100, 200, and 400 ng/mL) after gentle shaking for 50 min at
25 ◦C in the dark. After that, 30 μL MnO2 (1 mg/mL) was added, and the mixture was
incubated for 10 min and centrifuged at 10,000 rpm for 5 min. The fluorescence intensity of
the supernatant was measured and a standard curve was established with different SDZ
concentrations. The limit of detection (LOD) was acquired by calculating the 3SD/slope
(the slope of the linear curve), and the SD was the standard deviation from the fluorescence
values of a blank sample [29].
2.6. Determination of Selectivity
The selectivity was evaluated by adding 10 μL (100 ng/mL) of different structurally
related compounds, e.g., sulfaquinoxaline (SQX), sulfamethazine (SMZ), tetracycline (TC),
chlortetracycline (CTC), nitrofurazone (NFZ), furaltadone (FTD), norfloxacin (NOR) dis-
solved in 400 nM FAM-SDZ30-1 for 50 min to individual aptasensor against SDZ. After
centrifugation, the fluorescence intensity was measured in the supernatant. After taking
measurements for different antibiotics with relative fluorescence intensity F and the blank
sample with fluorescence intensity F0, the ΔF (ΔF = F − F0) was calculated [30].
2.7. Sample Preparation
All samples were prepared as previously described with slight modifications [31]. To
prepare soil samples, 2 g of soil was diluted 10 times with PBS (pH 7.4), centrifuged at
13,000 rpm for 20 min, and filtered through a 0.22 μm filter membrane. Lake and river
water samples were prepared and diluted 10 times as described above; 2 g of eggs were
5](https://image.slidesharecdn.com/27345891-250521081118-d54eec0f/85/Bioassays-And-Biosensors-For-Rapid-Detection-And-Analysis-Zhen-Zhang-18-320.jpg)
![Biosensors 2023, 13, 613
completely mixed with 4 mL of ethyl acetate and vortexed for 10 min, the supernatant
after centrifugation was evaporated under a stream of nitrogen at 40 ◦C to remove the
ethyl acetate from the mixture and finally dissolved in the buffer. Next, 5 g beef was
homogenized in a homogenizer, then 25 mL of acetonitrile was added, and the mixture
was vortexed and shaken for 15 min, ultrasonicated for 10 min, and then centrifuged at
12,000 rpm for 15 min. The supernatant was moved to 30 mL of acetonitrile-saturated
hexane and constantly stirred for 10 min to clear the fat. The organic solvent was removed
at 80 ◦C in a water bath, and the residue was dissolved in 5 mL of binding buffer, diluted
10 times, and filtered through a membrane.
2.8. Assay Validation
Five different samples were used to validate the properties of the aptasensor. First,
HPLC was used to confirm that all samples did not have SDZ, and then SDZ was added to
the processed sample buffer. The standard curves of the sample matrices were constructed
according to the aptasensor method described above. The precision and accuracy of the
fluorescent aptasensor were verified by analyzing the samples mentioned above with dif-
ferent SDZ concentrations (10, 20, 30 μg/mL), testing five times for each concentration. To
test the credibility of the aptasensor in food and environmental samples, the results of the
aptasensor and HPLC were compared using the same samples. Following the established
procedure, the HPLC analysis was performed on all spiked samples containing different
SDZ concentrations. The linear regression was used to calculate the correlation between
the results of aptasensor and HPLC. Recoveries were calculated as (measured concentra-
tion/known concentration) × 100%, and the coefficient of variation was determined [32].
Each spiked sample was analyzed five times.
3. Results and Discussion
3.1. Principle of the Aptasensor
The FAM-SDZ30-1 fluorescence was quenched by MnO2 through π-π stacking, elec-
trostatic adsorption, and electron-induced transfer in the absence of SDZ30-1 (Figure 1). In
contrast, in the presence of SDZ, FAM-SDZ30-1 selectively combines with SDZ instead of
being adsorbed by MnO2, preventing FAM-SDZ30-1 fluorescence quenching.
Figure 1. Schematic diagram of the aptasensor based on MnO2 for the detection of SDZ.
6](https://image.slidesharecdn.com/27345891-250521081118-d54eec0f/85/Bioassays-And-Biosensors-For-Rapid-Detection-And-Analysis-Zhen-Zhang-19-320.jpg)
![same report: how different from the strain in which her name had
once been breathed! Could the man who had destroyed her peace
and fame forgive himself? Travellers drawing near Bologna are
attracted by two very high towers; the one, however, leans so
obliquely as to create a sensation of alarm; vainly is it said to have
been built so, and to have lasted thus for centuries; its aspect is
irresistibly oppressive. Bologna boasts a great number of highly-
informed men; but the common people are disagreeable. Lucy
listened for the melodious Italian, of which she had been told; but
the Bolognese dialect painfully disappointed her. Nothing more harsh
can exist in the north. They arrived at the height of the Carnival, and
heard, both day and night, cries of joy that sounded like those of
rage. A population like that of the Lazzaroni, eat and sleep beneath
the numerous arcades that border the streets: during winter, they
carry a little fire in an earthen vessel. In cold weather, no nightly
music is heard in Italy: it is replaced in Bologna by a clamor truly
alarming to foreigners. The manners of the populace are much more
gross in some few southern states than can be found elsewhere. In-
door life perfects social order: the heat that permits people to live
thus in public engenders many savage habits.[1] Lord and Lady Nevil
could not walk forth without being assailed by beggars, the scourge
of Italy. As they passed the prisons, whose barred windows look
upon the streets, the captives demanded alms with immoderate
laughter. It is not thus, said Lucy, that our people show
themselves the fellow-citizens of their betters. O, Oswald! can such a
country please you?—Heaven forbid, he replied, that I should
ever forget my own! but when you have passed the Apennines you
will hear the Tuscans—meet intellectual and animated beings, who, I
hope, will render you less severe.
Italians, indeed must be judged according to circumstances.
Sometimes the evil that has been spoken of them seems but true; at
others, most unjust. All that has previously been described of their
governments and religion proves that much may be asserted against
them generally, yet that many private virtues are to be found
amongst them. The individuals chance throws on the acquaintance](https://image.slidesharecdn.com/27345891-250521081118-d54eec0f/85/Bioassays-And-Biosensors-For-Rapid-Detection-And-Analysis-Zhen-Zhang-21-320.jpg)
![of our travellers decide their notions of the whole race; such
judgment, of course, can find no basis in the public spirit of the
country. Oswald and Lucy visited the collections of pictures that
enrich Bologna. Among them was Domenichino's Sibyl; before which
Nevil unconsciously lingered so long, that his wife at last dared ask
him, if this beauty said more to his heart than Correggio's Madonna
had done. He understood, and was amazed at so significant an
appeal: after gazing on her for some time, he replied, The Sibyl
utters oracles no more: her beauty, like her genius, is gone; but the
angelic features I admired in Correggio have lost none of their
charms; and the unhappy wretch who so much wronged the one will
never betray the other. He left the place, to conceal his agitation.
[1] It was announced at Bologna that a solar eclipse would take
place one day at two. The people flocked to see it; and, impatient
at its delay, called on it to begin, as if it were an actor, who kept
them waiting. At last it commenced; but, as the cloudy weather
prevented its producing any great effect, they set up the most
violent hissings, angry that the spectacle fell so far short of their
expectations.
BOOK XX.
CONCLUSION.
CHAPTER I.
Oswald now, for the first time, comprehended that Lucy was aware
of his affection for her sister, and deemed that her coolness might
have sprung from secret disquietude: yet now he feared an](https://image.slidesharecdn.com/27345891-250521081118-d54eec0f/85/Bioassays-And-Biosensors-For-Rapid-Detection-And-Analysis-Zhen-Zhang-22-320.jpg)
![her husband; adding, I feel I may demand this of you now. Oswald
was fearfully agitated, wondering what subject she had chosen, and
whether she would recite herself: the bare possibility of looking on
her threw him into extreme confusion. The morning came, and
winter frowned on it with all the sternness of the north: the wind
howled, the rain beat violently against the windows, and by an
eccentricity more frequent in Italy than elsewhere, the thunder
added a sense of dread to all this gloom. Oswald could not speak:
everything around him increased the desolation of his soul. He
entered the hall with Lucy: it was immensely crowded. In an obscure
recess was placed a sofa, whereon Corinne was to recline, being too
ill to read her own verses. Dreading to show herself, changed as she
was, she had chosen those means of seeing Oswald unseen. As soon
as she knew that he was there, she veiled her face, and was
supported to this couch; from time to time staying to take breath, as
if that short space had been a painful journey: the last steps of life
are ever slow and difficult. Seating herself, her eyes sought Oswald,
found him, and involuntarily starting up, she spread her arms; but
instantly fell back, turning away her face, like Dido when she met
Æneas in a world which human passions should not penetrate.
