- Email: [email protected]
A ratiometric scheme for the fluorescent detection of protamine, a heparin antidote
Journal Pre-proof A ratiometric scheme for the fluorescent detection of protamine, a heparin antidote
Shrishti P. Pandey, Prabhat K. Singh PII:
S0167-7322(19)36869-2
DOI:
https://doi.org/10.1016/j.molliq.2020.112589
Reference:
MOLLIQ 112589
To appear in:
Journal of Molecular Liquids
Received date:
14 December 2019
Revised date:
20 January 2020
Accepted date:
26 January 2020
Please cite this article as: S.P. Pandey and P.K. Singh, A ratiometric scheme for the fluorescent detection of protamine, a heparin antidote, Journal of Molecular Liquids(2020), https://doi.org/10.1016/j.molliq.2020.112589
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
A Ratiometric Scheme for the Fluorescent Detection of Protamine, a Heparin antidote Shrishti P. Pandey†and Prabhat K. Singh,*
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Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085, INDIA
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*Authors for correspondence: Email: [email protected]; [email protected]
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Tel. 91-22-25590894, Fax: 91-22-5505151 Keywords: Protamine sensor; Ratiometry; Fluorescence sensor; Polystyrene sulfonate
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Abstract
Protamine is an arginine rich cationic protein and is known to be a very important pharmaceutical compound which is utilized in interventional radiology procedures, vascular surgery, and cardiac surgery, and is the only clinically approved antidote for Heparin overdose. Considering its importance in various biological processes, it is very important to develop sensor platforms for selective and sensitive detection of Protamine. Herein, we present a rarely reported ratiometric fluorescence detection scheme
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for Protamine, which is based on Protamine-induced disassembly of a dye-polyelectrolyte supramolecular assembly that causes a tuning of the monomer-aggregate equilibrium of the dye, and
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yields a sensitive ratiometric reponse for the Protamine. Our sensor system registers high sensitivity and
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selectivity for Protamine. Besides being simple, selective and sensitive, one of the prime features of this
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developed detection method is its ratiometric response which principally provides increased accuracy
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for quantitative analysis in complex samples due to inherent internal calibration of two bands. Another noteworthy feature associated with this detection scheme is that it employs commercially available
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probe molecule and components that avoid the heavy dependence on time-consuming and tedious
also
demonstrates
sensing
performance
in
complex
human
serum
matrix.
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scheme
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synthetic procedures involved with the previously existing methods. Importantly, our current sensing
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1. Introduction
Protamine is an arginine rich cationic protein which plays crucial biological roles in a variety of important biological events, for example, such as gene expression[1], gene transfer[2] and genesis of tissue.[3] Protamine also provides a highly compact configuration of chromatin in the nucleus of the sperm and binding to DNA.[4, 5] Protamine is extracted from the sperm of salmon roe fish. The high arginine content (up to 67%) in the poly-peptide sequence of Protamine leads to a high positive charge on the Protamine surface with an isoelectric point of about 13.8 units.[3, 6, 7] The molecular weight of
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Protamine is low (ca. 4500 Da), and under physiological conditions, it carries over 20 positive
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charges.[7-9] Apart from its various biological roles, Protamine is also known to be a very important
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pharmaceutical compound which is used in interventional radiology procedures, vascular surgery, and, cardiac surgery, and till date, it is the only clinically approved antidote for Heparin overdose.[10, 11]
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Heparin is a glycosaminoglycan (a copolymer of uronic or iduronic acids with alternate
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sulphated glucosamine residues) which bears an anionic rod-like structure.[12] Heparin plays a crucial
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role in different pathological and physiological procedures, for example, lipid transport, venous thromboembolism, cell growth, differentiation, metabolism, and blood circulation.[13-15] Importantly,
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Heparin is the most widely used anticoagulant drug in surgical procedures such as cardiopulmonary
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bypass surgery and extracorporeal blood circulation.[16-19] Heparin also acts as a co-factor for antithrombin III, an inhibitor present in the plasma.[20] However, excess dose as well as prolonged use of Heparin usually causes fatal bleeding complications. Therefore, the therapeutic levels of Heparin have to be monitored crucially to avoid the risk of complications such as thrombocytopenia and hemorrhage, which arise due to Heparin overdose. Heparin is administered in the range of 2–8 U mL-1 (17–67 µM) during cardiovascular surgery whereas a dose of 0.2–1.2 U mL-1 (1.7–10 µM) is recommended in postoperative conditions and long-term care.[21] Since Protamine is polycationic whereas Heparin is polyanionic, thus, a stable complex is formed due to rigid electrostatic interactions between Heparin and Protamine, which is devoid of anticoagulant activity. Protamine binds to Heparin competing with antithrombin III by the mechanism 3
Journal of charge neutralization.[20] Antithrombin III is Pre-proof thereby replaced from the Heparin-antithrombin III complex by Protamine which has a strong affinity towards Heparin, resulting in the efficient conversion of the anticoagulation activity.[8, 11] Protamine is well known and the only clinically approved drug till date, known to reverse the effects of Heparin.[22] However, Protamine may cause severe side effects which includes bradycardia, dyspnea, pulmonary hypertension, flushing and a feeling of warmth or sudden fall in blood pressure.[5, 11, 23] Therefore, there is an urgent need for developing methods for the fast, sensitive and selective detection of Protamine.
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The detection of Protamine is considered to be comparatively difficult due to the absence of
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aromatic amino acids in its structure.[24] This makes it inadequate for UV absorption measurements at 280nm, which are considered as the standard method for the quantification of proteins. Till now only a
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few methods for the sensing of Protamine have been reported, such as traditional colorimetric
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methods,[25, 26]High performance liquid chromatography,[27-29] electrochemical assay,[30, 31] etc.
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However, most of these methods fail to overcome certain limitations associated with Protamine sensing, such as moderate sensitivity and selectivity. Although these electrochemical and microgravimetric
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methods were successful to obtain a detection limit as low as 0.5 and 0.05 μg mL−1 respectively[24],
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these approaches suffer from certain disadvantages, such as, designing complicated electrochemical
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probes, time-consuming operational steps and requirement of sophisticated equipment. In addition, the previously reported methods also suffered from the problem of complicated synthetic design of employed probes, high cost, operational inconvenience, requirement of trained expertise, low interference ability with other biomolecules and so on. Therefore, it is important to develop rapid, selective and sensitive methods for the sensing of Protamine. In comparison to the other techniques, currently, there is an immense research focus on the development of fluorescent based sensors for the measurement of bioanalytes, because of the various powerful attractive attributes associated with the technique of fluorescence such as high sensitivity, operational simplicity, real-time detection and low cost.
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Journal Pre-proof Recently, a fluorescence based methods have been reported by Egawa et al for the determination of Protamine which is dependent on self-quenching of fluorescein-labeled protamine [32] while, Chem et al. reported a turn-on fluorescence based method for the determination of Heparin and Protamine which utilized the phenomenon of aggregation-induced emission enhancement.[33] However, these methods suffer from limited sensitivity and selectivity where protamine concentration could be detected only in the range of µg/mL. Therefore, to overcome these limitations, a method of fluorescence assay based on a fluorescein isothiocyanate (FITC)-labeled DNA probe was by Shu Pang et. al., where the
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detection limit of Protamine was obtained to be as low as 2.2 ng mL-1.[34] Further, N. Vasimalai et. al
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developed a sensor for the detection of Protamine based on the aggregation and de-aggregation of gold nanoparticles which was induced by polyionic protamine.[20] However, these methods, for the
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detection of Protamine still involves complicated methods to design the employed fluorescent probes
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and does not evaluate the sensing performance in the human serum matrix which restricts their
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application in real life detection of Protamine in serum samples. Further, most of these sensing schemes operate through signal measurement at single wavelength that make these methods susceptible to error
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due to variation in different analyte-independent factors such as change in the microenvironment of the
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probe, minute variation in experimental conditions such as pH of the medium, probe concentration,
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temperature, path length etc. In this regard, ratiometric sensors provide great advantage for sensor molecules which are exclusively based on intensity measurements, since the self-calibration of intensity at two distinct emission bands (distinct peak positions) excludes most or all of the uncertainties associated with small variation in experimental conditions which makes them more appropriate for quantitative measurements as well as for operation in complex environments.[35-37] To the best of our knowledge, there is no report on the ratiometric sensor primarily developed for Protamine. Thus, there is an urgent need for development of fluorescence based ratiometric methods for the detection of Protamine. In the present contribution, we report a fluorescence based ratiometric detection scheme for the selective and sensitive detection of Protamine. In the present system, the two distinct emission bands, 5
which is an essential requirement for theJournal design ofPre-proof ratiometric detection scheme, has been achieved by using a monomer-aggregate equilibrium system which is built up from an anionic polylelectrolyte induced aggregation of a cationic probe. The sensor operation is achieved as follows: (i) The cationic probe, pyrene methyl ammonium chloride (PMA), emits in the aqueous solution in the monomeric form (ii) The anionic polyelectrolyte, polystyrene sulfonate (PSS) induces aggregation of the cationic probe, PMA, leading to a monomer-aggregate system with two distinct emission bands. (iii) Protamine, being a cationic polyelectrolyte, interacts very strongly with the anionic polylectrolyte, PSS, and results into
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dissociation of PMA aggregates bound to PSS which significantly affects the monomer-aggregate
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equilibrium, and provides a sensitive ratiometric response for the protamine concentration. The detection scheme is simple, rapid, label free, sensitive, and selective. Importantly, the sensor platform
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includes the components which are all commercially available components, and thus provides the
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freedom from heavy dependence on complex and time-intensive synthetic procedures for the sensor
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probe production itself that significantly affects the practical applicability of many excellent sensing
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schemes in real-life applications.
