Ultrafine fluorene-pyridine oligoelectrolyte nanoparticles for supersensitive fluorescence sensing of heparin and protamine

Article abstract of DOI:10.1039/d1cc01969d

A new fluorene-pyridine oligoelectrolyte (OFP) is rationally proposed and readily synthesized via a simple one-pot Sonogashira approach. Hence, an unexpectedly small cationic oligomer nanosensor (i.e. OFPNPs, ～ 1.2 nm in diameter) was conveniently fabricated owing to the enhanced flexibility endowed by the meta-substituted pyridyl unit. Inspiringly, this facile nanoplatform with low cytotoxicity favors the ultrasensitive fluorescence assay for heparin and protamine with a detection limit (LOD, S/N = 3) as low as 1.2 ng mL-1 and 0.5 ng mL-1, respectively, involving heparin-induced aggregation of OFPNPs through electrostatic interaction or competitive rebinding of protamine to heparin. This journal is

Full text of DOI:10.1039/d1cc01969d

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Ultrafine fluorene–pyridine oligoelectrolyte
nanoparticles for supersensitive fluorescence
sensing of heparin and protamine†
Cite this: Chem. Commun., 2021,
57, 8304
Received 13th April 2021,
Accepted 17th July 2021
Huifeng Du,a Li Zhang,a Wensheng Mao,a Yaqian Zhao,a Hongmei Huang,
*
a
a
b
Yi Xiao,*a Youyu Zhang,
Xiaoxiao Heb and Kemin Wang
*
DOI: 10.1039/d1cc01969d
rsc.li/chemcomm
A new fluorene–pyridine oligoelectrolyte (OFP) is rationally proposed Nevertheless, a novel, simple and supersensitive technique for the
and readily synthesized via a simple one-pot Sonogashira approach. more convenient assaying of heparin and protamine is still intriguing.
Hence, an unexpectedly small cationic oligomer nanosensor (i.e.
Fluorescence technologies are powerful tools for the rational
OFPNPs, B 1.2 nm in diameter) was conveniently fabricated owing fabrication of new platforms owing to their simplicity, out-
to the enhanced flexibility endowed by the meta-substituted pyridyl standing sensitivity and selectivity.6 To date, a variety of fluor-
unit. Inspiringly, this facile nanoplatform with low cytotoxicity favors escent probes, such as small molecules like perylene,
the ultrasensitive fluorescence assay for heparin and protamine with a tetraphenylethylene (TPE) and tetraphenylbenzene derivatives,
detection limit (LOD, S/N = 3) as low as 1.2 ng mLÀ1 and 0.5 ng mLÀ1
,
rhodamine B-modified polyethyleneimine (RB-PEI), upconver-
respectively, involving heparin-induced aggregation of OFPNPs sion nanoparticles (UCNPs), CdTe-QDs or CdS QDs, and
through electrostatic interaction or competitive rebinding of prota- Tb-MOF, have been proposed for the efficient analysis of
mine to heparin.
heparin and protamine.2b3,4,7
Conjugated polymers (CPs) containing p-delocalized back-
Heparin (Hep), a linear polyanionic glycosaminoglycan, plays bones have attracted special attention due to their superior
vital roles in a wide range of biological processes including properties such as extraordinary amplification effect, low cost,
inflammatory response, tumour cell metastasis and virus infec- and good biocompatibility.8 In particular, fluorine-based CP
tion since it can bind numerous growth factors and cytokines.1 probes have been extensively studied due to their appealing
In particular, heparin possesses therapeutic functions as an performance. In 2008, Liu et al. developed a multicolor cationic
anticoagulant and antithrombotic agent through heparin– fluorene-2,1,3-benzothiadiazole conjugated polymer for naked-
antithrombin interactions.2 However, an overdose of heparin eye detection of heparin (LOD, 30 nM).9 It was also demon-
may induce severe complications, for instance, hemorrhage or strated by Han and co-workers that water-soluble polyfluorene
thrombocytopenia.3 Protamine (Pro), a highly cationic peptide with pendant glycoclusters could be employed for the fluores-
isolated from salmon sperm, is of clinical importance as an cence assaying of heparin (LOD, 100 nM).10 Recently, Huang
FDA-approved heparin antagonist in cardiac surgery despite and co-workers investigated efficient heparin sensing (LOD, 50
some possible negative impacts (e.g., bradycardia, pulmonary nM) using cationic polyfluorenes containing long-lived phos-
hypertension and dyspnea).4 Thus, various approaches, such as phorescent Ir(III) complexes (B50 or 80–100 nm in diameter).11
activated clotting time (ACT), chronopotentiometric res- Notably, conjugated polymer nanoparticles (CPNs) show size-
ponse, colorimetry, HPLC, ion chromatography, microgravimetric dependent sensing characteristics and smaller size may help to
methods, and resonance Rayleigh scattering, have been re- minimize non-specific interactions and to promote cellular
ported for the quantitative detection of heparin or protamine.5 penetration.12 In the past few years, the design of ultra-small
(e.g., particle diameter r 10 nm) CPNs or conjugated oligomer
nanoparticles (CONs) has therefore gained increasing interest.13
a Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research
However, a supersensitive fluorescent nanoprobe for the efficient
measurement of heparin and protamine based on ultra-small
CPNs or CONs remains relatively unexplored.