Castel Forte detained Lord Nevil, who now, utterly beside himself,
would have flown to fall at her feet: the Prince reminded him of the
respect he owed Corinne before the world.[1]
A young girl, dressed in white, and crowned with flowers, now
appeared on the stage which had been erected. Her meek and
peaceful face touchingly contrasting the sentiments she was about
to breathe; it was Corinne's taste, which thus mingled something
sweet with thoughts in themselves too dreary. Music nobly and
affecting prepared the auditors. The hapless Oswald could not tear
his eyes from Corinne: she was to him as an apparition that haunts a
night of fever: it was through his own deep sighs that he heard the
death-song of the swan, which the woman he had so much wronged
addressed to his heart.](https://image.slidesharecdn.com/27345891-250521081118-d54eec0f/85/Bioassays-And-Biosensors-For-Rapid-Detection-And-Analysis-Zhen-Zhang-34-320.jpg)
![Watering the dust which doth await me now.
I had accomplish'd all my destiny—
I had been worthy all the gifts of Heaven,
If I had only vow'd my sounding lyre
To celebrate that goodness all divine,
Made manifest throughout the universe.
And thou, my God!—Oh, thou wilt not reject
The offering of the mind; for poetry,
Its homage is religious, and the wings
Of thought but serve to draw more near to thee.
Religion has no limits, and no bonds;—
The vast, the infinite, and the eternal,
Never from her may Genius separate.
Imagination from its earliest flight,
Past o'er the bounds of life: and the sublime
Is the reflection of divinity.
Alas! my God, had I loved only thee;[2]
If I had raised my head aloft in heaven—
From passionate affections shelter'd there,
I had not now been crush'd before my time—
Phantoms had not displaced my brilliant dreams
Unhappy one, if yet my genius lives,
I only know it by my strength of grief:
Under the features of an enemy
I recognize it now.
Farewell, my birthplace! farewell, my own land!
Farewell, remembrances of infancy,
Farewell! Ah, what have ye to do with death?
And ye who in my writings may have found
Feelings, whose echo was within your soul,
Oh, friends of mine—where'er ye be—farewell!](https://image.slidesharecdn.com/27345891-250521081118-d54eec0f/85/Bioassays-And-Biosensors-For-Rapid-Detection-And-Analysis-Zhen-Zhang-36-320.jpg)
![Youth, hope, emotions of the heart—ye all
Are now no more. Far from me—vain regrets;
If I can yet obtain some falling tears,
If I can yet believe myself beloved,
It is because I am about to die.
Could I recall my fleeting life—that life,
Soon would it turn upon me all its stings.
And Rome! Rome, where my ashes will be borne!
Thou who hast seen so many die, forgive,
If, with a trembling step, I join the shades,
The multitude of your illustrious dead!
Forgive me for my pity of myself.[3]
Feelings, and noble thoughts, such thoughts perchance
As might have yielded fruit—expire with me.
Of all the powers of mind which nature gave,
The power of suffering has been the sole one,
Which I have used to its extent.
It matters not.—I do obey.—Whate'er
May be the mighty mystery of death,
That mystery at least must give repose.
Ye do not answer me, ye silent tombs!
Merciful God, thou dost not answer me!
I made my choice on earth, and now my heart
Has no asylum. Ye decide for me,
And such a destiny is best.
L. E. L.
Thus ended the last song of Corinne. The hall resounded with deep,
sad murmurs of applause. Lord Nevil could not support the violence
of his emotion, but fell senseless to the ground. Corinne, beholding
him in this condition, would have flown to him, but her strength
failed as she attempted to rise. She was borne home, and from that
hour no hopes were entertained of saving her. Lucy hastened to her,](https://image.slidesharecdn.com/27345891-250521081118-d54eec0f/85/Bioassays-And-Biosensors-For-Rapid-Detection-And-Analysis-Zhen-Zhang-38-320.jpg)
![too deep, too troubled, to be confided. She begged they would
remove her to a sofa, whence she could gaze upon the sky. Lucy
now came to her side; and the unhappy Oswald, following his wife,
fell at the feet of Corinne, who would have spoken to him, but her
voice failed: she raised her eyes to Heaven; the moon was covered
with just such a cloud as they had seen on their way to Naples.
Corinne pointed to it with a dying hand—one sigh—and that hand
sank powerless.
Oswald fell into such distraction that Lucy trembled for his life. He
followed the funeral pomp to Rome; then retired to Tivoli, where he
remained long, without seeing even his wife and child. At last, duty
and affection restored him to them; they returned to England. Lord
Nevil's domestic life became most exemplary: but did he ever pardon
his past conduct? Could the approving world console him? After the
fate he had enjoyed, could he content himself with common life? I
know not: nor will I, on that head, either absolve or condemn him.
[1] Not a word of what he owed his wife.—TR.
[2] Had I but served my God with half the zeal, c.—Wolsey.
(SHAKSPEARE.)
[3] J'a pitié de moi-même.—CORNEILLE.
THE END.
CONTENTS
Translator's Preface.
MADAME DE STAËL.
CORINNE
BOOK I.—OSWALD
CHAPTER I.
CHAPTER II.
CHAPTER III.](https://image.slidesharecdn.com/27345891-250521081118-d54eec0f/85/Bioassays-And-Biosensors-For-Rapid-Detection-And-Analysis-Zhen-Zhang-40-320.jpg)
Bioassays And Biosensors For Rapid Detection And Analysis Zhen Zhang
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- 5. mdpi.com/journal/biosensors Special Issue Reprint Bioassays and Biosensors for Rapid Detection and Analysis Edited by Zhen Zhang
- 6. Bioassays and Biosensors for Rapid Detection and Analysis
- 8. Bioassays and Biosensors for Rapid Detection and Analysis Editor Zhen Zhang Basel • Beijing • Wuhan • Barcelona • Belgrade • Novi Sad • Cluj • Manchester
- 9. Editor Zhen Zhang Jiangsu University Zhenjiang, China Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Biosensors (ISSN 2079-6374) (available at: https://www.mdpi.com/journal/biosensors/special issues/bioassay). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: Lastname, A.A.; Lastname, B.B. Article Title. Journal Name Year, Volume Number, Page Range. ISBN 978-3-0365-9082-0 (Hbk) ISBN 978-3-0365-9083-7 (PDF) doi.org/10.3390/books978-3-0365-9083-7 © 2023 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) license.
- 10. Contents About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Zhen Zhang Recent Progress in Biosensors Based on Biorecognition Molecules Reprinted from: Biosensors 2023, 13, 842, doi:10.3390/bios13090842 . . . . . . . . . . . . . . . . . 1 Xiaoling Zheng, Lulan Yang, Qi Sun, Lei Zhang and Tao Le Development and Validation of Aptasensor Based on MnO2 for the Detection of Sulfadiazine Residues Reprinted from: Biosensors 2023, 13, 613, doi:10.3390/bios13060613 . . . . . . . . . . . . . . . . . 3 Nuha Khalid Alekhmimi, Dana Cialla-May, Qasem Ramadan, Shimaa Eissa, Jürgen Popp, Khaled Al-Kattan and Mohammed Zourob Biosensing Platform for the Detection of Biomarkers for ALI/ARDS in Bronchoalveolar Lavage Fluid of LPS Mice Model Reprinted from: Biosensors 2023, 13, 676, doi:10.3390/ bios13070676 . . . . . . . . . . . . . . . . . 15 Kun Zeng, Jian Yang, Hao Su, Sheng Yang, Xinkai Gu, Zhen Zhang and Hongjun Zhao Enhanced Competitive Immunomagnetic Beads Assay Assisted with PAMAM-Gold Nanoparticles Multi-Enzyme Probes for Detection of Deoxynivalenol Reprinted from: Biosensors 2023, 13, 536, doi:10.3390/bios13050536 . . . . . . . . . . . . . . . . . 27 Daniela Dobrynin, Iryna Polischuk and Boaz Pokroy A Comparison Study of the Detection Limit of Omicron SARS-CoV-2 Nucleocapsid by Various Rapid Antigen Tests Reprinted from: Biosensors 2022, 12, 1083, doi:10.3390/bios12121083 . . . . . . . . . . . . . . . . . 41 Masanobu Iwanaga and Wanida Tangkawsakul Two-Way Detection of COVID-19 Spike Protein and Antibody Using All-Dielectric Metasurface Fluorescence Sensors Reprinted from: Biosensors 2022, 12, 981, doi:10.3390/bios12110981 . . . . . . . . . . . . . . . . . 49 Yueyue Huang, Yuanyuan Yao, Yueliang Wang, Lifen Chen, Yanbo Zeng, Lei Li and Longhua Guo Strategies for Enhancing the Sensitivity of Electrochemiluminescence Biosensors Reprinted from: Biosensors 2022, 12, 750, doi:10.3390/bios12090750 . . . . . . . . . . . . . . . . . 61 Benita Johannsen, Desirée Baumgartner, Lena Karkossa, Nils Paust, Michal Karpı́šek, Nagihan Bostanci, et al. ImmunoDisk—A Fully Automated Bead-Based Immunoassay Cartridge with All Reagents Pre-Stored Reprinted from: Biosensors 2022, 12, 413, doi:10.3390/bios12060413 . . . . . . . . . . . . . . . . . 79 Adeel Khan, Kaili Di, Haroon Khan, Nongyue He and Zhiyang Li Rapid Capturing and Chemiluminescent Sensing of Programmed Death Ligand-1 Expressing Extracellular Vesicles Reprinted from: Biosensors 2022, 12, 281, doi:10.3390/bios12050281 . . . . . . . . . . . . . . . . . 99 Hui Meng, Nannan Yao, Kun Zeng, Nuanfei Zhu, Yue Wang, Biying Zhao and Zhen Zhang A Novel Enzyme-Free Ratiometric Fluorescence Immunoassay Based on Silver Nanoparticles for the Detection of Dibutyl Phthalate from Environmental Waters Reprinted from: Biosensors 2022, 12, 125, doi:10.3390/bios12020125 . . . . . . . . . . . . . . . . . 113 v
- 11. Julia Niehues, Christopher McElroy, Alexander Croon, Jan Pietschmann, Martin Frettlöh and Florian Schröper Bacterial Lighthouses—Real-Time Detection of Yersinia enterocolitica by Quorum Sensing Reprinted from: Biosensors 2021, 11, 517, doi:10.3390/bios11120517 . . . . . . . . . . . . . . . . . 123 vi
- 12. About the Editor Zhen Zhang Zhen Zhang, he graduated from China Agricultural University with a bachelor’s degree or a master’s degree respectively in 2001 and 2006. He entered the ecological environment research center of the Chinese Academy of Sciences to study for a doctor’s degree in 2007, and joined the school of environmental and safety engineering of Jiangsu University with a doctor’s degree in 2010. He was a postdoctoral fellow at Changwon National University in Korea from 2012 to 2013, and a visiting scholar at North Carolina State University in the United States from 2016 to 2017. The research direction involves the establishment of rapid analysis methods for pollutants, equipment development and environmental behavior and risk assessment; Exploration of new bioanalytical methods based on nucleic acid functional materials and their applications in environment, food and health. vii