(A)
(B)
Scheme 1: Chemical structure of Polystyrene sulfonate (A) and Pyrene methyl ammonium chloride (B)
2. Results and Discussion Since the sensing platform for the current detection scheme is based on the formation of a dyepolyelectrolyte aggregate assembly, so we will first present the spectroscopic features of the dyepolyelectrolyte supramolecular assembly followed by its interaction with the Protamine. Figure 1 presents the steady-state emission spectra of the cationic probe, PMA, in the presence of increasing concentrations of an anionic polyelectrolyte, PSS. PMA, in the aqueous solution, emits in the monomer
6
Journal form with sharp emission bands at 376, 394 andPre-proof a weak shoulder at 410 nm which is in excellent agreement with the previous literature reports.[38] However, as evident from the figure, the gradual addition of PSS, to the aqueous solution of PMA, leads to a drastic reduction in the monomer emission bands along with the appearance of a new emission band at 480 nm that has been attributed to the formation of PMA aggregates induced by the anionic polyelectrolyte, PSS owing to the charge neutralization of the multiple cationic PMA molecules on the surface of negatively charged PSS.[39] It is well known that Pyrene based molecules displays characteristic emission band for its aggregated state
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or the excimer state, as compared to the emission band for the monomeric species of PMA.[40-43] Such
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charged polyelectrolyte induced aggregation of oppositely charged probe molecules have been amply
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reported in literature.[44, 45]
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Figure 1. Steady-state emission spectra of PMA in water (λexc=340 nm) at different concentrations of PSS (in M) (i) 0 (ii) 0.14 (iii) 0.19 (iv) 0.21 (v) 0.30. Inset: Variation in the ratio of emission intensity obtained at 376nm to 480nm respectively with increasing concentration of PSS.The indicated error bar is the standard deviation. The observation of two distinct emission bands for the monomer and the aggregate species of PMA molecules provides an ideal case for the fabrication of a ratiometric sensor,[46, 47] if a modulation in the above bands can be achieved in response to the targeted analyte. It has been also established that the major underlying interaction force in the formation of PMA-PSS aggregate assembly is the electrostatic interaction between the cationic PMA and the anionic PSS molecules.[39] Thus, if electrostatic
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Journal Pre-proofaggregate assembly, this will lead to the interaction forces can be modulated in this dye-polyelectrolyte modulation of the monomer-aggregate equilibrium in the system, and may provide a ratiometric response for a targeted analyte. In this regard, Protamine, which is the analyte of interest here, is polycationic in nature and is known to form a stable complex with polyanionic Heparin due to rigid electrostatic interactions between them, which is devoid of anticoagulant activity.[11]This makes the basis for using Protamine as the only pharmaceutical compound which is clinically approved for reversing the effect of Heparin, the most extensively employed blood anti-coagulant.[11] Since the
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present dye-polyelectrolyte assembly is built up on the surface of a highly negatively charged sulphated
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polyelectrolyte, so we envisaged that introduction of Protamine to the PSS-PMA assembly may disrupt the interaction between the PMA aggregate with the PSS owing to the relatively stronger interaction of
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polycationic Protamine with polyanionic PSS. This may lead to disassembly of aggregates of PMA from
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the surface of PSS, which, in turn, will lead to the manipulation in the monomer-aggregate equilibrium,
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and may yield an analytically significant signal in response to Protamine. To test this idea, we have performed steady-state emission measurements for the PSS-PMA system in the presence of increasing
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concentration of Protamine, and Figure 2 summarizes the results of these measurements. As it is clearly
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visible from the figure, the introduction of Protamine to the PMA-PSS solution, leads to reduction in the
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aggregate emission band at 480 nm together with a concomitant increase in the monomer emission band. This clearly indicates the dissociation of PMA aggregates from the anionic surface of PSS upon addition of Protamine that can be assigned to comparatively stronger interaction of polycationic protamine over monocationic PMA with polyanionic PSS. This Protamine-induced modulation in the monomer-aggregate equilibrium of PSS-PMA system thus generates a ratiometric signal, in response to Protamine concentration, (Figure 2B)
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Figure 2. (A) Steady–state emission spectra of PMA (35 M)-PSS (0.2 M) complex in presence of different concentrations of Protamine (in M) (i) 0 (ii) 1.04 (iii) 1.56 (iv) 2.08 (v) 2.59. (λexc=340 nm)
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(B) Variation of logarithmic ratio of emission intensity at 376 nm to 480 nm with varying concentration
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of Protamine. The experimental data points are represented by half-filled squares and the solid red line
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represents a linear fit to the data points. The indicated error bar is the standard deviation. To quantify the Protamine concentration dependent changes, the ratio of emission intensity of the monomer emission band (376 nm) to the aggregate emission band (480 nm) is plotted and has been presented in Figure 2B. The logarithm of the signal increases linearly with increase in concentration of protamine in the concentration range of 0-4 M with a linear regression equation of ln(I376/I480)=1.454(0.128) +1.72(0.27)[Prs/M] (R2=0.96). The limit of detection, based on 3σ/S,[48] was calculated to be 7.71 nM, where σ stands for the standard deviation of 10 blank measurements and S represents the slope of the calibration curve. Further, ratiometric detection scheme provides several advantages with respect to the single wavelength based detection measurements, as single wavelength
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based measurements are prone to error Journal by various Pre-proof analyte-independent factors such as variation in the microenvironment of the probe, small variation in the instrumental factors such as fluctuation in lamp light intensity, small variation in experimental parameters, such as, temperature, pathlength, probe concentration etc. Ratiometric detection provides immunity against these small but almost unavoidable variations in experimental conditions that makes ratiometric detection, a method of choice for accurate quantitative analysis as well as performance in complex medium.[35] Note that there is no report on the ratiometric detection scheme which is primarily developed for Protamine detection. Thus, the present
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sensing scheme is a unique contribution in the field of fluorescence sensing of Protamine (Table 1).
Analytical
Commercial
No
method
Availability
LOD
0.015 mg
HPLC
4
Fluorimetry
5
Colorimetry and
No
Yes
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Fluorimetry 6
Fluorimetry
No
7
Fluorimetry
No
Fluorimetry
No
Fluorimetry
No
9 10
11
12
[27]
2.0µg ml-1
-
No
[30]
15 µg ml-1
-
No
[28]
2.2 ng mL-1
No
No
[34]
0.1 μg mL−1
No
No
[24]
1.0 ng mL-1
No
Yes
[10]
No
Yes
[49]
mL−1
No
Yes
[50]
2.4 ng mL-1
No
Yes
[51]
No
Yes
[52]
No
Yes
[53]
Fluorimetry
No
Fluorimetry
No
Fluorimetry and
Yes
Yes
Colorimetry
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HPLC
Yes
serum matrix No
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No
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Electrochemistry
Human
-
ml-1
2
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Ratiometric Evaluation in Reference
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Table 1: Comparison of performance of the various sensors for Protamine detection
23.4 ng
0.4 mg mL1
0.13 μg mL−1 31.5 4.1 ng mL−1
This study
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Journal Pre-proof To explore the possibility of monitoring this Protamine-induced disintegration of PSS-PMA assembly colorimetrically, ground-state absorption measurements were also performed for the PMAPSS system in the presence of different concentration of Protamine, and Figure 3 summarizes the results of these measurements. PMA in aqueous solution displays absorption band at 340 nm, whereas in the presence of PSS, it shows a red-shifted absorption band at 352 nm (Figure S1, supplementary information) that characterizes the PMA aggregates in the presence of PSS.[39] However, as evident
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from figure 3, upon addition of Protamine, the absorbance at monomer absorption band (340 nm) starts recovering along with a reduction in absorbance at 352 nm. Figure S2 (supplementary material) presents
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the normalized absorption spectra for PMA-PSS complex the presence and absence of Protamine. It is
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clearly evident from the figure that the spectral features of PMA-PSS complex is widely different from
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that of PMA in water, however, in the presence of Protamine, it clearly resembles to that of PMA in
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water. Another spectroscopic analysis which is popularly performed with the help of absorption spectra of pyrene derivative, in connection to their state of aggregation is peak-to-valley ratio (PA) analysis.[54,
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55] PA is calculated as the ratio of the most intense peak to its adjacent minimum at the shorter
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wavelength. In the present system, the PA value for PMA in water is recorded as 2.1 which reduces to 1.08 in the presence of PSS which signifies a large extent of aggregation of PMA in the presence of
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PSS. However, upon addition of Protamine, the PA value recovers back to 2.01 which once again supports the recovery of free form of PMA. This clearly reflects the disassembly of PMA aggregates from the surface of PSS that can be attributed to the stronger interaction of polycationic Protamine with PSS as compared to the interaction of monocationic PMA with PSS. Thus, the ground-state absorption measurements nicely validate the proposal made from steady-state emission measurements.
