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Accepted Manuscript
Quantum dot–Cramoll lectin as novel conjugates to glycobiology
Cássia R.A. Cunha, Camila G. Andrade, Maria I.A. Pereira, Paulo
E. Cabral Filho, Luiz B. Carvalho, Luana C.B.B. Coelho, Beate S.
Santos, Adriana Fontes, Maria T.S. Correia
PII:
DOI:
Reference:
S1011-1344(17)30763-7
doi:10.1016/j.jphotobiol.2017.10.020
JPB 11023
To appear in:
Journal of Photochemistry & Photobiology, B: Biology
Received date:
Revised date:
Accepted date:
2 June 2017
22 October 2017
24 October 2017
Please cite this article as: Cássia R.A. Cunha, Camila G. Andrade, Maria I.A. Pereira,
Paulo E. Cabral Filho, Luiz B. Carvalho, Luana C.B.B. Coelho, Beate S. Santos, Adriana
Fontes, Maria T.S. Correia , Quantum dot–Cramoll lectin as novel conjugates to
glycobiology. The address for the corresponding author was captured as affiliation for all
authors. Please check if appropriate. Jpb(2017), doi:10.1016/j.jphotobiol.2017.10.020
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ACCEPTED MANUSCRIPT
Quantum Dot–Cramoll Lectin as Novel Conjugates to Glycobiology
Cássia R. A. Cunhaa,b, Camila G. Andradea,c, Maria I. A. Pereiraa, Paulo E. Cabral
Filhoa, Luiz B. Carvalho Jr.c, Luana C. B. B. Coelhob, Beate S. Santosd, Adriana
Fontesa,*, Maria T. S. Correiab,*
Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco, 50670-901, Recife,
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a
Brazil
Departamento de Bioquímica, Universidade Federal de Pernambuco, 50670-420, Recife, Brazil
c
Laboratório de Imunopatologia Keizo Asami, 50740-120, Recife, Pernambuco, Brazil
Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, 50740-120, Recife,
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b
Brazil
(Camila
G.
Andrade),
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E-mails addresses: cassia.ufpe@hotmail.com (Cássia R. A. Cunha), camila_galvao_@hotmail.com
mariaisabela.andrade@gmail.com
(Maria
I.
A.
Pereira),
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pauloeuzebio03@hotmail.com (Paulo E. Cabral Filho), lbcj.br@gmail.com (Luiz B. Carvalho Jr.),
lcbbcoelho@gmail.com (Luana C. B. B. Coelho), beate_santos@yahoo.com.br (Beate S. Santos),
adriana.fontes.biofisica@gmail.com (Adriana Fontes), mtscorreia@gmail.com (Maria T. S. Correia).
Corresponding authors: Adriana Fontes, Av. Prof. Moraes Rego, S/N, Departamento de Biofísica e
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*
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Radiobiologia, Centro de Biociências, UFPE, 50670-901, Recife, PE, Brazil, Phone: +55 81 21267818, email: adriana.fontes.biofisica@gmail.com; Maria Tereza dos Santos Correia, Av. Prof. Moraes Rego,
S/N, Departamento de Bioquímica, Centro de Biociências, Laboratório de Glicoproteínas, UFPE, 50670-
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420, Recife, PE, Brazil, Phone: + 55 81 2126854, e-mail: mtscorreia@gmail.com.
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Abstract: The optical properties of quantum dots (QDs) make them useful tools for
biology, especially when combined with biomolecules such as lectins. QDs conjugated
to lectins can be used as nanoprobes for carbohydrate expression analysis, which can
provide valuable information about glycosylation changes related to cancer and
pathogenicity of microorganisms, for example. In this study, we evaluated the best
strategy to conjugate Cramoll lectin to QDs and used the fluorescent labeling of
Candida albicans cells as a proof-of-concept. Cramoll is a mannose/glucose–binding
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lectin with unique biological properties such as immunomodulatory, antiparasitic, and
antitumor activities. We probed covalent coupling and adsorption as conjugation
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strategies at different pH values. QDs conjugated to Cramoll at pH 7.0 showed the best
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labeling efficiency in the fluorescence microscopy analysis. Moreover, QD-Cramoll
conjugates remained brightly fluorescent and preserved identical biological activity
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according to hemagglutination assays. Flow cytometry revealed that approximately 17%
of C. albicans cells were labeled after incubation with covalent conjugates, while
approximately 92% of cells were labeled by adsorption conjugates (both at pH 7.0).