- 14. Citation: Zhang, Z. Recent Progress in Biosensors Based on Biorecognition Molecules. Biosensors 2023, 13, 842. https://doi.org/ 10.3390/bios13090842 Received: 21 August 2023 Accepted: 23 August 2023 Published: 24 August 2023 Copyright: © 2023 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biosensors Editorial Recent Progress in Biosensors Based on Biorecognition Molecules Zhen Zhang School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China; zhangzhen@ujs.edu.cn Biosensors are considered a popular technology to rapidly detect targets, and are generally composed of biorecognition molecules that specifically capture analytes and signal elements. In terms of methodology, measurements are carried out in a limited time without sophisticated sample pre-treatments, implying obvious advantages over traditional instrument methods for rapid analysis [1]. Considering that biosensors are widely applied in environmental pollutant monitoring, detecting hazardous substances in foods and disease diagnosis [2–4], their advances and future trends should be highlighted, so we have organized this Special Issue entitled “Bioassays and Biosensors for Rapid Detection and Analysis”. Among the 10 papers published, 6 are articles, 3 are communications and 1 is a review. Huang et al. believe that electrogenerated chemiluminescence (ECL) is a powerful tool for sensitive and accurate detection of biological analytes and summarize the recent advances in this field. Moreover, some future trends and challenges of ECL biosensors are also discussed. They believe that after integration with low-cost photodetectors, ECL biosensors will have great potential in commercial applications [5]. Meanwhile, several works focus on contaminant determination in food and environments, like sulfadiazine (SDZ) [6], deoxynivalenol (DON) [7] and dibutyl phthalate (DBP) [8]. With regard to the detection systems, antibodies or aptamers were used to recognize the targets, which contributed to good selectivity. In addition, some novel strategies for signal amplification were employed to improve their analytical performance [8]. At the same time, sensitive biosensors were fabricated for the rapid detection of the biomarkers related to some diseases, which could be achieved easily [9,10]. Most studies focus on the sensitivity improvement of biosensors through introducing new functional materials or simplifying the measurement procedure, which are definitely important. However, more attention should be paid to real applications, increasing toler- ances against various matrices that potentially influence the performance of biosensors. Interestingly, in this Special Issue, two papers are related to commercial products using bioassays or biosensors [11,12]. In short, biosensors will be widely used in various fields after some problems have been overcome, including achieving satisfactory sensitivity and accuracy and maintaining enzyme activity in varied harsh environments. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. Biosensors 2023, 13, 842. https://doi.org/10.3390/bios13090842 https://www.mdpi.com/journal/biosensors 1
- 15. Biosensors 2023, 13, 842 References 1. Zhang, Z.; Zeng, K.; Liu, J. Immunochemical detection of emerging organic contaminants in environmental waters. TrAC Trends Anal. Chem. 2017, 87, 49–57. [CrossRef] 2. Pilolli, R.; Monaci, L.; Visconti, A. Advances in biosensor development based on integrating nanotechnology and applied to food-allergen management. TrAC Trends Anal. Chem. 2013, 47, 12–26. [CrossRef] 3. Iwanaga, M.; Tangkawsakul, W. Two-Way Detection of COVID-19 Spike Protein and Antibody Using All-Dielectric Metasurface Fluorescence Sensors. Biosensors 2022, 12, 981. [CrossRef] 4. Niehues, J.; McElroy, C.; Croon, A.; Pietschmann, J.; Frettlöh, M.; Schröper, F. Bacterial Lighthouses— Real-Time Detection of Yersinia enterocolitica by Quorum Sensing. Biosensors 2021, 11, 517. [PubMed] 5. Huang, Y.; Yao, Y.; Wang, Y.; Chen, L.; Zeng, Y.; Li, L.; Guo, L. Strategies for Enhancing the Sensitivity of Electrochemiluminescence Biosensors. Biosensors 2022, 12, 750. [CrossRef] [PubMed] 6. Zheng, X.; Yang, L.; Sun, Q.; Zhang, L.; Le, T. Development and Validation of Aptasensor Based on MnO2 for the Detection of Sulfadiazine Residues. Biosensors 2023, 13, 613. [CrossRef] [PubMed] 7. Zeng, K.; Yang, J.; Su, H.; Yang, S.; Gu, X.; Zhang, Z.; Zhao, H. Enhanced Competitive Immunomagnetic Beads Assay Assisted with PAMAM-Gold Nanoparticles Multi-Enzyme Probes for Detection of Deoxynivalenol. Biosensors 2023, 13, 536. [CrossRef] [PubMed] 8. Meng, H.; Yao, N.; Zeng, K.; Zhu, N.; Wang, Y.; Zhao, B.; Zhang, Z. A Novel Enzyme-Free Ratiometric Fluorescence Immunoassay Based on Silver Nanoparticles for the Detection of Dibutyl Phthalate from Environmental Waters. Biosensors 2022, 12, 125. [CrossRef] [PubMed] 9. Alekhmimi, N.K.; Cialla-May, D.; Ramadan, Q.; Eissa, S.; Popp, J.; Al-Kattan, K.; Zourob, M. Biosensing Platform for the Detection of Biomarkers for ALI/ARDS in Bronchoalveolar Lavage Fluid of LPS Mice Model. Biosensors 2023, 13, 676. [CrossRef] [PubMed] 10. Khan, A.; Di, K.; Khan, H.; He, N.; Li, Z. Rapid Capturing and Chemiluminescent Sensing of Programmed Death Ligand-1 Expressing Extracellular Vesicles. Biosensors 2022, 12, 281. [CrossRef] [PubMed] 11. Dobrynin, D.; Polischuk, I.; Pokroy, B. A Comparison Study of the Detection Limit of Omicron SARS-CoV-2 Nucleocapsid by Various Rapid Antigen Tests. Biosensors 2022, 12, 1083. [CrossRef] [PubMed] 12. Johannsen, B.; Baumgartner, D.; Karkossa, L.; Paust, N.; Karpíšek, M.; Bostanci, N.; Zengerle, R.; Mitsakakis, K. ImmunoDisk—A Fully Automated Bead-Based Immunoassay Cartridge with All Reagents Pre-Stored. Biosensors 2022, 12, 413. [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. 2
- 16. Citation: Zheng, X.; Yang, L.; Sun, Q.; Zhang, L.; Le, T. Development and Validation of Aptasensor Based on MnO2 for the Detection of Sulfadiazine Residues. Biosensors 2023, 13, 613. https://doi.org/ 10.3390/bios13060613 Received: 27 April 2023 Revised: 27 May 2023 Accepted: 1 June 2023 Published: 3 June 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biosensors Communication Development and Validation of Aptasensor Based on MnO2 for the Detection of Sulfadiazine Residues Xiaoling Zheng, Lulan Yang, Qi Sun, Lei Zhang and Tao Le * College of Life Sciences, Chongqing Normal University, Chongqing 401331, China; 2021110513064@stu.cqnu.edu.cn (X.Z.); 2020110513031@stu.cqnu.edu.cn (L.Y.); sunqi2017@cqnu.edu.cn (Q.S.); 20132133@cqnu.edu.cn (L.Z.) * Correspondence: letao@cqnu.edu.cn Abstract: The monitoring of sulfadiazine (SDZ) is of great significance for food safety, environmental protection, and human health. In this study, a fluorescent aptasensor based on MnO2 and FAM- labeled SDZ aptamer (FAM-SDZ30-1) was developed for the sensitive and selective detection of SDZ in food and environmental samples. MnO2 nanosheets adsorbed rapidly to the aptamer through its electrostatic interaction with the base, providing the basis for an ultrasensitive SDZ detection. Molecular dynamics was used to explain the combination of SMZ1S and SMZ. This fluorescent aptasensor exhibited high sensitivity and selectivity with a limit of detection of 3.25 ng/mL and a linear range of 5–40 ng/mL. The recoveries ranged from 87.19% to 109.26% and the coefficients of variation ranged from 3.13% to 13.14%. In addition, the results of the aptasensor showed an excellent correlation with high-performance liquid chromatography (HPLC). Therefore, this aptasensor based on MnO2 is a potentially useful methodology for highly sensitive and selective detection of SDZ in foods and environments. Keywords: aptamer; MnO2; aptasensor; sulfadiazine 1. Introduction Sulfadiazine (SDZ) is an antibiotic of the sulfonamide family, which is widely applied to prevent and treat bacterial infections in livestock [1,2]. However, SDZ can lead to residues in animal-derived foods when overused. These residues can not only accumulate in humans through the food chain and cause serious health problems, but can also pollute soil or water by the pathway of animal excretion, leading to ecotoxicological contamination [3,4]. After discovering its harmful effects, the maximum residue level of SDZ has been set at 100 μg/kg in the EU, the USA, and China. Therefore, monitoring the residues of SDZ in food and the environment is of great significance [5,6]. In previous studies, SDZ was detected by HPLC, capillary electrophoresis, enzyme-linked immunosorbent assay, and time-resolved immunoassay [7–11]. Although these approaches are highly sensitive, the necessity of specialized operators, high costs, weak stability of antibodies, and the potential of cross-reactivity limit their application. Therefore, a simple, effective, and economical method is required to measure SDZ in food and environmental risk monitoring. Aptamer development as a new type of recognition probe provides new ideas for the development of its detection. Aptamers are single-stranded DNA (ssDNA) or RNA that specifically bind to target molecules. Aptamers have superior stability, excellent affinity, strong specificity, and easy in vitro screening compared to antibodies [12–14]. With these advantages, various aptamer-based sensors have been widely developed for environmental monitoring and food safety inspection (e.g., fluorescent, electrochemical, surface-enhanced Raman scattering, and photoelectrochemical sensing signals) [15–17]. In comparison, flu- orescent aptasensors have been widely used in the field of detection for their ability of sensitivity, low cost, and selectivity, which make them particularly useful in monitoring Biosensors 2023, 13, 613. https://doi.org/10.3390/bios13060613 https://www.mdpi.com/journal/biosensors 3