Oligofluorene holds the potential for the fabrication of ultra-
fine nanoparticles (in particular, sub À5 nm in size), which is
important for improved biosafety owing to better tissue and
cellular penetration.14 However, small-sized oligofluorene
(Ministry of Education), College of Chemistry and Chemical Engineering,
Hunan Normal University, Changsha 410081, P. R. China.
E-mail: huanghongmei@hunnu.edu.cn, xiaoyi@hunnu.edu.cn
b State Key Laboratory of Chemo/Biosensing and Chemometrics,
College of Chemistry and Chemical Engineering, College of Biology,
Hunan University, Changsha 410082, P. R. China. E-mail: kmwang@hnu.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1cc01969d
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Fig. 1 TEM images of OFPNPs (A), OFPNPs/Hep (B) and OFPNPs/Hep/Pro
(C). Zeta potentials of OFPNPs under different conditions (D).
OFPNPs exhibit a maximum emission peak at B 419 nm
(Fig. S5, ESI,† curve a). Obviously, the addition of heparin will
induce a sharp decrease in the FL intensity of OFPNPs (Fig. S5,
ESI,† curve b, ‘‘off state’’), implying the strong interactions
between OFPNPs and heparin. As observed in Fig. 1D, OFPNPs
are positively charged possessing a zeta potential of approxi-
mately 41.4 mV. Given the fact that heparin is highly negatively
charged, Hep will tend to bind OFPNPs, triggering aggregation
and subsequent aggregation-induced fluorescence quenching
Scheme 1 One-pot synthesis of oligoelectrolyte OFP and the fluores-
cence switching via ultra-small OFPNPs.
nanoparticles have been rarely reported in practical applications.15 of OFPNPs.5d Furthermore, protamine is a highly positive
Here, a new cationic conjugated fluorene–pyridine oligoelectrolyte, protein and thus can form a stable ion-pair complex with
OFP (Fig. S1–S3, ESI†), was designed and conveniently synthesized heparin via affinitive interactions.4 Indeed, changes of the zeta
by a facile one-pot Sonogashira route, thereby avoiding tedious potential (i.e., decrease from 41.4 to 9.9 mV, and then increase
stepwise synthesis. Thus, unexpectedly small OFPNPs (B1.2 nm) to 27.2 mV) caused by heparin binding or release on the surface
were readily prepared by a modified reprecipitation method for of OFPNPs were well consistent with the smart ‘‘off–on’’
the ultrasensitive sensing of heparin and protamine using OFP fluorescence switching. Therefore, the addition of protamine
(Mn = 1662, Mw/Mn = 2.2; QY: 10.1%) due to the enhanced backbone to the OFPNP-heparin system may render the release of the
flexibility and amphiphilicity of OFP.13a,16 The principle of our aggregated OFPNPs due to the stronger attraction between
strategy is demonstrated in Scheme 1. OFP featuring a conjugated heparin and protamine, thus causing the fluorescence recovery
oligofluorene backbone containing meta-substituted pyridyl can be (Fig. S5, ESI,† curve c, ‘‘on state’’).