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Inhibition assays confirmed QD-Cramoll specificity, which reduced the labeling to at
most 3%. Therefore, the conjugates obtained by adsorption (pH 7.0) proved to be
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promising and versatile fluorescent tools for glycobiology.
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Keywords: nanoparticles; bioconjugation; fluorescence; glycoconjugate; proteins.
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1 Introduction
Carbohydrate expression profiles in biological systems have shown to be
associated with pathogenic microorganisms and their drug resistance [1, 2], as well as
with diseases, such as cancer, making studies related to glycobiology quite relevant [3].
The specific recognition of glycidic residues by lectins make these proteins, of nonimmune origin [4], important tools to elucidate the role of carbohydrates in the progress
and maintenance of a variety of biological processes.
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The association of fluorescence-based techniques with nanotechnology has
allowed the development of novel nanoprobes, such as quantum dots (QDs), to
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investigate carbohydrate expression profiles with high sensitivity and specificity. QDs
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have shown great potential for biological research due to their unique physicochemical
properties, especially their exceptional resistance to photobleaching and their active
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surface, which enables conjugation with a large variety of biomolecules, such as the
lectins, which are proteins that bind specifically and reversibly to carbohydrates [5-8].
QDs conjugated to lectins have already been used to characterize and identify
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carbohydrate rich structures in blood, tissues, and cells [2, 9, 10]. Moreover, some
authors have also applied QDs in optical and electrochemical biosensors [3, 11, 12],
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providing valuable information for glycobiology. For example, Tenório and co-workers
reported the conjugation of Concanavalin A (ConA) lectin to cadmium telluride (CdTe)
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QDs for investigating microbial infections and monitoring their pathogenicity [2].
Cramoll is a mannose/glucose–binding lectin extracted from the plant Cratylia
mollis, which belongs to the same family as ConA and shares similar structural
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characteristics and comparable carbohydrate recognition [13]. When compared to
ConA, studies have demonstrated that Cramoll has a higher immunomodulatory effect
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and an improved ability to induce IL-6, IL-17A, IL-22, and IL-23 cytokine production
in vitro [14-16]. In addition, Cramoll induces immunologic memory generation, which
makes it a potential biotechnological tool in Th17 pathway studies [16]. Other Cramoll
applications involve: (i) treatment of thermal burns [17]; (ii) antioxidant activity by
blocking hydrogen peroxide production [18]; (iii) development of biosensors to
recognize serum glycoproteins from different serotypes and stages of infection by
dengue fever based on the recognition of viral glycoproteins [19]; (iv) antiparasitic
actions [20]; (v) antitumoral activities [21], and (vi) molecular probes in breast [22] and
prostate cancer studies [23].
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The valuable properties of Cramoll have encouraged us to develop QDCramoll bioconjugates as versatile targeting probes for glycobiology research.
Considering the challenges of preserving biomolecule function and QD optical features
after the bioconjugation procedure, the aim of this work was to establish an effective
protocol to conjugate carboxyl-coated CdTe QDs to Cramoll lectin. In order to
accomplish successful bioconjugation, two types of interaction strategies – adsorption
and covalent coupling – were employed at different pH values and evaluated through
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the labeling of Candida albicans cell walls, rich in glucose and mannose, as a proof-of-
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may be useful as novel tools for glycobiology studies.