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Figure 3.(A) Ground-state absorption spectra of PMA (35 M)-PSS (0.2 M) complex in presence of
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different concentrations of Protamine (in M) (i) 0 (ii) 0.52 (iii) 1.10 (iv) 1.56 (v) 2.08 (vi) 2.59 (vii) 3.10 (viii) 4.11. (B) Changes in the ratio of absorbance at 340 to 352 nm with increasing concentration
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of Protamine. The experimental data points have been represented by square, whereas the solid red line
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is a fit to the data points according to the linear model. The indicated error bar is the standard deviation. A ratiometric analysis of the titration data from ground-state absorption measurements suggest that the logarithm of the ratio of monomer to aggregate absorption band, represented by OD340/OD352, changes linearly with increasing concentration of Protamine in a dynamic range of 0-4 M with a linear regression equation of ln(OD340/OD352)= 0.49 (0.03)[Prs/M]-0.078(0.015), (R2=0.97). The detection limit calculated, from this analysis was found to be 9.31.5 nM. Thus, these measurements clearly suggest that PMA-PSS system can function as a ratiometric sensor for Protamine. Time-resolved emission measurements are also quite responsive towards the molecular state of a probe molecule such as monomer, aggregate etc as the state of association of probe molecules generally
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Journal56, Pre-proof affects the excited-state relaxation processes.[22, 57] Thus, to further demonstrate the phenomenon of Protamine induced disintegration of PMA-PSS aggregate system, time-resolved emission measurements were also performed for the PSS-PMA system, in the presence of various concentrations of Protamine, and Figure 4 summarizes the results of these measurements. PMA in aqueous solution displays a long excited–state lifetime of ~124 ns, whereas in the presence of PSS, it displays a dominant short component (Figure S3, Supplementary material) which characterizes the formation of aggregates
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of PMA in the presence of PSS. [39, 55, 58]
Figure 4.(A) Transient decay traces of PMA (35 M)-PSS (0.2 M) complex (λex = 339nm, λem =376 nm) in presence of various concentrations of Protamine (in M) (i) 0 (ii) 0.52 (iii) 1.04 (iv) 6.11. The instrument response function (IRF) is represented by black dotted lines whereas the decay trace represented by light blue line stands for PMA in water (B) The variation of average excited state lifetime (τavg) with increasing concentration of Protamine. The indicated error bar is the standard deviation.
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Pre-proof However, as evident from Figure 4, Journal when Protamine is gradually added to the PSS-PMA system, there is a gradual reduction in the contribution of faster component in transient decay traces, and in the presence of a relatively higher concentration of Protamine, the excited-state decay traces for PMA reproduces the same situation as observed for PMA in aqueous solution. This is more evident from the trend in the variation of average excited-state lifetime which has been presented in Figure 4B. This observation in the time-resolved emission measurements can be appreciated in relation to disintegration of aggregates of PMA from the surface of PSS, when Protamine is added to the solution which is
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ascribed to the relatively stronger interaction of Protamine with PSS when compared to that of PMA.
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Thus, time-resolved emission measurements are in excellent concurrence with the steady-state emission and ground-state absorption measurements.
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Thus, all the above measurements clearly suggested that the addition of Protamine to the
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PSS-PMA system leads to disassembly of aggregates of PMA from the surface of PSS owing to
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relatively stronger electrostatic interaction of the polycationic Protamine with the polyanionic PSS when compared to that of monocationic PMA with PSS. This Protamine induced disassembly of PMA-PSS
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system leads to significant changes in the photophysical features of the PMA-PSS system and provides
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a unique opportunity to detect Protamine in a sensitive and ratiometric fashion.
Scheme2. Schematic diagram of Protamine induced disassembly of aggregates of PMA from Polystyrene sulfonate surface. Apart from the sensitive detection of a targeted analyte, another important parameter to be assessed for a sensor is its selectivity towards the targeted analyte. To assess the selectivity parameter for our sensing system, we have chosen a variety of proteins with different iso-electric points, different 14
Journal Pre-proof amino acid composition, different molecular weight, and also we have chosen basic amino acids and their response to our sensing platform has been summarized in Figure 5. It is quite evident from the figure that the current sensing platform shows very high selectivity towards Protamine which can be ascribed to the arginine rich composition of the Protamine which provides a very high positive charge to the Protaminei.e., over 20 positive charges under physiological condition. Because of its polycationic nature, Protamine undergoes multiple electrostatic interaction with the polyanionic PSS which makes their interaction very stronger as compared to the other proteins which either carries less positive charge
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or are either neutral or anionic at physiological pH that leads to relatively much weaker interaction with
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the PSS and fails to dissociate the PSS-PMA assembly.
Another valuable parameter to evaluate for a new sensing scheme is its pH range of response. A
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good sensor system should be efficient over a broad pH range including physiological pH. Thus, to
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evaluate this aspect, we have also tested the response of the PMA-PSS complex as a function of pH and
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the results have been presented in figure S4 (Supplementary materia). As evident from the figure the response of the PMA-PSS system largely remains invariant over a pH range of 4-9 which satisfies the
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requirement of a broad pH range of operation.
Figure 5.Response plot for I376 for various proteins and analytes at a concentration of 4M. The indicated error bar is the standard deviation.
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Journal Pre-proof To evaluate the practical utility of the PSS-PMA platform towards Protamine sensing, we have also attempted to test the response of the present system in human serum samples. In diluted human serum samples, the PSS-PMA system could nicely respond to the spiked Protamine which has been presented in Figure 6. As evident from the figure, the ratiometric signal shows a nice linear variation with
Protamine
concentration
with
a
linear
regression
equation
of
ln(I376/I480)=0.52
(0.02)[Prs/M]+0.19 (0.09) (R2=0.985). The LOD value calculated for the serum sample is found to be 505 nM which is slightly higher than that obtained in pure aqueous system (7.71 nM) and can be
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ascribed to competition from the other components of the serum matrix.
Figure 6. Steady–state emission spectrum of PMA (35 M)-PSS (0.2 M) complex (λexc=340 nm) in
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Human serum matrix (1%) with increasing concentrations of Protamine (in M) (i) 0 (ii) 2.06 (iii) 3.08
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(iv) 4.09 (v) 9.11. Inset: Changes in logarithmic ratio of emission intensities at 376 to 480 nm with increasing concentrations of Protamine. The experimental data points are represented by red triangles, whereas the solid blue line is a linear fit to the data points. The indicated error bar in the inset is the standard deviation. Ground-state absorption measurements were also performed for the PSS-PMA system in the human serum samples with spiked Protamine and the results have been summarized in Figure S5 (Supplementary material). Here also we registered a linear response for the ratiometric signal with a linear regression equation ln(OD340/OD352)=0.169(0.04)[Prs/M]+0.32(0.10), (R2=0.966). The
16
detection limit was calculated to be 31.5Journal nM. Thus,Pre-proof these measurements suggest that the current sensing system has the potential to be employed for detecting Protamine in real life samples.