- 17. Biosensors 2023, 13, 613 food and environmental samples [18,19], and fluorescent aptamer sensors based on nano- materials (quantum dots, graphene oxide, nanosilicon, etc.) have attracted considerable attraction [20–22]. In particular, manganese dioxide (MnO2) has attracted much attention for its low cost, strong molar extinction coefficient, environmental tolerance, and non- toxicity, which has been widely used in biomedical and biosensing fields [22,23]. As a constantly developing two-dimensional nanomaterial, MnO2 nanosheets are easy to pre- pare on a large scale and possess broad absorption peaks that can overlap with the emission spectra of many fluorescent materials, making them considered to be quencher candidates. In addition, because MnO2 has a large specific surface area and good biocompatibility, it can greatly improve the detection of targets. Xu et al. reported that MnO2 nanosheets adsorb ssDNA through van der Waals forces interacting with bases, thus exhibiting strong absorption and efficiency of fluorescence burst [24]. Consequently, Qin et al. established an upconversion MnO2-based biosensor for the detection of carbendazim pesticides in food [25]. Li et al. constructed an open fluorescence sensor based on carbon dot-labeled oligodeoxyribonucleotide and MnO2 nanosheets for the detection of mercury(II) [26]. How- ever, MnO2-based fluorescent aptasensors for the detection of SDZ residues have not been reported so far. In this research, we constructed an aptasensor for selective detection of SDZ using FAM-labeled aptamer (FAM-SDZ30-1) and MnO2 nanosheets as donor-acceptor pair for the first time. Molecular dynamics simulations were performed to study the reason for the high specificity for SDZ by SDZ30-1. The performance of the established aptasensor was tested in food and environmental samples by adding different concentrations of SDZ to five samples, including soil, lake water, river water, egg, and beef. Finally, we compared the results of this method with HPLC and explored their correlation, demonstrating that the aptasensor can detect SDZ in a variety of samples. 2. Materials and Methods 2.1. Chemicals and Materials The SDZ 30-1 (5-AACCCAATGGGAT-3, Kd = 65.72 nM) was selected in our labo- ratory, which was purified using HPLC and synthesized by Sangon Biotech, Shanghai, China [24]. All antibiotics, e.g., SDZ, tetracycline (TC), furaltadone (FTD), and norfloxacin (OFL) were bought from Sigma-Aldrich, St. Louis, MO, USA. KMnO4, hexadecyl trimethyl ammonium bromide (CTAB), buffer, and other reagents were purchased from Aladdin Co., Ltd., Ontario, CA, USA. The accuracy of the method was verified by HPLC on LC-20A (Shimadzu, Kyoto, Japan). Fluorescence intensity measurements were performed at the excitation wavelength of 492 nm and emission wavelength of 518 nm using Varioskan LUX. 2.2. Synthesis of MnO2 The MnO2 nanosheets were prepared as previously described [23]. Specifically, 0.5 g KMnO4 was dissolved in 450 mL of water and stirred for 30 min, then 1.5 g of CATB was added to form a stable emulsion. Next, 50 mL of 0.1 M MES (pH 6.0) was added to the above mixture and stirred for 12 h until a black color was formed. The product was centrifuged at 8000 rpm for 10 min and then washed thrice alternately with water and alcohol. Finally, the collected product was dried under a vacuum at 60 ◦C for 12 h. MnO2 was characterized by transmission electron microscope images (TEM), the UV-Vis, EDS elemental mapping and Fourier transform infrared spectrometer (FTIR). 2.3. Molecular Dynamics Simulation The interactions between aptamers and different targets were compared through molecular dynamics simulations [27,28]. Firstly, the structures and parameters of the aptamer and target molecules were obtained from SwissParam. Subsequently, implicit solvation simulations were performed for the aptamer to target under constant temperature, volume, pressure, etc., while the obtained structures were subjected to an explicit solvent model of molecular dynamics simulations. Finally, the stability was measured using the 4
- 18. Biosensors 2023, 13, 613 root mean squared deviation (RMSD) produced by the molecular dynamics simulations trajectory. When the RMSD curve showed a slope upward tendency, the conformation of the system might undergo significant movement, and the smooth oscillation of the RMSD curve around a certain height indicated that the system had reached equilibrium. Other than that, a lower RMSD value means that the structural deviation of the binomial has small and higher stability. 2.4. Optimization of Aptasensor Conditions We optimized the detection conditions to obtain high sensitivity and selectivity with the established aptasensor. First, to determine the optimal concentration of FAM-SDZ30-1 with MnO2, different concentrations of MnO2 (0, 25, 50, 75, 100, 125, 150, 175, 200 μg/mL) were incubated with 50 nM aptamer. After centrifugation, the fluorescence intensity of the supernatant was measured, and the optimal concentration of MnO2 was determined. Subsequently, at the optimal MnO2 concentration, different concentrations of aptamer (50, 100, 200, 400, 600, 800 nM) were incubated with 100 ng/mL SDZ, followed by the addition of MnO2 to the mixture, incubation, and centrifugation to determine the optimal aptamer concentration based on fluorescence intensity. To identify the optimal reaction time of this sensor, MnO2 was added to the FAM-SDZ30-1 and incubated for 0, 2, 4, 6, 8, 10, 12, and 14 min, and the optimal time of the aptamer with MnO2 was determined by the value of fluorescence. To find the effects of the reaction time of SDZ with the FAM-aptamer on the fluorescence intensity, the aptamer was then incubated with SDZ for 10, 20, 30, 40, 50, and 60 min. The fluorescence intensity of the supernatant was measured, and the best incubation time of the aptamer with the target was determined. Finally, to explore the effects of the binding buffer on the aptasensor, 400 nM of FAM-SDZ30-1 was incubated with 100 ng/mL of SDZ with four different buffers (HEPES, TE, PBS, Tris-HCl), followed by the addition of 150 μg/mL of MnO2, incubated and centrifuged, resulting in the determination of the optimized buffer according to the fluorescence in the supernatant. Similarly, the aptasensor was kept at different pH values (6.4, 6.9, 7.4, 7.9, 8.4) and operated as described above to determine the optimal pH based on the fluorescence intensity. 2.5. Standard Curves of the Aptasensor Briefly, 40 μL of FAM-labeled aptamer (2 μM) was mixed with SDZ at various con- centrations (5, 10, 20, 40, 80, 100, 200, and 400 ng/mL) after gentle shaking for 50 min at 25 ◦C in the dark. After that, 30 μL MnO2 (1 mg/mL) was added, and the mixture was incubated for 10 min and centrifuged at 10,000 rpm for 5 min. The fluorescence intensity of the supernatant was measured and a standard curve was established with different SDZ concentrations. The limit of detection (LOD) was acquired by calculating the 3SD/slope (the slope of the linear curve), and the SD was the standard deviation from the fluorescence values of a blank sample [29]. 2.6. Determination of Selectivity The selectivity was evaluated by adding 10 μL (100 ng/mL) of different structurally related compounds, e.g., sulfaquinoxaline (SQX), sulfamethazine (SMZ), tetracycline (TC), chlortetracycline (CTC), nitrofurazone (NFZ), furaltadone (FTD), norfloxacin (NOR) dis- solved in 400 nM FAM-SDZ30-1 for 50 min to individual aptasensor against SDZ. After centrifugation, the fluorescence intensity was measured in the supernatant. After taking measurements for different antibiotics with relative fluorescence intensity F and the blank sample with fluorescence intensity F0, the ΔF (ΔF = F − F0) was calculated [30]. 2.7. Sample Preparation All samples were prepared as previously described with slight modifications [31]. To prepare soil samples, 2 g of soil was diluted 10 times with PBS (pH 7.4), centrifuged at 13,000 rpm for 20 min, and filtered through a 0.22 μm filter membrane. Lake and river water samples were prepared and diluted 10 times as described above; 2 g of eggs were 5
- 19. Biosensors 2023, 13, 613 completely mixed with 4 mL of ethyl acetate and vortexed for 10 min, the supernatant after centrifugation was evaporated under a stream of nitrogen at 40 ◦C to remove the ethyl acetate from the mixture and finally dissolved in the buffer. Next, 5 g beef was homogenized in a homogenizer, then 25 mL of acetonitrile was added, and the mixture was vortexed and shaken for 15 min, ultrasonicated for 10 min, and then centrifuged at 12,000 rpm for 15 min. The supernatant was moved to 30 mL of acetonitrile-saturated hexane and constantly stirred for 10 min to clear the fat. The organic solvent was removed at 80 ◦C in a water bath, and the residue was dissolved in 5 mL of binding buffer, diluted 10 times, and filtered through a membrane. 2.8. Assay Validation Five different samples were used to validate the properties of the aptasensor. First, HPLC was used to confirm that all samples did not have SDZ, and then SDZ was added to the processed sample buffer. The standard curves of the sample matrices were constructed according to the aptasensor method described above. The precision and accuracy of the fluorescent aptasensor were verified by analyzing the samples mentioned above with dif- ferent SDZ concentrations (10, 20, 30 μg/mL), testing five times for each concentration. To test the credibility of the aptasensor in food and environmental samples, the results of the aptasensor and HPLC were compared using the same samples. Following the established procedure, the HPLC analysis was performed on all spiked samples containing different SDZ concentrations. The linear regression was used to calculate the correlation between the results of aptasensor and HPLC. Recoveries were calculated as (measured concentra- tion/known concentration) × 100%, and the coefficient of variation was determined [32]. Each spiked sample was analyzed five times. 3. Results and Discussion 3.1. Principle of the Aptasensor The FAM-SDZ30-1 fluorescence was quenched by MnO2 through π-π stacking, elec- trostatic adsorption, and electron-induced transfer in the absence of SDZ30-1 (Figure 1). In contrast, in the presence of SDZ, FAM-SDZ30-1 selectively combines with SDZ instead of being adsorbed by MnO2, preventing FAM-SDZ30-1 fluorescence quenching. Figure 1. Schematic diagram of the aptasensor based on MnO2 for the detection of SDZ. 6