endowed with excellent flexibility and thus facilitates the formation
In order to achieve the optimal conditions for the ultra-small
of untrafine OFPNPs.16 Meanwhile, lower concentration of OFP led OFPNP nanosensor, the influence of several parameters (e.g., pH
to an apparent decrease in OFPNP diameter under ultrasonication. value, incubation time, and the concentrations of the probe and
Therefore, when the dosage of CH3OH solvent is increased from heparin) was investigated. We first examined the effect of pH on
1.0 mL to 8.0 mL, the size of OFPNPs can be tuned from 2.0 nm the detection of heparin. As shown in Fig. S6 (ESI†), slightly acidic
(QY: 7.9%; Fig. S4 and Table S1, ESI†) to 1.2 nm (QY: 8.4%; Fig. 1A), environments (pH = 4.5–5.4) are favorable for heparin sensing,
as confirmed by the TEM results. The negatively charged heparin perhaps due to the fact that the deprotonation of carboxylic groups
can bind OFPNPs bearing cationic quaternary ammonium groups on the surface of OFPNPs may occur under alkaline environments,
by electrostatic interaction,7a,c thus causing fluorescence quenching thus preventing the formation of OFPNP/heparin aggregates via
of OFPNPs due to the heparin-induced aggregation (B20 nm in electrostatic repulsion between COOÀ and anionic heparin.17
diameter, Fig. 1B). In the presence of protamine, aggregated However, the presence of a large amount of H+ may also be
OFPNPs are redispersed (B2–6 nm, Fig. 1C) owing to the highly unfavorable for heparin-induced aggregation of OFPNPs since the
specific affinity between protamine and heparin, leading to super- competing hydrogen ions can hinder the assembly of an OFPNP-
sensitive fluorescence ‘‘turn-on’’ sensing of protamine. In particu- heparin composite.7a The fluorescence quenching efficiency
lar, ultra-small OFPNPs with bright blue fluorescence and low reached a maximum at a pH of 5.0. Therefore, the pH value used
cytotoxicity can be used for imaging of living cells.
in the following experiments was set at 5.0. Then, we evaluated the
Fig. S5 (ESI†) shows the fluorescence emission spectra of effect of OFPNP concentration on the fluorescence quenching
ultra-small OFPNPs in the presence of heparin and protamine. efficiency toward heparin. In the presence of 2.0 Â 10À7 g mLÀ1
As a typical blue-emitting oligofluorene-based nanoprobe, heparin, 0.5 mg mLÀ1 OFPNPs gave the best response when the
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fluorescence quenching efficiency and linear range were taken into also observed in the range of 6.0 Â 10À9–7.5 Â 10À7 g mLÀ1
account, as exhibited in Fig. S7 (ESI†). From Fig. S8 (ESI†), we can (Fig. 2D). Interestingly, an extremely high sensitivity to heparin
see that the fluorescence quenching efficiency is abruptly elevated (LOD, 1.2 ng mLÀ1) or protamine (LOD, 0.5 ng mLÀ1) can be
and then remains constant after 16 min; hence the spectral data achieved, as listed in Tables S2 and S3 (ESI†). Moreover, these
were recorded after 16 min of incubation in the following experi- results revealed that extremely small OFPNPs can be used to
ments. Fig. S9 (ESI†) shows the effect of heparin concentration on measure Hep and Pro quantitatively.
the fluorescence recovery efficiency. It was found that the fluores-
To evaluate the selective response of the OFPNP nanosensor
cence recovery efficiency increased sharply and then decreased towards heparin, several soluble anions and biomacromolecule
rapidly with increasing heparin concentration, reaching a max- analogs are investigated in detail, including FÀ, ClÀ, BrÀ, IÀ,
imum at a heparin concentration of 2.0 Â 10À7 g mLÀ1. Finally, we SO42À, NO3À, CH3COOÀ, PO43À, S2O52À, S2O82À, SCNÀ, chon-
studied the effect of incubation time on the detection of protamine. droitin sulfate (Chs), dextran (Dex) and hyaluronan (HA). As
As illustrated in Fig. S10 (ESI†), the fluorescence recovery efficiency shown in Fig. 3A, when 6.0 Â 10À8 g mLÀ1 heparin was added to
tends to be stable after 10 min, and thus an optimal reaction time the OFPNP nanosystem, a change in the fluorescence signal
of 10 min is chosen for the analysis of protamine.
was clearly observed. In contrast, the existence of other sub-
We then investigated the fluorescence response of ultrafine stances only resulted in a slight change in the fluorescence
OFPNPs to heparin. As anticipated, the fluorescence of OFPNPs intensity, thus indicating that OFPNPs have excellent selectivity
around 419 nm gradually decreased with increasing Hep concen- during heparin detection. To test the selectivity for sensing of
tration owing to the strong electrostatic interaction-induced protamine, the ultra-small OFPNP-based nanoprobe was stu-
aggregation (Fig. 1B and 2A). The fluorescence intensity was died with different analytes such as Na+, Mg2+, K+, Ca2+, Mn2+,
found to be linear with the logarithm of Hep concentration in Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, fructose, glucose, collagen,
the range from 6.0 Â 10À9 to 6.0 Â 10À7 g mLÀ1 (1 À F/F0
=
IgG and BSA. As demonstrated in Fig. 3B, only protamine
3.8178 + 0.4615 log cHep) with a correlation coefficient (R2) of caused clear fluorescence recovery, since it has the ability to
0.9975, as depicted in Fig. 2B. In Fig. 2C, we can see that the specifically bind to heparin, thereby verifying that the extremely
fluorescence intensity of OFPNPs would be recovered in the small OFPNP-based nanosensor possesses outstanding selec-
presence of protamine due to the stronger affinity between Pro tivity towards protamine.