3. Conclusions In summary, we have developed a fluorescence based ratiometric sensing system for Protamine which is the only clinically approved antidote for the widely employed blood anti-coagulant Heparin. The detection scheme is based on the Protamine-induced disintegration of PMA-PSS aggregate assembly which hosts a monomer-aggregate equilibrium system. This Protamine induced disintegration brings out
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a modulation in the monomer-aggregate equilibrium and yields a very sensitive ratiomeric response for
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the detection of Protamine. Apart from being sensitive, the sensor system also shows very high
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selectivity towards Protamine, when compared to various proteins. The sensor system provides several advantages such as dual read out of sensor response both in terms of fluorimetry and colorimetry, high
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sensitivity and selectivity, consists of all commercially available components which provides a freedom
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from tedious and time-consuming synthetic efforts that significantly impacts the prospect of many
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developed sensors in application scenario. More importantly, the sensor system provides a ratiometric response which is a unique advantage in terms of robustness of its performance over previously reported
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Protamine sensors that operate through single wavelength based measurements. Importantly, our sensor
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system also shows response towards Protamine in human serum samples which suggest that this novel ratiometric sensor for Protamine may find potential applications in future.
ASSOCIATED CONTENT Supporting Information. The ground-state absorption spectra of PMA in the presence of various concentration of PSS; Normalized ground-state absorption spectra of PMA in water and of PMA-PSS complex in presence and absence of Protamine; Transient decay traces of PMA in water and in presence of PSS; Response of PMA-PSS as a function of pH of the medium, Ground-state absorption spectra of PMA-PSS complex in the presence of Protamine in human serum matrix (Figs S1-S5) AUTHOR INFORMATION 17
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Corresponding Author
*Email: [email protected]; [email protected]
Notes The authors declare no competing financial interest. †
On M. Sc. research project from Viva College, Virar, Palghar 401303, India
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Acknowledgments: PKS acknowledges the financial support from Department of Atomic Energy for carrying out this work. The authors also acknowledge the constant encouragement and support from
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Dr. H. Pal, Dr. S. Adhikari, and Dr. S. Kapoor, during the course of this work. S.P.P. gratefully thank
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Human Resource Development Division, Bhabha Atomic Research Centre, for providing the
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opportunity for this project internship.
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References [1] L. Zhou, H. Matsumura, M. Mezawa, H. Takai, Y. Nakayama, M. Mitarai, et al., Protamine stimulates bone sialoprotein gene expression, Gene, 516(2013) 228. [2] F.L. Sorgi, S. Bhattacharya, L. Huang, Protamine sulfate enhances lipid-mediated gene transfer, Gene Ther, 4(1997) 961-8. [3] S. Taylor, J. Folkman, Protamine is an inhibitor of angiogenesis, Nature, 297(1982) 307-12. [4] G. Fuentes-Mascorro, H. Serrano, A. Rosado, Sperm chromatin, Arch Androl, 45(2000) 215–25. [5] M.L. Meistrich, B. Mohapatra, C.R. Shirley, M. Zhao, Roles of transition nuclear proteins in
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spermiogenesis, Chromosoma, 111(2003) 483–8.
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[6] Y. Okamoto, K. Ogawa, T. Motohiro, N. Nishi, E. Muta, S. Ota, Primary Structure of Scombrine γ, Protamine Isolated from Spotted Mackerel (Scomber australasicus), J Biochem, 113(1993) 658-64.
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[10] A.A. Ensafi, N. Kazemifard, B. Rezaei, A simple and rapid label-free fluorimetric biosensor for 71(2015) 243–8.
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protamine detection based on glutathione-capped CdTe quantum dots aggregation, Biosens Bioelectron, [11] J.A. Carr, N. Silverman, The heparin-protamine interaction: a review, JCardiovascSurg, 40(1999) 659–66.
[12] I. Capila, R.J. Linhardt, Heparin–Protein Interactions, Angew Chem Int Ed, 41(2002) 390–412. [13] N. Mackman, Triggers, targets and treatments for thrombosis, Nature, 451(2008) 914–8. [14] J. Fareed, D.A. Hoppensteadt, R.L. Bick, An update on heparins at the beginning of the new millennium, Semin Thromb Hemostasis, 26(2000) 5–21. [15] J.M. Whitelock, R.V. Iozzo, Heparan sulfate: a complex polymer charged with biological activity, Chem Rev, 105(2005) 2745–64. [16] B. Casu, Structure and biological activity of heparin, Adv Carbohydr Chem Biochem, 43(1985) 51–134. [17] L.B. Jaques, Heparins--anionic polyelectrolyte drugs, Pharmacol Rev, 31(1979) 99–166.
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Journal [18] D.L. Rabenstein, Heparin and heparan sulfate: Pre-proof structure and function, Nat Prod Rep, 19(2002) 312– 31. [19] T.K. Toby, C.D. Sommers, D.A. Keire, Detection of native chondroitin sulfate impurities in heparin sodium with a colorimetric micro-plate based assay, Anal Methods, 4(2012) 1488–91. [20] N. Vasimalai, S.A. John, Aggregation and de-aggregation of gold nanoparticles induced by polyionic drugs: spectrofluorimetric determination of picogram amounts of protamine and heparin drugs in the presence of 1000-fold concentration of major interferences, J Mat Chem B, 1(2013) 5620-7. [21] R. Zhan, Z. Fang, B. Liu, Naked-Eye Detection and Quantification of Heparin in Serum with a Cationic Polythiophene, Anal Chem, 82(2010) 1326-33. [22] N.H. Mudliar, P.K. Singh, Emissive H-Aggregates of an Ultrafast Molecular Rotor: A Promising
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[23] T.W. Wakefield, H.C. B., B. Lindblad, W.M.J. Whitehouse, J.C. Stanley, Protamine pretreatment attenuation of hemodynamic and hematologic effects of heparin–protamine interaction: a prospective
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randomized study in human beings undergoing aortic reconstructive surgery, J Vasc Surg, 3(1986) 885– 9.
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[24] Z. Yao, W. Ma, Y. Yang, X. Chen, L. Zhang, C. Linc, et al., Colorimetric and fluorescent detection
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of protamines with an anionic polythiophene derivative, Org Biomol Chem, 11(2013) 6466-9. [25] C.W. Chan, J.W. Thompson, T.A. Gill, Quantitative determination of protamines by coomassie
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blue G assay, Anal Biochem, 226(1995) 191.
[26] S. Sakaguchi, A new method for the colorimetric determination of arginine, J Biochem, 37(1950).
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[27] D. Awotwe-Otoo, C. Agarabi, P.J. Faustino, M.J. Habib, S. Lee, M.A. Khan, et al., Application of quality by design elements for the development and optimization of an analytical method for protamine
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sulfate, J Pharmaceut Biomed, 62(2012) 61-7. [28] A. Snycerski, J. Dudkiewicz-Wilczynska, J. Tautt, Determination of protamine sulphate in drug formulations using high performance liquid chromatography, J Pharm Biomed Anal, 18(1998) 907–10. [29] A. Hvass, B. Skelbaek-Pedersen, Determination of protamine peptides in insulin drug products using reversed phase high performance liquid chromatography, J Pharm Biomed Anal, 37(2005) 551–7. [30] V.P.Y. Gadzekpo, K.P. Xiao, H. Aoki, P. Buhlmann, Y. Umezawa, Voltammetric detection of the polycation protamine by the use of electrodes modified with self-assembled monolayers of thioctic acid, Anal Chem, 71(1999) 5109. [31] K.P. Xiao, B.Y. Kim, M.L. Bruening, Detection of protamine and heparin using electrodes modified with poly (acrylic acid) and its amine derivative, Electroanalysis, 13(2001) 1447–53. [32] Y. Egawa, R. Hayashida, T. Seki, J. Anzai, Fluorometric determination of heparin based on selfquenching of fluorescein-labeled protamine, Talanta, 76(2008) 736–41.
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Journal Pre-proof [33] G. Herzog, D.W.M. Arrigan, Electrochemical Strategies for the Label-free Detection of Amino acids, Peptides and Proteins, Analyst, 132(2007) 615–32. [34] S. Pang, S. Liu, X. Su, A fluorescence assay for the trace detection of protamine and heparin, RSC Adv, 4(2014) 25857–62. [35] M.H. Lee, J.S. Kim, J.L. Sessler, Small Molecule-based Ratiometric Fluorescence Probes for Cations, Anions, and Biomolecules, Chem Soc Rev, 44(2015) 4185-91. [36] A.M. Pettiwala, P.K. Singh, Supramolecular Dye Aggregate Assembly Enables Ratiometric Detection and Discrimination of Lysine and Arginine in Aqueous Solution, ACS Omega, 2(2017) 8779−87. [37] A.M. Pettiwala, P.K. Singh, A molecular rotor based ratiometric sensor for basic amino acids.,
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Spectrochim Acta Mol Biomol Spectrosc, (2017).