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- 21. same report: how different from the strain in which her name had once been breathed! Could the man who had destroyed her peace and fame forgive himself? Travellers drawing near Bologna are attracted by two very high towers; the one, however, leans so obliquely as to create a sensation of alarm; vainly is it said to have been built so, and to have lasted thus for centuries; its aspect is irresistibly oppressive. Bologna boasts a great number of highly- informed men; but the common people are disagreeable. Lucy listened for the melodious Italian, of which she had been told; but the Bolognese dialect painfully disappointed her. Nothing more harsh can exist in the north. They arrived at the height of the Carnival, and heard, both day and night, cries of joy that sounded like those of rage. A population like that of the Lazzaroni, eat and sleep beneath the numerous arcades that border the streets: during winter, they carry a little fire in an earthen vessel. In cold weather, no nightly music is heard in Italy: it is replaced in Bologna by a clamor truly alarming to foreigners. The manners of the populace are much more gross in some few southern states than can be found elsewhere. In- door life perfects social order: the heat that permits people to live thus in public engenders many savage habits.[1] Lord and Lady Nevil could not walk forth without being assailed by beggars, the scourge of Italy. As they passed the prisons, whose barred windows look upon the streets, the captives demanded alms with immoderate laughter. It is not thus, said Lucy, that our people show themselves the fellow-citizens of their betters. O, Oswald! can such a country please you?—Heaven forbid, he replied, that I should ever forget my own! but when you have passed the Apennines you will hear the Tuscans—meet intellectual and animated beings, who, I hope, will render you less severe. Italians, indeed must be judged according to circumstances. Sometimes the evil that has been spoken of them seems but true; at others, most unjust. All that has previously been described of their governments and religion proves that much may be asserted against them generally, yet that many private virtues are to be found amongst them. The individuals chance throws on the acquaintance
- 22. of our travellers decide their notions of the whole race; such judgment, of course, can find no basis in the public spirit of the country. Oswald and Lucy visited the collections of pictures that enrich Bologna. Among them was Domenichino's Sibyl; before which Nevil unconsciously lingered so long, that his wife at last dared ask him, if this beauty said more to his heart than Correggio's Madonna had done. He understood, and was amazed at so significant an appeal: after gazing on her for some time, he replied, The Sibyl utters oracles no more: her beauty, like her genius, is gone; but the angelic features I admired in Correggio have lost none of their charms; and the unhappy wretch who so much wronged the one will never betray the other. He left the place, to conceal his agitation. [1] It was announced at Bologna that a solar eclipse would take place one day at two. The people flocked to see it; and, impatient at its delay, called on it to begin, as if it were an actor, who kept them waiting. At last it commenced; but, as the cloudy weather prevented its producing any great effect, they set up the most violent hissings, angry that the spectacle fell so far short of their expectations. BOOK XX. CONCLUSION. CHAPTER I. Oswald now, for the first time, comprehended that Lucy was aware of his affection for her sister, and deemed that her coolness might have sprung from secret disquietude: yet now he feared an
- 23. explanation as much as she had done; and now she would have told him all, had he required it; but it would have cost him too much to speak of Corinne, just as he was about to rejoin her, especially with a person whose character he so imperfectly knew. They crossed the Apennines, and regained the sweet climate of Italy. The sea-breeze, so glowing in summer, now spread a gentle heat. The turf was green, the autumn hardly over, and yet the spring already peeping forth. The markets teemed with oranges and pomegranates. The Tuscan tongue was audible; and all Oswald's dearest memories revived, though now unmixed with hope. The mild air would have rendered Lucy confiding, had he encouraged her. Had a Corinne been with them, she would soon have learned their secrets; but the more congenial they were, in natural and national reserve, the less easy was it for them to break the ice which kept their hearts asunder. CHAPTER II. As soon as they arrived in Florence, Nevil wrote to Castel Forte; and in a few minutes the Prince came to him. It was some time ere either spoke; at last Nevil asked for Corinne. I have none but sad news for you, said her friend: she grows weaker every day; sees no one but myself, and can scarce attempt any occupation; yet I think she has been calmer since we learned you were in Italy; though I cannot disguise from you, that at first her emotions on that intelligence caused her a relapse of fever. She has not told me her intentions, for I carefully avoid your name.—Have the goodness, Prince, said Oswald, to give her the letter I wrote you nearly five years since: it contained a detail of all the circumstances that prevented my hearing of her journey to Scotland before I married. When she has read it, ask her to receive me. I long to justify myself with her, if possible. Her esteem is essential to me, though I can no longer pretend to more.—I will obey your desires, my Lord, said
- 24. Castel Forte, and wish that I may in any way be of service. Lady Nevil now entered the room. Oswald made her known to his friend. She met him coldly. He gazed on her with much attention, sighed, thought of Corinne, and took leave. Oswald followed him. Lady Nevil is very beautiful, said the Prince: so fresh and young! Alas! my poor love is no longer so; yet forget not, my Lord, that she was a brilliant creature when you saw her first.—Forget! exclaimed Oswald: no, nor ever forgive myself. He could utter no more, and for the rest of the day was gloomily silent. Lucy sought not to disturb him: her forbearance was unlucky; for he only thought: Had Corinne beheld me sad, she would have striven to console me. The next morning his anxiety early led him to Castel Forte. Well! he cried, what says she?—That she will not see you, answered the Prince.—And her motives?—I found her yesterday, in spite of her weakness, pacing the room all agitation, her paleness sometimes giving way to a vivid blush, that faded as suddenly as it rose. I told her your request: after some instants' silence, she said—if you exact from me her own words: 'That man has done me too much wrong already; but the foe who threw me into prison, banished and proscribed me has not yet brought my spirit quite so low as he may think. I have suffered more than woman ever endured beside— alternate fondness and indignation making thought a perpetual torture. Oswald should remember that I once told him it would cost me more to renounce my admiration than my love. He has despoiled the object of my worship: he deceived me, voluntarily or otherwise— no matter: he is not what I believed him. He sported for nearly a year with my affection; and, when he ought to have defended me, when his actions should have proved he had a heart, how did he treat me? Can he boast of having made one generous sacrifice? No! he is happy now, possessing all the advantages best appreciated by the world. I am dying, let him leave me in peace!'—These words are very harsh, sighed Oswald.—She is changed by suffering, admitted Castel Forte; yet I have often found her so charitable, that, let me own, she has defended you against me.—You think me unpardonable, then?—If you permit me to say so. The injuries we may do women hurt not us in public opinion. The fragile idol of
- 25. to-day may be broken to-morrow, without finding one protector; for that very reason do I respect the sex, whose moral welfare can find its safety but in our bosoms. A mortal stab is punished by the law; but breaking a tender heart is a theme for jest. I would forgive murder by poniard soonest.—Believe me, cried Nevil, I, too, have been wretched—that is my sole extenuation; but formerly she would have listened to it, now it avails me nothing; yet I will write to her: I still believe, in spite of all that parts us, she may yet understand me.—I will bear your letter, my Lord; but I entreat you temper it well; you guess not what you are to her. Years can but deepen an impression, when no new idea has divided its empire. Would you know in what state she is at present? A fantasy, from which my prayers could not divert her, enables me to show you. He opened the door of another room; and Nevil first beheld a portrait of Corinne as she appeared in Juliet, on the night, of all others, when he felt most enamored of her. The confidence of happiness breathed from each feature. The memories of that festal time came back on Oswald's heart; but as he yielded to them, the Prince took his hand, drew aside a crape from another picture, and showed him Corinne, painted that same year, in the black dress, such as she had never abandoned since her return from England. Her lost lover recollected the figure which had passed him in the Park: but above all was he struck with the total change in her appearance. The long black lashes veiled her languid eyes, and threw a shadow over the tintless cheek: beneath was written this line, from the Pastor Fido:—— A pena si pudò dir: 'Questa fu rosa!' Scarcely can we now say: 'This was a rose!' How! cried Lord Nevil; looks she like this?—-Within the last fortnight still worse, returned the Prince; and Oswald rushed from him, as if distracted.