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2 Experimental Section
2.1 QD synthesis and characterization
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concept [2]. Given the special properties of Cramoll lectin, QD-Cramoll bioconjugates
Experiments were performed with water-dispersed CdTe nanocrystals
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synthesized using 3-mercaptosuccinic acid (MSA) as a stabilizing/functionalizing agent,
according to previously reported methodology [24]. In brief, a Te2− aqueous solution
was obtained by reducing metallic tellurium with sodium borohydride (NaBH4) at pH
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>10 under inert atmosphere (N2). Afterwards, Te2− ions were added into a cadmium
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perchlorate [Cd(ClO4)2] solution at pH 10 containing MSA. The reaction proceeded
under constant stirring and heating at 90 °C for 8 h in order to allow the growth of
nanocrystals for achieving red emission. QDs were prepared at a molar ratio of 5:1:6
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(Cd:Te:MSA). After synthesis, optical characterization was performed by electronic
absorption and emission spectroscopies using a UV–Vis 1800 (Shimadzu)
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spectrophotometer and an LS 55 spectrometer (PerkinElmer, at λexc = 488 nm),
respectively.
2.2 Lectin purification and protein dosage
C. mollis ex. Benth is a predominantly shrubby species that retains its foliage
even in severe drought conditions with good capacity for regrowth, and serves as a
reserve of important proteins for animal forage, especially during drought periods [25].
C. mollis seeds were collected in the Caatinga biome in Ibimirim city in the State of
Pernambuco, Northeast of Brazil [13]. The purification of Cramoll 1,4 isolectins
(Cramoll) was performed using a previously established protocol using Sephadex G-75
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affinity chromatography [26]. The protein dosage was performed using Pierce™ BCA
Protein Assay, Thermo Fisher Scientific kit.
2.3 Hemagglutination assay
The hemagglutination activity (HA) was performed according to Correia and
Coelho [26]. A lectin aliquot (50 µL at the original purified lectin concentration) was
serially diluted in NaCl 0.15 mol∙L-1, and after that a suspension (2.5% v/v in NaCl 0.15
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mol∙L-1) of glutaraldehyde-treated rabbit erythrocytes was added. HA was defined as the
last dilution that showed hemagglutination and was expressed in hemagglutinating units
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(HAU), the inverse of the highest dilution still capable of displaying visible
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hemagglutination. The procedure was also performed for bare QDs and QD-Cramoll
conjugates in duplicate.
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2.4 Preparation and characterization of conjugates
The lectin was conjugated to QDs through two different strategies: (1) by
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adsorption between QDs and proteins and (2) by a covalent bond, using coupling agents
to provide a chemical bond between carboxylic moieties of QDs and the amino
terminals of proteins.
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The adsorption process was performed by adapting a protocol previously
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published [2, 10] with modifications. This protocol was formerly developed to promote
the binding of ConA (280 µg∙mL-1) to QDs at pH 8.0. For Cramoll, this conjugation
process was evaluated at two different pH values, 7.0 and 8.0. Briefly, the QD pH was
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adjusted to 7.0 or 8.0 using an MSA solution (6.8 mg∙mL-1), and Cramoll (10 µL, 28
mg∙mL-1) was added to the QD suspension (1 mL, 2.6 µmol∙L-1) under slow stirring for
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2 h at room temperature (RT, approximately 25 °C).
In order to conjugate QDs to Cramoll by covalent binding, 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide sodium
salt (Sulfo-NHS) were used as coupling agents. One milliliter of QD suspension (2.6
µmol∙L-1, pH = 5.5 adjusted with MSA 6.8 mg∙mL-1) was mixed with 500 µL of EDC
(0.4 mg∙mL-1) and 500 µL of sulfo-NHS (1.1 mg∙mL-1). After 15 min, 20 µL of Cramoll
(28 mg∙mL-1) was added to the sample to obtain the same protein concentration of the
adsorption bioconjugation. After the conjugation process, the pH was adjusted with
NaOH to 6.0, 6.5, 7.0, or 7.4, and systems were maintained for 24 h at 4 °C.
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Conjugates were purified with ultracentrifugal filters (Pierce Concentrators; cutoff 30K MWCO; Thermo Scientific) in order to remove residues from the synthesis that
could interfere in the analysis in four centrifugation cycles (908 × g for 6 min). The
optical properties of the bioconjugates were also evaluated by electronic absorption and
emission spectroscopies under the same condition and concentration than bare QDs.
The biological activity of bioconjugates was evaluated by HA and by the labeling of C.
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albicans.
2.5 Candida albicans labeling
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C. albicans cells were cultivated according to Tenório and co-workers [2].