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[38] C. Wang, Q. Sun, Z. Tong, X. Liu, F. Zeng, S. Wu, Interaction of cetyltrimethylammonium bromide and ploy(2-(acrylamido)-2-methylpropanesulfonic acid) in aqueous solutions determined by
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excimer fluorescence, Colloid Polym Sci, 279(2001) 664-70.
[39] S.P. Pandey, P.K. Singh, A Polyelectrolyte based Ratiometric Optical Sensor for Arginine and
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Lysine, Sens Actuators, B, (2019) 10.1016/j.snb.2019.127182.
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[40] R. Zhang, D. Tang, P. Lu, X. Yang, D. Liao, Y. Zhang, et al., Nucleic Acid-Induced Aggregation and Pyrene Excimer Formation, Org Lett, 11(2009) 4302-5.
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[41] J.B. Birks, Photophysics of Aromatic Molecules, New York: Wiley; 1970. [42] J.B. Birks, Excimers, Rep Prog Phys 38(1975) 903–74.
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[43] F.C. De Schryver, P. Collart, J. Vandendriessche, R. Goedeweeck, A. Swinnen, M. Van Der Auweraer, Intramolecular excimer formation in bichromophoric molecules linked by a short flexible
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chain, Acc Chem Res, 20(1987) 159–66. [44] J. Mondek, F. Mravec, T. Halasova, Z. Hnyluchova, M. Pekar, Formation and Dissociation of the Acridine Orange Dimer as a Tool for Studying Polyelectrolyte–Surfactant Interactions, Langmuir, 30(2014) 8726–34. [45] A.B. Fradj, R. Lafi, S.B. Hamouda, L. Gzara, A.H. Hamzaoui, A. Hafiane, Effect of chemical parameters on the interaction between cationic dyes and poly (acrylic acid), Journal of Photochemistry and Photobiology A: Chemistry, 284(2014) 49–54. [46] A.P. de Silva, H.Q.N. Gunaratne, T. Gunnlaugsson, A.J.M. Huxley, C.P. McCoy, J.T. Rademacher, et al., Signaling Recognition Events with Fluorescent Sensors and Switches, Chem Rev, 97(1997) 151566. [47] J.S. Kim, D.T. Quang, Calixarene-Derived Fluorescent Probes, Chem Rev, 107(2007) 3780-99.
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Journal Pre-proof [48] IUPAC Compendium of Analytical Nomenclature: Definitive Rules, Oxford: Pergamon Press; 1981. [49] Y. Gao, K. Wei, J. Li, Y. Li, J. Hu, A facile four-armed AIE fluorescent sensor for heparin and protamine, Sensors & Actuators: B Chemical, 277(2018) 408–14. [50] G. Guan, J. Sha, D. Zhu, Heparin-MPA dual modified CdS quantum dots used as a simple and rapid label-free fluorescent sensor for protamine and hemin detection, Microchemical Journal, 133(2017) 391–7. [51] H. Li, X. Yang, Bovine serum albumin-capped CdS quantum dots as inner-filter effect sensor for rapid detection and quantification of protamine and heparin, Anal Methods, 7(2015) 8445-52 [52] J. Liu, M. Xu, B. Wang, Z. Zhou, L. Wang, Fluorescence sensor for detecting protamines based on
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protamines and aminated graphene oxide, RSC Adv, 7(2017) 1432–8.
[53] Y. Liu, F. Zhang, X. He, P. Ma, Y. Huang, S. Tao, et al., A novel and simple fluorescent sensor
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based on AgInZnS QDs for the detection of protamine and trypsin and imaging of cells, Sensors & Actuators: B Chemical, 294(2019) 263–9.
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[54] F.M. Winnik, N. Tamai, J. Yonezawa, Y. Nishimura, I. Yamazaki, Temperature-induced phase
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[55] F.M. Winnik, Photophysics of Preassociated Pyrenes in Aqueous Polymer Solutions and in Other Organized Media, Chem Rev, 93(1993, ) 587-614.
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[56] A.A. Awasthi, P.K. Singh, Stimulus-Responsive Supramolecular Aggregate Assembly of Auramine O Templated by Sulfated Cyclodextrin, J Phys Chem B, 121(2017) 6208−19.
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[57] N.H. Mudliar, P.K. Singh, Fluorescent H-aggregates Hosted by a Charged Cyclodextrin Cavity, Chem Euro J, 22(2016) 7394-8.
[58] T. Costa, J.S. de Melo, C.S. Castro, S. Gago, M. Pillinger, I.S. Goncalves, Picosecond Dynamics of Dimer Formation in a Pyrene Labeled Polymer, J Phys Chem B, 114(2010) 12439-47.
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Declaration of competing interests
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The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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Journal Pre-proof Author statement
Shrishti P. Pandey: Conceptualization, data collection and Analysis, Writing- Original draft preparation
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Prabhat K. Singh: Conceptualization, Supervision, Writing- Reviewing and Editing
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Journal Pre-proof Graphical abstract
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A fluorescent ratiometric sensor for Protamine has been devised which involves Protamine induced disintegration of a supramolecular assembly which is built-up from a negatively charged polyelctrolyte, and yields a sensitive and selective sensor for a convenient and economic ratiometric detection of Protamine, a Heparin antidote.
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Journal Pre-proof Highlights
1. The sensor works via Protamine-induced disruption of a dye-polyelectrolyte system. 2. The sensor system displays an advantageous ratiometric response towards Protamine. 3. The sensor system is highly selective for Protamine as compared to other proteins. 4. The sensor system employs a commercially available probe molecule.
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5. The sensor system also shows good response in serum samples.
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Shrishti P. Pandey, Prabhat K. Singh PII:
S0167-7322(19)36869-2
DOI:
https://doi.org/10.1016/j.molliq.2020.112589
Reference:
MOLLIQ 112589
To appear in:
Journal of Molecular Liquids
Received date:
14 December 2019
Revised date:
20 January 2020
Accepted date:
26 January 2020
Please cite this article as: S.P. Pandey and P.K. Singh, A ratiometric scheme for the fluorescent detection of protamine, a heparin antidote, Journal of Molecular Liquids(2020), https://doi.org/10.1016/j.molliq.2020.112589
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
A Ratiometric Scheme for the Fluorescent Detection of Protamine, a Heparin antidote Shrishti P. Pandey†and Prabhat K. Singh,*
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Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085, INDIA
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*Authors for correspondence: Email: [email protected]; [email protected]
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Tel. 91-22-25590894, Fax: 91-22-5505151 Keywords: Protamine sensor; Ratiometry; Fluorescence sensor; Polystyrene sulfonate
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Abstract
Protamine is an arginine rich cationic protein and is known to be a very important pharmaceutical compound which is utilized in interventional radiology procedures, vascular surgery, and cardiac surgery, and is the only clinically approved antidote for Heparin overdose. Considering its importance in various biological processes, it is very important to develop sensor platforms for selective and sensitive detection of Protamine. Herein, we present a rarely reported ratiometric fluorescence detection scheme
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for Protamine, which is based on Protamine-induced disassembly of a dye-polyelectrolyte supramolecular assembly that causes a tuning of the monomer-aggregate equilibrium of the dye, and
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yields a sensitive ratiometric reponse for the Protamine. Our sensor system registers high sensitivity and
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selectivity for Protamine. Besides being simple, selective and sensitive, one of the prime features of this
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developed detection method is its ratiometric response which principally provides increased accuracy
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for quantitative analysis in complex samples due to inherent internal calibration of two bands. Another noteworthy feature associated with this detection scheme is that it employs commercially available
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probe molecule and components that avoid the heavy dependence on time-consuming and tedious
also
demonstrates
sensing
performance
in
complex
human
serum
matrix.