- 26. CHAPTER III. The unhappy man shut himself in his room. At the dinner hour, Lucy, leading Juliet by the hand, tapped gently at his door; he opened it, saying: Think not the worse of me, my dear, for begging that I may be left to myself to-day. His wife raised her child in her arms, and retired without a word. He now looked at the letter he had written to Corinne, and, bursting into tears exclaimed: Shall I, then, make poor Lucy wretched too? What is my life worth, if it serves but to render all who love me miserable? Letter from Lord Nevil to Corinne. Were you not the most generous of human beings, what could I say to you, who might weigh me so low by reproaches, or still lower by your griefs? I have done such ill to her I loved, that I almost believe myself a monster. Am I, Corinne? I suffer so much, that I cannot think myself an utter barbarian! You know, when first I met you, I was a prey to despair, that nearly brought me to the grave: I sought not happiness, but struggled long against your attraction; even when it triumphed, presentiments of misfortune lingered still. Sometimes I believed you destined by my father to make me once more feel myself as well beloved as I had been by him; then did I fear to disobey his will, in marrying a foreigner. On my return to England, this sentiment prevailed, sanctioned as it was by parental authority. Had he still lived, I should have felt a right to combat it; but the dead cannot hear us, and the irrevocable commands of those now powerless, possess a touching and a sacred force.—Once more surrounded by the ties of country, I met your sister, selected for me by my sire, and well according with my wish for a regular, a quiet life. My weakness makes me dread some kinds of agitation: my mind is easily seduced by new hopes; but my sick soul shrinks from resolves that interfere with its original habits or affections. Yet, Corinne, had I known you were in England, that proof of tenderness would have decided me. Ah!
- 27. wherefore vaunt I what I would have done? Should we have been content? Am I capable of being so? Could I ever have chosen any one fate, without still pining after some other? When you restored my liberty, I fell into the common error, telling myself that so superior a woman might easily be estranged from me. Corinne, I have wounded your heart, I know; but I thought mine the only sacrifice: I deemed you would forget me. I cannot deny that Lucy is worthy of a still warmer attachment than I could give her; but since I learned your voyage to England, and the sorrow I had dealt you, my life has been a perpetual pain. I sought for death, certain that when you heard I was no more, you would forgive me. Doubtless, you can oppose to this years of fidelity and regret, such as my ingratitude ill merits; yet think—a thousand complicated circumstances invade the constancy of man. Imagine, if possible, that I have neither given nor received felicity; that my heart has been lonely since I left you, scarce daring even to commune with itself; that the mother of my child, who has so many titles to my love, is a stranger to my history and feelings; in truth, that my habitual sadness has reduced me to the state from which your cares, Corinne once extracted me. If I have returned to Italy, not for my health (you cannot suspect me of any love for life), but to bid you farewell, can you refuse to see me but once more? I wish it, because I think that it would benefit you; my own sufferings less prompt this desire. What use were it that I am miserable, that a dreadful weight presses upon my heart, if I came hither without obtaining pardon from you? I ought to be unhappy, and am sure of being so; but I feel certain that you would be solaced, if you could think upon me as your friend, and read, in Oswald's looks and accents, how dear you are to the criminal whose fate is far more altered than his heart. I respect the ties I have formed, and love your sister; but the human breast, wild and inconsistent as it is, can reconcile that tenderness with what I feel for you. I have nothing to say for myself that can be written; all I might explain would but condemn me; yet, if you saw me prostrate before
- 28. you, through all my faults and duties, you would perceive what you are to me still, and that conversation would leave a balm for both. Our health is failing: Heaven may not accord us length of days. Let, then, whichever may be destined to precede the other, feel regretted by the dear friend left behind. The innocent alone deserve such joy: but may it not be granted to the guilty? Corinne, sublime soul! you who can read all hearts, guess what I cannot add, and comprehend me, as you used to do. Let me but see you; let my pallid lips touch your weak hand! It was not I alone who wrought this ruin. No; the same sentiment consumed us both: destiny struck two hearts, devoting one to crime; that one, Corinne, may not be the least pitiable. Answer. If I required but to see and pardon you, I could not for an instant refuse. Why is it that I do not feel resentment, although the pangs you have caused me are so dreadful? I must still love you, not to hate. Religion alone would not disarm me thus. There have been moments when my reason has left me; others, far sweeter, when I hoped to die before the day could end; and some in which I have doubted even virtue: you were to me its image here below: there was no guide for either my thoughts or feelings, when the same blow struck both my admiration and my love. What would have become of me without Heaven's help? Everything in this world was poisoned by your image: one sole asylum was left, and God received me. My strength decays, but not that supporting enthusiasm. I joy to think that the best aim in life is to become worthy of eternity: our bliss, our bane, alike tend to this purpose: and you were chosen to uproot the too strong hold I had on earth. Yet, when I saw your handwriting, learned that you were but on the other side of the river, a fearful tumult rose within me: incessantly was I obliged to tell myself, 'My sister is his wife.' To see you again appeared felicity: I will not deny that my heart, inebriated afresh, preferred these indefinite raptures to an age of calm: but
- 29. Providence has not abandoned me in this peril. Are you not the husband of another? What then have I to say to you? Is it for me to die in your arms? What would my conscience suffer, if I made no sacrifice? if I permitted myself another hour with you? I can only appear before my God with anything like confidence by renouncing it. This resolution may appease my soul. Such happiness as I felt while you loved me is not in harmony with our mortal state; it agitates us, because we feel its fleetness: but religious meditation, that aims at self-improvement, and refers every cause to duty, is a state of peace; and I know not what ravages the mere sound of your voice would make on the repose I believe I have regained. Why do you tell me that your health is impaired? Alas! I am no longer your nurse; but still, I suffer with you. May God bless and prolong your days, my Lord! Be happy, but be so through piety. A secret communion with Divinity gives us in ourselves the power of confiding to a being who consoles us: it makes two friends of one spirit. Do you still seek for what the world calls happiness? Where will you find more than my tenderness would have bestowed? Know you that in the deserts of the New World I should have blessed my lot had you permitted me to follow you? I could have served you like a slave, have knelt before you as a heavenly being, had you but loved me truly. What have you done with so much faith? You have changed it into an affliction peerless as itself. Outrage me not, then, by one hope of happiness, except in prayer: let our thoughts meet in heaven! Yet when I feel myself about to die, perhaps I will be taken somewhere whence I may behold you pass. Assuredly, when my failing eyes can see no more, your image will be with me; but might not a recent review of your features render it more distinct? Deities of old were never present at the hour of death, so I forbid you mine; but I should like to see you perfectly when Oswald, Oswald! behold how weak I am, when abandoned to your recollection! Why has not Lucy sought me? Though she is your wife, she is still my sister. I have some kind and even generous things to tell her. And your child—I ought not to meet you; but you are surrounded by my
- 30. family. Do they disown me still? or fear ye that poor little Juliet would be scared at seeing me? Ghost as I look, I yet could smile upon your daughter. Adieu, my Lord, adieu! Remember that I might call you brother. At least you will mourn for me externally, and, as a kinsman, follow my remains to Rome: let them be borne by the road where my car passed; and pause upon the spot where you restored my crown. Yet no, I am wrong, Oswald: I could exact nothing that could afflict you, only one tear, and sometimes a fond look towards the heaven where I shall soon await you. CHAPTER IV. Many days elapsed ere Oswald could regain his composure: he avoided the presence of his wife, and passed whole hours on the banks of the river that separated him from Corinne; often tempted to plunge amid its waves, that they might bear his body to the abode he never must enter living. Amazed as he was at Corinne's wish to see her sister, he longed to gratify it; yet how introduce the subject? He saw that Lucy was hurt by his distress, and hoped that she would question him; but she forbore, merely expressing a desire to visit Rome or Naples: he always begged a brief delay, and Lucy, with cold dignity, was silent. Oswald, at least, could secure Corinne the presence of his little daughter, and secretly bade the nurse take Juliet to her. He met them on their return, and asked the child how she had enjoyed her visit. She replied by an Italian phrase, and with an accent so resembling Corinne's that her father started. Who taught you that, dear? he asked.—The lady, she replied.—And how did she behave to you?—Oh, she kissed me, and cried; I don't know why; but it made her worse, for she looks very ill, papa.—Do you love her, darling?—That I do. I'll go to her every day. She has promised