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Briefly, Sabouraud dextrose broth was used for culturing C. albicans cells (ATCC
10231) for 24 h at 37 °C under constant shaking at 75 rpm. After centrifugation (1680 ×
g for 2 min), yeast cells were resuspended in phosphate–buffered saline solution (PBS
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1×, from now on called PBS). Using the optical density of 0.16 at 540 nm, the cell
concentration was adjusted to about 1 × 106 CFU∙mL-1.
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Cell suspensions (1 × 106 CFU∙mL-1) were incubated with 100 µL of
fluorescent probe (bare QD or QD-Cramoll) at a ratio of 1:1 for 1 h at RT. Before
incubation with cells, carboxylic moieties of covalent conjugates were blocked using
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TRIS base (1 mM in a proportion of 50 μL∙mL-1) for 1.5 h in order to reduce non-
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specific binding [24]. Afterwards, to remove QD-Cramoll conjugates that did not bind
to cells, suspensions were centrifuged at 1200 × g for 30 s, washed, and resuspended in
PBS. To confirm the specificity of labeling, an inhibition assay [10] was performed by
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incubating the conjugates with methyl-α-D-mannopyranoside (0.4 mol∙L-1) for 30 min
before addition to cells.
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Cell labeling was evaluated by two complementary methods: fluorescence
microscopy (DMI 4000B, Leica) and flow cytometry (BD Accuri™ C6, Becton
Dickinson). While fluorescence microscopy provides information on cell morphology
and spatial localization of the target molecules, flow cytometry allows for fast
quantification of a large number of labeled cells. For fluorescence microscopy, an
excitation band-pass (BP) filter at 560/40 and an emission BP filter at 645/75 nm were
used. Flow cytometry analysis was performed with excitation at 488 nm and emission
was acquired using a BP filter 675/25 (FL4-H). Assays were performed using the same
parameters for all systems.
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3 Results and Discussion
3.1 QD optical characterization
CdTe QDs used in our study had a characteristic absorption spectrum and a
maximum emission at 660 nm with a full width at a half maximum (FWHM) of about
70 nm as depicted in Figure 1. Using the first peak of the absorption spectrum, we
estimated the QD diameter as approximately 3.6 nm [27, 28]. The QD concentration
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was approximately 2.6 µmol∙L-1 based on Lambert–Beer’s law and on the extinction
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coefficient provided by Yu and co-authors [29].
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Figure 1. CdTe QD emission (filled line) and absorption (dashed line) spectra, λexc =
488 nm.
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3.2 Hemagglutination assay
Purified lectin in 0.15 M NaCl had a concentration of 9.5 mg∙mL-1 (R2 =
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0.9964), which was used for two strategies of conjugation – covalent coupling and
adsorption. All conjugates were translucent and remained stable for over 7 days, except
the QD-Cramoll system obtained by covalent coupling at pH 6.0, which showed phase
separation after 48 h. This system was therefore excluded from further analyses.
Since the first experiment conducted by Stillmark [30], which highlighted the
potential of lectins to agglutinate erythrocytes, many studies have used agglutination for
screening the presence of lectins in a variety of samples. Additionally, this test also
provides a way of monitoring the biological activity of lectins after exposure to possible
denaturant conditions. Carvalho and co-authors [31] showed that QDs can be efficiently
conjugated to lectins, but this process may cause changes in the biological properties of
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some biomolecules. Thus, the HAU can be a simple tool to assist this type of
investigation.
The HA of the lectin and all QD-Cramoll conjugates revealed that there was
no change in the protein activity after the bioconjugation (for all systems the result was
512-1 HAU). For both conjugation strategies (covalent or adsorption), the specific HA
was 53.7 HAU∙mg-1. As expected, bare QDs were unable to agglutinate erythrocytes,
which indicates that the bioconjugate HA activity is related only to the presence of the
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lectin.
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3.3 Bioconjugation evaluation
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C. albicans is a well-studied model with high relevance for biomedical
research. C. albicans is a type of pathogenic opportunistic fungi that can affect humans
[32], and is known to have a high resistance profile when organized in biofilms, where
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yeasts and hyphae are entangled in an extracellular matrix, which protects them against
some therapies [1, 2].