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scheme
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synthetic procedures involved with the previously existing methods. Importantly, our current sensing
2
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1. Introduction
Protamine is an arginine rich cationic protein which plays crucial biological roles in a variety of important biological events, for example, such as gene expression[1], gene transfer[2] and genesis of tissue.[3] Protamine also provides a highly compact configuration of chromatin in the nucleus of the sperm and binding to DNA.[4, 5] Protamine is extracted from the sperm of salmon roe fish. The high arginine content (up to 67%) in the poly-peptide sequence of Protamine leads to a high positive charge on the Protamine surface with an isoelectric point of about 13.8 units.[3, 6, 7] The molecular weight of
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Protamine is low (ca. 4500 Da), and under physiological conditions, it carries over 20 positive
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charges.[7-9] Apart from its various biological roles, Protamine is also known to be a very important
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pharmaceutical compound which is used in interventional radiology procedures, vascular surgery, and, cardiac surgery, and till date, it is the only clinically approved antidote for Heparin overdose.[10, 11]
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Heparin is a glycosaminoglycan (a copolymer of uronic or iduronic acids with alternate
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sulphated glucosamine residues) which bears an anionic rod-like structure.[12] Heparin plays a crucial
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role in different pathological and physiological procedures, for example, lipid transport, venous thromboembolism, cell growth, differentiation, metabolism, and blood circulation.[13-15] Importantly,
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Heparin is the most widely used anticoagulant drug in surgical procedures such as cardiopulmonary
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bypass surgery and extracorporeal blood circulation.[16-19] Heparin also acts as a co-factor for antithrombin III, an inhibitor present in the plasma.[20] However, excess dose as well as prolonged use of Heparin usually causes fatal bleeding complications. Therefore, the therapeutic levels of Heparin have to be monitored crucially to avoid the risk of complications such as thrombocytopenia and hemorrhage, which arise due to Heparin overdose. Heparin is administered in the range of 2–8 U mL-1 (17–67 µM) during cardiovascular surgery whereas a dose of 0.2–1.2 U mL-1 (1.7–10 µM) is recommended in postoperative conditions and long-term care.[21] Since Protamine is polycationic whereas Heparin is polyanionic, thus, a stable complex is formed due to rigid electrostatic interactions between Heparin and Protamine, which is devoid of anticoagulant activity. Protamine binds to Heparin competing with antithrombin III by the mechanism 3
Journal of charge neutralization.[20] Antithrombin III is Pre-proof thereby replaced from the Heparin-antithrombin III complex by Protamine which has a strong affinity towards Heparin, resulting in the efficient conversion of the anticoagulation activity.[8, 11] Protamine is well known and the only clinically approved drug till date, known to reverse the effects of Heparin.[22] However, Protamine may cause severe side effects which includes bradycardia, dyspnea, pulmonary hypertension, flushing and a feeling of warmth or sudden fall in blood pressure.[5, 11, 23] Therefore, there is an urgent need for developing methods for the fast, sensitive and selective detection of Protamine.
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The detection of Protamine is considered to be comparatively difficult due to the absence of
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aromatic amino acids in its structure.[24] This makes it inadequate for UV absorption measurements at 280nm, which are considered as the standard method for the quantification of proteins. Till now only a
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few methods for the sensing of Protamine have been reported, such as traditional colorimetric
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methods,[25, 26]High performance liquid chromatography,[27-29] electrochemical assay,[30, 31] etc.
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However, most of these methods fail to overcome certain limitations associated with Protamine sensing, such as moderate sensitivity and selectivity. Although these electrochemical and microgravimetric
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methods were successful to obtain a detection limit as low as 0.5 and 0.05 μg mL−1 respectively[24],
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these approaches suffer from certain disadvantages, such as, designing complicated electrochemical
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probes, time-consuming operational steps and requirement of sophisticated equipment. In addition, the previously reported methods also suffered from the problem of complicated synthetic design of employed probes, high cost, operational inconvenience, requirement of trained expertise, low interference ability with other biomolecules and so on. Therefore, it is important to develop rapid, selective and sensitive methods for the sensing of Protamine. In comparison to the other techniques, currently, there is an immense research focus on the development of fluorescent based sensors for the measurement of bioanalytes, because of the various powerful attractive attributes associated with the technique of fluorescence such as high sensitivity, operational simplicity, real-time detection and low cost.
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Journal Pre-proof Recently, a fluorescence based methods have been reported by Egawa et al for the determination of Protamine which is dependent on self-quenching of fluorescein-labeled protamine [32] while, Chem et al. reported a turn-on fluorescence based method for the determination of Heparin and Protamine which utilized the phenomenon of aggregation-induced emission enhancement.[33] However, these methods suffer from limited sensitivity and selectivity where protamine concentration could be detected only in the range of µg/mL. Therefore, to overcome these limitations, a method of fluorescence assay based on a fluorescein isothiocyanate (FITC)-labeled DNA probe was by Shu Pang et. al., where the
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detection limit of Protamine was obtained to be as low as 2.2 ng mL-1.[34] Further, N. Vasimalai et. al
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developed a sensor for the detection of Protamine based on the aggregation and de-aggregation of gold nanoparticles which was induced by polyionic protamine.[20] However, these methods, for the
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detection of Protamine still involves complicated methods to design the employed fluorescent probes
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and does not evaluate the sensing performance in the human serum matrix which restricts their
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application in real life detection of Protamine in serum samples. Further, most of these sensing schemes operate through signal measurement at single wavelength that make these methods susceptible to error
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due to variation in different analyte-independent factors such as change in the microenvironment of the
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probe, minute variation in experimental conditions such as pH of the medium, probe concentration,
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temperature, path length etc. In this regard, ratiometric sensors provide great advantage for sensor molecules which are exclusively based on intensity measurements, since the self-calibration of intensity at two distinct emission bands (distinct peak positions) excludes most or all of the uncertainties associated with small variation in experimental conditions which makes them more appropriate for quantitative measurements as well as for operation in complex environments.[35-37] To the best of our knowledge, there is no report on the ratiometric sensor primarily developed for Protamine. Thus, there is an urgent need for development of fluorescence based ratiometric methods for the detection of Protamine. In the present contribution, we report a fluorescence based ratiometric detection scheme for the selective and sensitive detection of Protamine. In the present system, the two distinct emission bands, 5
which is an essential requirement for theJournal design ofPre-proof ratiometric detection scheme, has been achieved by using a monomer-aggregate equilibrium system which is built up from an anionic polylelectrolyte induced aggregation of a cationic probe. The sensor operation is achieved as follows: (i) The cationic probe, pyrene methyl ammonium chloride (PMA), emits in the aqueous solution in the monomeric form (ii) The anionic polyelectrolyte, polystyrene sulfonate (PSS) induces aggregation of the cationic probe, PMA, leading to a monomer-aggregate system with two distinct emission bands. (iii) Protamine, being a cationic polyelectrolyte, interacts very strongly with the anionic polylectrolyte, PSS, and results into
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dissociation of PMA aggregates bound to PSS which significantly affects the monomer-aggregate
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equilibrium, and provides a sensitive ratiometric response for the protamine concentration. The detection scheme is simple, rapid, label free, sensitive, and selective. Importantly, the sensor platform
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includes the components which are all commercially available components, and thus provides the
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freedom from heavy dependence on complex and time-intensive synthetic procedures for the sensor
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probe production itself that significantly affects the practical applicability of many excellent sensing
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schemes in real-life applications.