- 31. to teach me all she knows; and says, that she will make me grow like Corinne: what's that, pa? the lady did not tell me. Lord Nevil could not answer: he withdrew, to conceal his agitation, but bade the nurse take Juliet daily to Corinne. Perhaps he erred in disposing of his child without her mother's consent; but in a few days the young pupil's progress was astonishing: her masters for Italian and music were all amazed. Nothing had ever pained Lucy more than her sister's influence over Juliet's education. The child informed her that, ill as the lady seemed, she took great pains with her. Lucy's heart would have melted, could she have seen in all this anything but a design to win Nevil back. She was divided between the natural wish of being sole directress for her daughter, and self-reproach at the idea of withholding her from such valuable instructions. One day Oswald came in as Juliet was practising a music lesson. She held a lyre proportioned to her size; and her pretty arms fell into Corinne's own attitude so perfectly, that he felt gazing on the miniature copy of a fine picture, with the added grace of childish innocence. He could not speak, but sank, trembling, on a seat. Juliet then played the Scotch air which he had heard at Tivoli, before the design from Ossian; he listened breathlessly. Lucy, unseen, stole behind him: as Juliet ceased, her father took her on his knee, and said: The lady on the banks of the Arno taught you this, did she not?—Yes, papa; but it hurt her very much: she was so ill while she taught me, that I begged her to leave off, but she would not. She made me promise to play you that tune every year, on a particular day, I believe it was the 17th of November.—My God! cried Oswald bursting into tears. Lucy now stepped forward, and, taking Juliet by the hand, said, hastily: My Lord, it is too much to rob me of my child's affection; that solace, at least, is due to my misfortunes. She retired. Oswald would have followed her, but was refused. At the dinner hour he was told that she had been out for some time, not saying where. He was fearfully alarmed at her absence; but she shortly returned, with a calm and gentle air, such as he little expected. He would now have confided in her, and gained her pardon by sincerity, but she replied: Explanation, indeed, is needful to us both; yet, my dear Lord, permit me still to defer it: you will soon know my motives for this
- 32. request. Her address, he perceived, was more animated than usual; and every day its warmth, its interest, increased. He could not understand this change: its cause is soon told. And that Lucy so long had hidden in her heart escaped in the brief reproach she made her husband; and, as usually happens to persons who suddenly break from their habitual character, she now ran into extremes, resolving to seek Corinne, and ask her if she had determined perpetually to disturb her wedded peace; but, as she arrived at her sister's door, her diffidence returned; nor would she have had courage to enter, had not the invalid, who saw her from a window, sent Thérésina to entreat her. Lucy ascended to the sick chamber, and all her anger vanished at sight of its occupant. The sisters embraced in tears. Corinne then set an example of frankness which it was impossible for Lucy not to follow. Such was that mind's ascendency over every one, that, in her presence, neither dissimulation nor constraint could be preserved. Pallor and weakness confirmed her assertion, that she had not long to live: this sad truth added weight to her counsels. All Castel Forte had told her, and all she had guessed from Oswald's letters, proved that reserve and coldness separated the Nevils from each other. She entered very simply on this delicate subject: her perfect knowledge of the husband's character enabled her to point out why he required to find spontaneously in those he loved the confidence which he could not solicit, and to be received with cheerfulness proportioned to his own susceptibility of discouragement. She described her past self impartially, as if speaking of another, and showed how agreeable it must be for a man to find, united with moral conduct, that desire to please which is often inspired by a wish to atone for the loss of virtue. Many women, she said, have been beloved, not merely in spite of, but for the sake of their very errors; because they strove to extort a pardon by being ever agreeable, and having so much need of indulgence dared impose no laws on others. Therefore, dear sister, pride not in your perfections; let your charms consist in seeming to forget them; be Corinne and Lucy in one: nor let your own worth excuse to you a moment's neglect of your graces, nor your self- respect render your manners repulsive. Were your dignity ill
- 33. founded, it might wound him less; for an over-exertion of certain rights chills the heart more than do unjust pretensions. Love delights in paying more than is due, where nothing is exacted. Lucy thanked her sister with much tenderness for the interest thus generously evinced in her welfare; and Corinne resumed: If I were doomed to live, I might not be capable of it; but now my only selfish wish is, that Oswald should find some traces of my influence in you and in his child; nor ever taste one rapture that reminds him not of Corinne. Lady Nevil returned to her every day, and with the most amiable delicacy, studied to resemble the being so dear to her Lord. His curiosity increased, as he remarked the fresh attractions she thus acquired: he knew that she must owe them to Corinne; yet Lucy having promised to keep the secret of their meetings, no explanation occurred. The sufferer proposed yet to see the wedded pair together, but not till she was assured that she had but a few moments to live; but she involved this plan in so much mystery, that Lucy knew not in what manner it was to be accomplished. CHAPTER V. Corinne desired to bid Nevil and Italy such a farewell as might recall the days on which her genius shone with its full splendor. A pardonable weakness. Love and glory were ever blended in her mind; and, at that moment when her heart was about to resign all earthly ties, she wished Oswald to feel, once more, that it was the greatest woman of her day he had destroyed—the woman who best knew how to love and think—whose brilliant success he had obscured in misery and death. She had no longer the strength required by an improvisatrice; but in solitude, since Oswald's return, had resumed her zest for writing poetry; she therefore named a day for assembling in one of the galleries all who desired to hear her verses, begging Lucy to bring
- 34. her husband; adding, I feel I may demand this of you now. Oswald was fearfully agitated, wondering what subject she had chosen, and whether she would recite herself: the bare possibility of looking on her threw him into extreme confusion. The morning came, and winter frowned on it with all the sternness of the north: the wind howled, the rain beat violently against the windows, and by an eccentricity more frequent in Italy than elsewhere, the thunder added a sense of dread to all this gloom. Oswald could not speak: everything around him increased the desolation of his soul. He entered the hall with Lucy: it was immensely crowded. In an obscure recess was placed a sofa, whereon Corinne was to recline, being too ill to read her own verses. Dreading to show herself, changed as she was, she had chosen those means of seeing Oswald unseen. As soon as she knew that he was there, she veiled her face, and was supported to this couch; from time to time staying to take breath, as if that short space had been a painful journey: the last steps of life are ever slow and difficult. Seating herself, her eyes sought Oswald, found him, and involuntarily starting up, she spread her arms; but instantly fell back, turning away her face, like Dido when she met Æneas in a world which human passions should not penetrate. Castel Forte detained Lord Nevil, who now, utterly beside himself, would have flown to fall at her feet: the Prince reminded him of the respect he owed Corinne before the world.[1] A young girl, dressed in white, and crowned with flowers, now appeared on the stage which had been erected. Her meek and peaceful face touchingly contrasting the sentiments she was about to breathe; it was Corinne's taste, which thus mingled something sweet with thoughts in themselves too dreary. Music nobly and affecting prepared the auditors. The hapless Oswald could not tear his eyes from Corinne: she was to him as an apparition that haunts a night of fever: it was through his own deep sighs that he heard the death-song of the swan, which the woman he had so much wronged addressed to his heart.