and Hep, since Hep and Pro could form a stable ion-pair complex
To examine the applicability of the newly developed nanop-
to release OFPNPs (Fig. 1C). A good linearity (a correlation robe, the heparin and protamine levels in real blood serum
coefficient of R2 = 0.9958) between (F/F0 À 1) and log cPro was samples were determined according to the experimental pro-
cedure. As shown in Tables S4 and S5 (ESI†), good recovery of
94%–110% or 93%–106% for the highly sensitive fluorescent
sensing could be obtained via the ultrafine OFPNP-based
nanosensor, indicating that the proposed approach can be
simple and practical during the assay for heparin and prota-
mine in real samples.
Low cytotoxicity and high fluorescence intensity are significant
for the potential applications in cell imaging. The cytotoxicity of
OFPNPs was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay using different concentrations
Fig. 2 (A) Fluorescence emission spectra (lex = 365 nm) of OFPNPs in the
presence of different concentrations of heparin in a pH 5.0 (HAc–NaAc)
buffer solution. Curves (a to n): 0, 2.0 Â 10À9, 4.0 Â 10À9, 6.0 Â 10À9
,
,
Fig. 3 (A) Fluorescence response of OFPNPs containing different sub-
8.0 Â 10À9, 1.0 Â 10À8, 2.0 Â 10À8, 4.0 Â 10À8, 6.0 Â 10À8, 8.0 Â 10À8
stances at pH 5.0:(1) FÀ, (2) ClÀ, (3) BrÀ, (4) IÀ, (5) SO42À, (6) NO3
,
À
1.0 Â 10À7, 2.0 Â 10À7, 4.0 Â 10À7, and 6.0 Â 10À7 g mLÀ1. (B) The plot of
1 À F/F0 versus the logarithm of heparin concentration. (C) The emission
spectra (lex = 365 nm) of OFPNPs/Hep in the presence of various
(7) CH3COOÀ, (8) PO43À, (9) S2O52À, (10) S2O82À, (11) SCNÀ, (12) Chs, (13)
Dex, (14) HA, and (15) Hep. The concentration of various substances was
6.0
Â
10À8 mLÀ1
g . (B) Fluorescence responses of OFPNPs/Hep
[protamine] in a pH = 5.0 buffer solution. Curves (a to l): 0, 8.0 Â 10À10
,
,
(2.0 Â 10À7 g mLÀ1) containing protamine (5.0 Â 10À7 g mLÀ1) or other
3.0 Â 10À9, 6.0 Â 10À9, 9.0 Â 10À9, 2.5 Â 10À8, 5.0 Â 10À8, 7.5 Â 10À8
substances (5.0 Â 10À5 g mLÀ1) in a pH 5.0 buffer solution: (1) Na+,
1.0 Â 10À7, 2.5 Â 10À7, 5.0 Â 10À7, and 7.5 Â 10À7 g mLÀ1; [heparin] =
(2) Mg2+, (3) K+, (4) Ca2+, (5) Mn2+, (6) Fe2+, (7) Fe3+, (8) Co2+, (9) Ni2+
,
2.0 Â 10À7 g mLÀ1. (D) The plot of F/F0 À 1 versus the logarithm of
(10) Cu2+, (11) Zn2+, (12) fructose, (13) glucose, (14) collagen, (15) IgG,
protamine concentration. [OFPNPs] = 5.0 Â 10À7 g mLÀ1
.
(16) BSA, and (17) Pro. [OFPNPs] = 5.0 Â 10À7 g mLÀ1
.
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convenient construction of potent probes for feasible clinical
applications.
Financial support for this research from the National Natural
Science Foundation of China (21475042), the Natural Science
Foundation of Hunan Province (2016JJ4060), the Open Fund
Project of State Key Laboratory of Chemo/Biosensing and Chemo-
metrics in Hunan University (201103) and Program for Science
and Technology Innovative Research Team in Higher Educational
Institutions of Hunan Province is gratefully acknowledged.
Conflicts of interest
There are no conflicts to declare.
Notes and references
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