(A)
(B)
Scheme 1: Chemical structure of Polystyrene sulfonate (A) and Pyrene methyl ammonium chloride (B)
2. Results and Discussion Since the sensing platform for the current detection scheme is based on the formation of a dyepolyelectrolyte aggregate assembly, so we will first present the spectroscopic features of the dyepolyelectrolyte supramolecular assembly followed by its interaction with the Protamine. Figure 1 presents the steady-state emission spectra of the cationic probe, PMA, in the presence of increasing concentrations of an anionic polyelectrolyte, PSS. PMA, in the aqueous solution, emits in the monomer
6
Journal form with sharp emission bands at 376, 394 andPre-proof a weak shoulder at 410 nm which is in excellent agreement with the previous literature reports.[38] However, as evident from the figure, the gradual addition of PSS, to the aqueous solution of PMA, leads to a drastic reduction in the monomer emission bands along with the appearance of a new emission band at 480 nm that has been attributed to the formation of PMA aggregates induced by the anionic polyelectrolyte, PSS owing to the charge neutralization of the multiple cationic PMA molecules on the surface of negatively charged PSS.[39] It is well known that Pyrene based molecules displays characteristic emission band for its aggregated state
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or the excimer state, as compared to the emission band for the monomeric species of PMA.[40-43] Such
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charged polyelectrolyte induced aggregation of oppositely charged probe molecules have been amply
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reported in literature.[44, 45]
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Figure 1. Steady-state emission spectra of PMA in water (λexc=340 nm) at different concentrations of PSS (in M) (i) 0 (ii) 0.14 (iii) 0.19 (iv) 0.21 (v) 0.30. Inset: Variation in the ratio of emission intensity obtained at 376nm to 480nm respectively with increasing concentration of PSS.The indicated error bar is the standard deviation. The observation of two distinct emission bands for the monomer and the aggregate species of PMA molecules provides an ideal case for the fabrication of a ratiometric sensor,[46, 47] if a modulation in the above bands can be achieved in response to the targeted analyte. It has been also established that the major underlying interaction force in the formation of PMA-PSS aggregate assembly is the electrostatic interaction between the cationic PMA and the anionic PSS molecules.[39] Thus, if electrostatic
7
Journal Pre-proofaggregate assembly, this will lead to the interaction forces can be modulated in this dye-polyelectrolyte modulation of the monomer-aggregate equilibrium in the system, and may provide a ratiometric response for a targeted analyte. In this regard, Protamine, which is the analyte of interest here, is polycationic in nature and is known to form a stable complex with polyanionic Heparin due to rigid electrostatic interactions between them, which is devoid of anticoagulant activity.[11]This makes the basis for using Protamine as the only pharmaceutical compound which is clinically approved for reversing the effect of Heparin, the most extensively employed blood anti-coagulant.[11] Since the
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present dye-polyelectrolyte assembly is built up on the surface of a highly negatively charged sulphated
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polyelectrolyte, so we envisaged that introduction of Protamine to the PSS-PMA assembly may disrupt the interaction between the PMA aggregate with the PSS owing to the relatively stronger interaction of
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polycationic Protamine with polyanionic PSS. This may lead to disassembly of aggregates of PMA from
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the surface of PSS, which, in turn, will lead to the manipulation in the monomer-aggregate equilibrium,
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and may yield an analytically significant signal in response to Protamine. To test this idea, we have performed steady-state emission measurements for the PSS-PMA system in the presence of increasing
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concentration of Protamine, and Figure 2 summarizes the results of these measurements. As it is clearly
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visible from the figure, the introduction of Protamine to the PMA-PSS solution, leads to reduction in the
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aggregate emission band at 480 nm together with a concomitant increase in the monomer emission band. This clearly indicates the dissociation of PMA aggregates from the anionic surface of PSS upon addition of Protamine that can be assigned to comparatively stronger interaction of polycationic protamine over monocationic PMA with polyanionic PSS. This Protamine-induced modulation in the monomer-aggregate equilibrium of PSS-PMA system thus generates a ratiometric signal, in response to Protamine concentration, (Figure 2B)
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Figure 2. (A) Steady–state emission spectra of PMA (35 M)-PSS (0.2 M) complex in presence of different concentrations of Protamine (in M) (i) 0 (ii) 1.04 (iii) 1.56 (iv) 2.08 (v) 2.59. (λexc=340 nm)
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(B) Variation of logarithmic ratio of emission intensity at 376 nm to 480 nm with varying concentration
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of Protamine. The experimental data points are represented by half-filled squares and the solid red line
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represents a linear fit to the data points. The indicated error bar is the standard deviation. To quantify the Protamine concentration dependent changes, the ratio of emission intensity of the monomer emission band (376 nm) to the aggregate emission band (480 nm) is plotted and has been presented in Figure 2B. The logarithm of the signal increases linearly with increase in concentration of protamine in the concentration range of 0-4 M with a linear regression equation of ln(I376/I480)=1.454(0.128) +1.72(0.27)[Prs/M] (R2=0.96). The limit of detection, based on 3σ/S,[48] was calculated to be 7.71 nM, where σ stands for the standard deviation of 10 blank measurements and S represents the slope of the calibration curve. Further, ratiometric detection scheme provides several advantages with respect to the single wavelength based detection measurements, as single wavelength
9
based measurements are prone to error Journal by various Pre-proof analyte-independent factors such as variation in the microenvironment of the probe, small variation in the instrumental factors such as fluctuation in lamp light intensity, small variation in experimental parameters, such as, temperature, pathlength, probe concentration etc. Ratiometric detection provides immunity against these small but almost unavoidable variations in experimental conditions that makes ratiometric detection, a method of choice for accurate quantitative analysis as well as performance in complex medium.[35] Note that there is no report on the ratiometric detection scheme which is primarily developed for Protamine detection. Thus, the present
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sensing scheme is a unique contribution in the field of fluorescence sensing of Protamine (Table 1).
Analytical
Commercial
No
method
Availability
LOD
0.015 mg
HPLC
4
Fluorimetry
5
Colorimetry and
No
Yes
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Fluorimetry 6
Fluorimetry
No
7
Fluorimetry
No
Fluorimetry
No
Fluorimetry
No
9 10
11
12
[27]
2.0µg ml-1
-
No
[30]
15 µg ml-1
-
No
[28]
2.2 ng mL-1
No
No
[34]
0.1 μg mL−1
No
No
[24]
1.0 ng mL-1
No
Yes
[10]
No
Yes
[49]
mL−1
No
Yes
[50]
2.4 ng mL-1
No
Yes
[51]
No
Yes
[52]
No
Yes
[53]
Fluorimetry
No
Fluorimetry
No
Fluorimetry and
Yes
Yes
Colorimetry
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HPLC
Yes
serum matrix No
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3
No
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Electrochemistry
Human
-
ml-1
2
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Ratiometric Evaluation in Reference
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Table 1: Comparison of performance of the various sensors for Protamine detection
23.4 ng
0.4 mg mL1
0.13 μg mL−1 31.5 4.1 ng mL−1
This study
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Journal Pre-proof To explore the possibility of monitoring this Protamine-induced disintegration of PSS-PMA assembly colorimetrically, ground-state absorption measurements were also performed for the PMAPSS system in the presence of different concentration of Protamine, and Figure 3 summarizes the results of these measurements. PMA in aqueous solution displays absorption band at 340 nm, whereas in the presence of PSS, it shows a red-shifted absorption band at 352 nm (Figure S1, supplementary information) that characterizes the PMA aggregates in the presence of PSS.[39] However, as evident
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from figure 3, upon addition of Protamine, the absorbance at monomer absorption band (340 nm) starts recovering along with a reduction in absorbance at 352 nm. Figure S2 (supplementary material) presents
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the normalized absorption spectra for PMA-PSS complex the presence and absence of Protamine. It is
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clearly evident from the figure that the spectral features of PMA-PSS complex is widely different from
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that of PMA in water, however, in the presence of Protamine, it clearly resembles to that of PMA in
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water. Another spectroscopic analysis which is popularly performed with the help of absorption spectra of pyrene derivative, in connection to their state of aggregation is peak-to-valley ratio (PA) analysis.[54,
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55] PA is calculated as the ratio of the most intense peak to its adjacent minimum at the shorter
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wavelength. In the present system, the PA value for PMA in water is recorded as 2.1 which reduces to 1.08 in the presence of PSS which signifies a large extent of aggregation of PMA in the presence of
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PSS. However, upon addition of Protamine, the PA value recovers back to 2.01 which once again supports the recovery of free form of PMA. This clearly reflects the disassembly of PMA aggregates from the surface of PSS that can be attributed to the stronger interaction of polycationic Protamine with PSS as compared to the interaction of monocationic PMA with PSS. Thus, the ground-state absorption measurements nicely validate the proposal made from steady-state emission measurements.
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Figure 3.(A) Ground-state absorption spectra of PMA (35 M)-PSS (0.2 M) complex in presence of
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different concentrations of Protamine (in M) (i) 0 (ii) 0.52 (iii) 1.10 (iv) 1.56 (v) 2.08 (vi) 2.59 (vii) 3.10 (viii) 4.11. (B) Changes in the ratio of absorbance at 340 to 352 nm with increasing concentration
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of Protamine. The experimental data points have been represented by square, whereas the solid red line
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is a fit to the data points according to the linear model. The indicated error bar is the standard deviation. A ratiometric analysis of the titration data from ground-state absorption measurements suggest that the logarithm of the ratio of monomer to aggregate absorption band, represented by OD340/OD352, changes linearly with increasing concentration of Protamine in a dynamic range of 0-4 M with a linear regression equation of ln(OD340/OD352)= 0.49 (0.03)[Prs/M]-0.078(0.015), (R2=0.97). The detection limit calculated, from this analysis was found to be 9.31.5 nM. Thus, these measurements clearly suggest that PMA-PSS system can function as a ratiometric sensor for Protamine. Time-resolved emission measurements are also quite responsive towards the molecular state of a probe molecule such as monomer, aggregate etc as the state of association of probe molecules generally
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Journal56, Pre-proof affects the excited-state relaxation processes.[22, 57] Thus, to further demonstrate the phenomenon of Protamine induced disintegration of PMA-PSS aggregate system, time-resolved emission measurements were also performed for the PSS-PMA system, in the presence of various concentrations of Protamine, and Figure 4 summarizes the results of these measurements. PMA in aqueous solution displays a long excited–state lifetime of ~124 ns, whereas in the presence of PSS, it displays a dominant short component (Figure S3, Supplementary material) which characterizes the formation of aggregates
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of PMA in the presence of PSS. [39, 55, 58]
Figure 4.(A) Transient decay traces of PMA (35 M)-PSS (0.2 M) complex (λex = 339nm, λem =376 nm) in presence of various concentrations of Protamine (in M) (i) 0 (ii) 0.52 (iii) 1.04 (iv) 6.11. The instrument response function (IRF) is represented by black dotted lines whereas the decay trace represented by light blue line stands for PMA in water (B) The variation of average excited state lifetime (τavg) with increasing concentration of Protamine. The indicated error bar is the standard deviation.