- 35. THE LAST SONG OF CORINNE. Take ye my solemn farewell! O, my friends, Already night is darkening on my eyes;— But is not heaven most beautiful by night? Thousands of stars shine in the kindling sky, Which is an azure desert during day. Thus do the gathering of eternal shades Reveal innumerable thoughts, half lost In the full daylight of prosperity. But weaken'd is the voice which might instruct; The soul retires within itself, and seeks To gather round itself its failing fire. From my first days of youth, my inward hope Was to do honor to the Roman name; That name at which the startled heart yet beats. Ye have allow'd me fame, O generous land! Ye banished not a woman from the shrine! Ye do not sacrifice immortal gifts To passing jealousies, Ye who still yield Applause to Genius in its daring flight; Victor without the vanquished—Conqueror, Yet without spoil;—who, from eternity, Draws riches for all time. Nature and Life! with what deep confidence Ye did inspire me! I deem'd all grief arose For what we did not feel, or think enough: And that we might, even on this our earth, Beforehand taste that heavenly happiness, Which is—but length in our enthusiasm, But constancy in love. No, I repent it not, this generous faith; No, that caused not the bitter tears I've shed,
- 36. Watering the dust which doth await me now. I had accomplish'd all my destiny— I had been worthy all the gifts of Heaven, If I had only vow'd my sounding lyre To celebrate that goodness all divine, Made manifest throughout the universe. And thou, my God!—Oh, thou wilt not reject The offering of the mind; for poetry, Its homage is religious, and the wings Of thought but serve to draw more near to thee. Religion has no limits, and no bonds;— The vast, the infinite, and the eternal, Never from her may Genius separate. Imagination from its earliest flight, Past o'er the bounds of life: and the sublime Is the reflection of divinity. Alas! my God, had I loved only thee;[2] If I had raised my head aloft in heaven— From passionate affections shelter'd there, I had not now been crush'd before my time— Phantoms had not displaced my brilliant dreams Unhappy one, if yet my genius lives, I only know it by my strength of grief: Under the features of an enemy I recognize it now. Farewell, my birthplace! farewell, my own land! Farewell, remembrances of infancy, Farewell! Ah, what have ye to do with death? And ye who in my writings may have found Feelings, whose echo was within your soul, Oh, friends of mine—where'er ye be—farewell!
- 37. Corinne has suffer'd much—but suffer'd not In an unworthy cause: she has not lost At least her claim on pity. Beautiful Italy! it is in vain To promise me your loveliness; my heart Is worn and wasted; what can ye avail? Would ye revive my hopes, to edge my griefs! Would ye recall my happiness, and thus Make me revolt against my fate? Meekly I do submit myself. Oh, ye Who may survive me—when the spring returns, Remember how I loved its loveliness! How oft I sung its perfume and its air. I pray you sometimes to recall a line From out my songs—my soul is written there: But fatal Muses, love and misery, Taught my best poetry. When the designs of mighty Providence Are work'd in us, internal music marks The coming of the angel of the grave: Nor fearful, nor yet terrible he spreads His white wings; and, though compass'd by night, A thousand omens tell of his approach. If the wind murmurs, then they seem to hear His voice; and when night falls, the shadows round Seem the dark foldings of his sweeping robe. At noon, when life sees only the clear sky, Feels only the bright sun, the fated one Whom Death hath called, upon the distance marks The heavy shade is so soon to shroud All nature from their eyes.
- 38. Youth, hope, emotions of the heart—ye all Are now no more. Far from me—vain regrets; If I can yet obtain some falling tears, If I can yet believe myself beloved, It is because I am about to die. Could I recall my fleeting life—that life, Soon would it turn upon me all its stings. And Rome! Rome, where my ashes will be borne! Thou who hast seen so many die, forgive, If, with a trembling step, I join the shades, The multitude of your illustrious dead! Forgive me for my pity of myself.[3] Feelings, and noble thoughts, such thoughts perchance As might have yielded fruit—expire with me. Of all the powers of mind which nature gave, The power of suffering has been the sole one, Which I have used to its extent. It matters not.—I do obey.—Whate'er May be the mighty mystery of death, That mystery at least must give repose. Ye do not answer me, ye silent tombs! Merciful God, thou dost not answer me! I made my choice on earth, and now my heart Has no asylum. Ye decide for me, And such a destiny is best. L. E. L. Thus ended the last song of Corinne. The hall resounded with deep, sad murmurs of applause. Lord Nevil could not support the violence of his emotion, but fell senseless to the ground. Corinne, beholding him in this condition, would have flown to him, but her strength failed as she attempted to rise. She was borne home, and from that hour no hopes were entertained of saving her. Lucy hastened to her,
- 39. so afflicted by her husband's grief, that she threw herself at her sister's feet, imploring her to admit him; but Corinne refused. I forgive him, she said, for having broken my heart. Men know not what they do; society persuades them that it is sport to fill a heart with rapture, and then consign it to despair; but God's free grace has given me back composure. The sight of Oswald would revive sensations that ill befit a death-bed. Religion only possesses the secret clue through this terrific labyrinth. I pardon the being I so loved, she continued, with a failing voice; may he be happy with you! but when in his turn he is called on to die, then may he recollect the poor Corinne. She will watch over him, if Heaven permits; for those never cease to love, whose love has had the strength to cost them life. Oswald stood at her door, sometimes about to enter, spite her prohibition, sometimes motionless with sorrow. Lucy passed from one to the other, like an angel of peace, between despair and death. One evening Corinne appeared more easy, and the parents went for a short time to their child, whom they had not seen for three days. During their absence the dying woman performed all the duties of religion; then said to the reverend man who received her last solemn confession: Now, father, you know my fate. Judge me! I have never taken vengeance on my foes; the griefs of others never asked my sympathy in vain; my faults sprung but from passions not guilty in themselves, though human pride and weakness led them to excess and error. Think you, my father—you who have so much longer experience than I—that God will pardon me?—Yes, child, I hope so; is not your heart now wholly his?—I believe it, father; take away this portrait, it is Oswald's; lay on my breast the image of Him who descended to this life—not for the powerful, nor the inspired, but for the sufferer, the dying; they need his mercy. She then perceived Castel Forte, who wept beside her bed, and holding out her hand to him, exclaimed: My friend! you only are beside me now. I lived for love; yet, but for you, should die alone. Her tears fell as she spoke, yet she added: There is no help for such a moment; friends can but follow us to the brink; there begin thoughts
- 40. too deep, too troubled, to be confided. She begged they would remove her to a sofa, whence she could gaze upon the sky. Lucy now came to her side; and the unhappy Oswald, following his wife, fell at the feet of Corinne, who would have spoken to him, but her voice failed: she raised her eyes to Heaven; the moon was covered with just such a cloud as they had seen on their way to Naples. Corinne pointed to it with a dying hand—one sigh—and that hand sank powerless. Oswald fell into such distraction that Lucy trembled for his life. He followed the funeral pomp to Rome; then retired to Tivoli, where he remained long, without seeing even his wife and child. At last, duty and affection restored him to them; they returned to England. Lord Nevil's domestic life became most exemplary: but did he ever pardon his past conduct? Could the approving world console him? After the fate he had enjoyed, could he content himself with common life? I know not: nor will I, on that head, either absolve or condemn him. [1] Not a word of what he owed his wife.—TR. [2] Had I but served my God with half the zeal, c.—Wolsey. (SHAKSPEARE.) [3] J'a pitié de moi-même.—CORNEILLE. THE END. CONTENTS Translator's Preface. MADAME DE STAËL. CORINNE BOOK I.—OSWALD CHAPTER I. CHAPTER II. CHAPTER III.
- 41. CHAPTER IV. CHAPTER V. BOOK II.—CORINNE AT THE CAPITOL. CHAPTER I. CHAPTER II. CHAPTER III. BOOK III.—CORINNE. CHAPTER I. CHAPTER II. CHAPTER III. BOOK IV.—ROME. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. CHAPTER V. CHAPTER VI. BOOK V.—THE TOMBS, CHURCHES, AND PALACES. CHAPTER I. CHAPTER II. CHAPTER III. BOOK VI.—ON ITALIAN CHARACTER AND MANNERS. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. BOOK VII.—ITALIAN LITERATURE. CHAPTER I. CHAPTER II. CHAPTER III. BOOK VIII.—THE STATUES AND PICTURES. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. BOOK IX.—ON THE CARNIVAL, AND ITALIAN MUSIC. CHAPTER I. CHAPTER II. CHAPTER III. BOOK X.—PASSION WEEK. CHAPTER I. CHAPTER II. CHAPTER III.
- 42. CHAPTER V. CHAPTER VI. BOOK XI.—NAPLES, AND THE HERMITAGE OF ST. SALVADOR. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. BOOK XII.—HISTORY OF LORD NEVIL. CHAPTER I. CHAPTER II. BOOK XIII.—VESUVIUS, AND THE CAMPAGNA OF NAPLES. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. CHAPTER V. CHAPTER VI. CHAPTER VII. BOOK XIV.—HISTORY OF CORINNE. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. BOOK XV.—THE ADIEU TO ROME, AND JOURNEY TO VENICE. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. CHAPTER V. CHAPTER VI. CHAPTER VII. CHAPTER VIII. CHAPTER IX. BOOK XVI.—PARTING AND ABSENCE. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. CHAPTER V. CHAPTER VI. CHAPTER VII. CHAPTER VIII. BOOK XVII.—CORINNE IN SCOTLAND.
- 43. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. CHAPTER V. CHAPTER VI. CHAPTER VII. CHAPTER VIII. CHAPTER IX. BOOK XVIII.—THE SOJOURN AT FLORENCE. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. BOOK XIX.—OSWALD'S RETURN TO ITALY. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. CHAPTER V. CHAPTER VI. CHAPTER VII. BOOK XX.—CONCLUSION. CHAPTER I. CHAPTER II. CHAPTER III. CHAPTER IV. CHAPTER V. THE END.
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