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Approximately 80–90% of C. albicans yeast cell wall consists of carbohydrates
such as β-glucans, chitin, and mannoproteins [2, 33]. Because Cramoll binds
specifically to α-D-mannose and α-D-glucose, C. albicans yeast cells were used in this
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work as a proof-of-concept to evaluate the maintenance of the carbohydrate recognition
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ability of the lectin following bioconjugation in order to select the best conjugation
strategy. The localization of the bioconjugates on labeled C. albicans cells was initially
observed by fluorescence microscopy and further analyzed by flow cytometry. Flow
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cytometry allows quick, objective, and quantitative analysis of a large number of cells
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in suspension [34]
3.4 Fluorescence microscopy analysis
We analyzed by fluorescence microscopy yeast cells incubated with QD-
Cramoll bioconjugates covalently prepared at pH = 6.5, 7.0, and 7.4. According to
microscopy images, the system with pH 7.0 promoted a brighter labeling profile as
shown in Fig. 2. On the other hand, heterogeneous labeling with some bioconjugate
aggregates on the cell walls was also observed as indicated by the arrows in Fig. 2B.
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Figure 2. C. albicans yeasts labeled with QD-Cramoll conjugated by covalent coupling
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at various pH values: (A) pH 6.5, (B) pH 7.0, and (C) pH 7.4. Scale bar: 10 µm.
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We observed a highly homogeneous labeling for most of the cells for
bioconjugates obtained by the adsorption process (Fig. 3). Moreover, the bioconjugated
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system at pH 7.0 (Fig. 3A) provided brighter fluorescence images compared with that at
pH 8.0 (Fig. 3B). Tenório and co-authors [2] obtained CdTe QD-ConA conjugates at an
optimal pH of 8.0, which showed an intense staining of C. albicans cells. Cramoll is a
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lectin of the same Diocleinae subtribe as ConA, and both proteins not only recognize
specifically mannose/glucose carbohydrates but also present monomers that are
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structurally similar [13]. Thus, we can conclude that the adsorption conjugate (pH 7.0)
provided a more efficient and homogeneous labeling pattern, and the covalent conjugate
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(at the same pH) promoted a more heterogeneous profile.
Figure 3. C. albicans yeasts labeled with QD-Cramoll bioconjugates obtained by
adsorption at different pH values: (A) pH 7.0, and (B) pH 8.0. Scale bar: 10 μm.
Although Cramoll and ConA have many structural and carbohydrate
recognition similarities [16, 35], these lectins are distinct, and therefore, may not behave
in the same way under the conditions tested. One difference between these lectins is
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their isoelectric point (pI), which has proven to be an important aspect in the
development of bioconjugation strategies.
The influence of pI in bioconjugation was already reported by Wang and coauthors [36], who studied the interaction between mercaptopropionic acid (MPA)
stabilized CdTe QDs with five different proteins (Streptavidin, Erbitux antibody,
Peroxidase, and Mouse Anti-Human Alpha-Fetoprotein (AFP) Antibodies, AFP1A6 and
AFP2A5) via covalent binding. The authors reported that the conjugation efficiency was
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considerably different according to the protein pI. By optimization of the buffer pH,
proteins with different pI values were efficiently conjugated with QDs using EDC and
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Sulfo-NHS as coupling agents, such as the Erbitux antibody (pI 7.5–8.0), which had
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almost 100% conjugation when the pH was adjusted from 7.7 to 6.3.
Luo and co-authors [37] evaluated non-specific interactions between the
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following proteins: bovine serum albumin (BSA), immunoglobulin G (IgG), and wheat
germ agglutinin (WGA) – presenting different isoelectric points – with CdSe/ZnS QDs.
The authors noted that the conjugate with the WGA lectin (pI 9.0) had higher bond
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strength at low pH values, lower than their pI. Because the net charge of the protein is
positive at a pH below the pI and carboxyl QDs (QD-COOH) are negatively charged in
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the same situation, the authors theorized that electrostatic interactions favored
conjugation.