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Pre-proof However, as evident from Figure 4, Journal when Protamine is gradually added to the PSS-PMA system, there is a gradual reduction in the contribution of faster component in transient decay traces, and in the presence of a relatively higher concentration of Protamine, the excited-state decay traces for PMA reproduces the same situation as observed for PMA in aqueous solution. This is more evident from the trend in the variation of average excited-state lifetime which has been presented in Figure 4B. This observation in the time-resolved emission measurements can be appreciated in relation to disintegration of aggregates of PMA from the surface of PSS, when Protamine is added to the solution which is
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ascribed to the relatively stronger interaction of Protamine with PSS when compared to that of PMA.
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Thus, time-resolved emission measurements are in excellent concurrence with the steady-state emission and ground-state absorption measurements.
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Thus, all the above measurements clearly suggested that the addition of Protamine to the
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PSS-PMA system leads to disassembly of aggregates of PMA from the surface of PSS owing to
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relatively stronger electrostatic interaction of the polycationic Protamine with the polyanionic PSS when compared to that of monocationic PMA with PSS. This Protamine induced disassembly of PMA-PSS
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system leads to significant changes in the photophysical features of the PMA-PSS system and provides
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a unique opportunity to detect Protamine in a sensitive and ratiometric fashion.
Scheme2. Schematic diagram of Protamine induced disassembly of aggregates of PMA from Polystyrene sulfonate surface. Apart from the sensitive detection of a targeted analyte, another important parameter to be assessed for a sensor is its selectivity towards the targeted analyte. To assess the selectivity parameter for our sensing system, we have chosen a variety of proteins with different iso-electric points, different 14
Journal Pre-proof amino acid composition, different molecular weight, and also we have chosen basic amino acids and their response to our sensing platform has been summarized in Figure 5. It is quite evident from the figure that the current sensing platform shows very high selectivity towards Protamine which can be ascribed to the arginine rich composition of the Protamine which provides a very high positive charge to the Protaminei.e., over 20 positive charges under physiological condition. Because of its polycationic nature, Protamine undergoes multiple electrostatic interaction with the polyanionic PSS which makes their interaction very stronger as compared to the other proteins which either carries less positive charge
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or are either neutral or anionic at physiological pH that leads to relatively much weaker interaction with
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the PSS and fails to dissociate the PSS-PMA assembly.
Another valuable parameter to evaluate for a new sensing scheme is its pH range of response. A
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good sensor system should be efficient over a broad pH range including physiological pH. Thus, to
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evaluate this aspect, we have also tested the response of the PMA-PSS complex as a function of pH and
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the results have been presented in figure S4 (Supplementary materia). As evident from the figure the response of the PMA-PSS system largely remains invariant over a pH range of 4-9 which satisfies the
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requirement of a broad pH range of operation.
Figure 5.Response plot for I376 for various proteins and analytes at a concentration of 4M. The indicated error bar is the standard deviation.
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Journal Pre-proof To evaluate the practical utility of the PSS-PMA platform towards Protamine sensing, we have also attempted to test the response of the present system in human serum samples. In diluted human serum samples, the PSS-PMA system could nicely respond to the spiked Protamine which has been presented in Figure 6. As evident from the figure, the ratiometric signal shows a nice linear variation with
Protamine
concentration
with
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regression
equation
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ln(I376/I480)=0.52
(0.02)[Prs/M]+0.19 (0.09) (R2=0.985). The LOD value calculated for the serum sample is found to be 505 nM which is slightly higher than that obtained in pure aqueous system (7.71 nM) and can be
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ascribed to competition from the other components of the serum matrix.
Figure 6. Steady–state emission spectrum of PMA (35 M)-PSS (0.2 M) complex (λexc=340 nm) in
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Human serum matrix (1%) with increasing concentrations of Protamine (in M) (i) 0 (ii) 2.06 (iii) 3.08
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(iv) 4.09 (v) 9.11. Inset: Changes in logarithmic ratio of emission intensities at 376 to 480 nm with increasing concentrations of Protamine. The experimental data points are represented by red triangles, whereas the solid blue line is a linear fit to the data points. The indicated error bar in the inset is the standard deviation. Ground-state absorption measurements were also performed for the PSS-PMA system in the human serum samples with spiked Protamine and the results have been summarized in Figure S5 (Supplementary material). Here also we registered a linear response for the ratiometric signal with a linear regression equation ln(OD340/OD352)=0.169(0.04)[Prs/M]+0.32(0.10), (R2=0.966). The
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detection limit was calculated to be 31.5Journal nM. Thus,Pre-proof these measurements suggest that the current sensing system has the potential to be employed for detecting Protamine in real life samples.
3. Conclusions In summary, we have developed a fluorescence based ratiometric sensing system for Protamine which is the only clinically approved antidote for the widely employed blood anti-coagulant Heparin. The detection scheme is based on the Protamine-induced disintegration of PMA-PSS aggregate assembly which hosts a monomer-aggregate equilibrium system. This Protamine induced disintegration brings out
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a modulation in the monomer-aggregate equilibrium and yields a very sensitive ratiomeric response for
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the detection of Protamine. Apart from being sensitive, the sensor system also shows very high
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selectivity towards Protamine, when compared to various proteins. The sensor system provides several advantages such as dual read out of sensor response both in terms of fluorimetry and colorimetry, high
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sensitivity and selectivity, consists of all commercially available components which provides a freedom
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from tedious and time-consuming synthetic efforts that significantly impacts the prospect of many
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developed sensors in application scenario. More importantly, the sensor system provides a ratiometric response which is a unique advantage in terms of robustness of its performance over previously reported
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Protamine sensors that operate through single wavelength based measurements. Importantly, our sensor
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system also shows response towards Protamine in human serum samples which suggest that this novel ratiometric sensor for Protamine may find potential applications in future.
ASSOCIATED CONTENT Supporting Information. The ground-state absorption spectra of PMA in the presence of various concentration of PSS; Normalized ground-state absorption spectra of PMA in water and of PMA-PSS complex in presence and absence of Protamine; Transient decay traces of PMA in water and in presence of PSS; Response of PMA-PSS as a function of pH of the medium, Ground-state absorption spectra of PMA-PSS complex in the presence of Protamine in human serum matrix (Figs S1-S5) AUTHOR INFORMATION 17
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Corresponding Author
*Email: [email protected]; [email protected]
Notes The authors declare no competing financial interest. †
On M. Sc. research project from Viva College, Virar, Palghar 401303, India
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Acknowledgments: PKS acknowledges the financial support from Department of Atomic Energy for carrying out this work. The authors also acknowledge the constant encouragement and support from
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Dr. H. Pal, Dr. S. Adhikari, and Dr. S. Kapoor, during the course of this work. S.P.P. gratefully thank
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Human Resource Development Division, Bhabha Atomic Research Centre, for providing the
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opportunity for this project internship.
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Declaration of competing interests
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The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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Journal Pre-proof Author statement
Shrishti P. Pandey: Conceptualization, data collection and Analysis, Writing- Original draft preparation
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Prabhat K. Singh: Conceptualization, Supervision, Writing- Reviewing and Editing
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Journal Pre-proof Graphical abstract
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A fluorescent ratiometric sensor for Protamine has been devised which involves Protamine induced disintegration of a supramolecular assembly which is built-up from a negatively charged polyelctrolyte, and yields a sensitive and selective sensor for a convenient and economic ratiometric detection of Protamine, a Heparin antidote.
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Journal Pre-proof Highlights
1. The sensor works via Protamine-induced disruption of a dye-polyelectrolyte system. 2. The sensor system displays an advantageous ratiometric response towards Protamine. 3. The sensor system is highly selective for Protamine as compared to other proteins. 4. The sensor system employs a commercially available probe molecule.
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5. The sensor system also shows good response in serum samples.
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