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Similar to WGA, Cramoll is a basic pH lectin (pI 8.5–8.6) [26], which suggests
that at a pH lower than the pI, it has a positive net charge. According to our results, the
best condition to bioconjugate QDs to Cramoll was obtained at pH 7.0, and thus, we
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believe that electrostatic interactions are the driving forces for the adsorption of this
lectin on the surface of the negatively charged (MSA) QDs. At pH 8.0, near the pI, QD–
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Cramoll electrostatic interactions become weaker, which leads to a less effective
adsorption conjugation. Luo and co-authors [37] noted that electrostatic forces are
mainly affected by the nature and pI of the proteins.
Studies with ConA lectin have shown high efficiency for the bioconjugation
process at pH 8.0 with MSA-CdTe QDs [2, 10], and more recently using ZnSe:Mn QDs
[38]. Because of the ConA pI (5.7) [39], this lectin, at pH 8.0, has a negative charge as
well as the QDs, which suggests that other interaction forces may be involved in this
process. For example, at this pH, ConA exists as a tetramer, which increases the
possibility of interaction by hydrogen bonds. Yan and co-authors [39] also showed by
applying Resonance Rayleigh Scattering that at pH 6.0 (near the pI) ConA was properly
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adsorbed on the surface of Ag2Se QDs stabilized with thioglycolic acid. They suggested
that because of the negative charge of both lectins and QDs, at this pH, electrostatic
interaction is not so important, but other interactions, such as hydrogen bonds, could be
the main driving force between the lectin and nanoparticles.
Our results showed that pH is an important factor for conjugation and that minor
changes in its value may affect the potential of conjugates to interact efficiently with the
biological system. Analysis of pH for both adsorption and covalent strategies was
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considered useful for selecting the best method for conjugating QDs to lectins as in
other studies [36, 37, 40]. Therefore, the best conjugates were those obtained at pH 7.0
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by the two strategies used – adsorption and covalent binding.
3.5 QD-Cramoll bioconjugates optical characterization
Another important parameter to be analyzed after bioconjugation is the
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maintenance of optical properties. The covalent conjugate (pH 7.0) had an FWHM of
approximately 85 nm and a shift in the emission spectrum to the red of approximately 7
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nm, while the conjugate obtained by adsorption (also at pH 7.0) had an FWHM of 75
nm and a discreet shift in the emission spectrum of 2 nm to the blue (Fig. 4A). These
changes in bandwidth and spectral shifts are ascribed to surface modifications related to
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similar nanosystems [41, 42].
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the bioconjugation with the lectin and were already mentioned in the literature for
Figure 4. Emission (A, λexc = 488 nm) and absorption (B) spectra of bare QDs (filled
line), QD-Cramoll conjugated by covalently coupling (dotted line) or by adsorption
(dashed line).
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Moreover, both systems did not have considerable changes in the absorption
spectra (Fig. 4B). The conjugates had a decrease in emission intensity when compared
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to bare QDs, but remained brightly fluorescent, as can also be observed in Figure 5.
Figure 5. QD-Cramoll bioconjugates obtained by adsorption (A) and covalent binding
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(B), both at pH 7.0, observed under UV light (λex = 365 nm).
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3.6 Flow cytometry
According to the flow cytometry analysis (Fig. 6), covalently bound QDCramoll bioconjugates labeled about 17% of C. albicans cells (Fig. 6B). After an
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inhibition assay with methyl-α-D-mannopyranoside, labeling was reduced to about 2%
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(Fig. 6D), which revealed the specificity of QD-Cramoll bioconjugates for C. albicans
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as summarized in Table 1.
Figure 6. Histogram profiles of C. albicans cells labeled by QD-Cramoll conjugated by
covalent bonding at pH 7.0. (A) non-labeled C. albicans cells as a control; (B) cells
after incubation with bare QDs; (C) cells after incubation with QD-Cramoll
bioconjugates, and (D) cells incubated with QD-Cramoll bioconjugates inhibited by
specific carbohydrates.
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The intense cell labeling profile, already observed by fluorescence microscopy
after incubation with CdTe QD-Cramoll conjugated by adsorption at pH 7.0 (Fig. 5),
was confirmed by flow cytometry analysis as shown in Fig. 7. This bioconjugate was
able to label approximately 92% of the cells and, when inhibited with methyl-α-Dmannopyranoside, the labeling decreased to about 3% as shown in Fig. 7C and 7D,
respectively, and summarized in Table 1. The two peaks observed in the cytometry
histograms of Fig. 7C correspond to the heterogeneity of C. albicans cell morphologies
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with the possible presence of yeast cells, shoots, and the formation of germ tubes and
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hyphae [2].
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Figure 7. Histogram profiles of C. albicans cells labeled by QD-Cramoll conjugated by
the adsorption process at pH 7.0. (A) non-labeled C. albicans cells as a control; (B)
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cells after incubation with bare QDs; (C) yeast cells after incubation with QD-Cramoll
bioconjugates, and (D) cells incubated with QD-Cramoll bioconjugates inhibited by
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specific carbohydrates.
By flow cytometry it was possible to evaluate in a quantitative manner the more
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effective conjugation protocol. Bioconjugates obtained by adsorption provided the
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highest quantity of labeled cells, even after nearly a year of preparation (Table 1).
Table 1. Average percentage of C. albicans labeled with QD-Cramoll conjugates.
Systems
Labeled cells (%)
QDs
QD-Cramoll adsorption, pH 7.0
QD-Cramoll adsorption, pH 7.0 [a]
QD-Cramoll adsorption, pH 7.0 inhibited
QD-Cramoll covalent, pH 7.0
QD-Cramoll covalent, pH 7.0 inhibited
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92
62
3
17
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[a] System after 1 year of bioconjugation.
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Therefore, similarly to previously reported studies that showed successful
conjugation of ConA to CdTe MSA QDs by adsorption, this strategy (at pH 7.0) was
also the best for the Cramoll lectin because it provided the most homogenous, effective,
and brightest labeling. Adsorption of biomolecules on the surface of nanoparticles is a
common strategy for conjugation [8, 43, 44]. This approach has been employed to
attach a variety of engineered proteins, such as maltose binding protein (MBP) and G
Protein, to the QDs’ surface [2, 8, 10, 31, 45, 46]. The native pI of a protein can drive
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its association with oppositely charged QDs by controlling the reaction pH [47].
Moreover, adsorption strategies are simpler than covalent ones because they do not
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involve other chemical steps to conjugate molecules to the QD surface [8]. Taking into
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account the characteristics and functions of carbohydrates, CdTe QD-Cramoll
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conjugates may be versatile and excellent tools for glycobiology [4].
4 Conclusions
CdTe QDs were successfully conjugated to Cramoll lectin by adsorption at pH
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7.0, and caused no considerable changes in hemagglutination activity of the lectin or
optical properties of the nanocrystals. These QD-Cramoll nanosystems remained
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labeling cells for at least one year after bioconjugation, which suggests the potential of
this system for various applications. Because of the rich amount of glycidic residues on
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C. albicans walls, these cells have proved to be a simple and effective model to evaluate
the best strategy for obtaining QD-Cramoll conjugates. The overall results showed that
the developed method is an efficient strategy for bioconjugation of CdTe QDs to
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Cramoll to target glycidic residues, which can be used to investigate microbial
infections, monitor pathogenicity of microorganisms, and assist in the elucidation of
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malignant processes of tumor cells and metastasis-related changes in carbohydrate
expression profiles.
Acknowledgements
The Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) is
acknowledged for fellowships (LBCJ, LCBBC, AF and MTSC) and grants. The authors
are also grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) and the Fundação de Amparo à Ciência e Tecnologia do Estado de
Pernambuco (FACEPE). The National Institute of Photonics (INCT de Fotônica) is also
recognized.
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Graphical abstract
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Highlights
- Covalent coupling or adsorption was probed to conjugate QD with Cramoll lectin;
- The best conjugation strategy was evaluated by the labeling of C. albicans cells;
- The adsorption at pH 7.0 was a superior strategy for QD-Cramoll lectin conjugation;
- QD-Cramoll conjugates provided a homogenous, effective and bright labeling;
- QD-Cramoll conjugates are versatile tools for carbohydrate expression studies.
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