A Study on Plasminogen-Ag Nanoparticles Interaction and its Application in Sensor Development

The interaction between plasminogen and Ag nanoparticles is studied using different techniques and applied for plasminogen sensor development. Ag nanoparticles are synthesized using an algal extract and their absorbance, emission, and electrochemical oxidation signals are detected and monitored as a function of plasminogen concentration. The variation in the optical and electrochemical properties of Ag nanoparticles is correlated with the changes in the hydrodynamic size of the bioconjugate at different plasminogen concentrations. A steady decrease in the absorbance and electrochemical oxidation peak of Ag nanoparticles is observed, while a threshold plasminogen concentration results in increasing the emission of Ag nanoparticles followed by a steady decrease in signal. The decrease in the optical and electrochemical oxidation signal of Ag nanoparticles agrees with the plasminogen-induced Ag nanoparticle agglomeration shown by dynamic light scattering. Calibration curves are established based on the absorbance, emission, and voltammetric studies obtaining a limit of detection as low as 0.740 nM with a wide linear range of 0.942-18.2 nM, which is a very promising analytical system for plasminogen detection, facilitating its progress as a biomarker for different biomedical applications.


Introduction
Plasminogen (Plg) is a plasma glycoprotein with a molecular weight of 92 kDa, present at a concentration of 2 µM with a half-life of around two days.It is a multifaceted zymogenic protein that needs to be activated to plasmin, a key fibrinolytic serine protease.The plasminogen-activating system is conventionally studied for its role in hemostasis.However, recently, there has been growing evidence of a much bigger role of this system in the human body.The multitude of Plg receptors allows it to exert other functions that affect the immune system, inflammatory processes, wound repair, protein aggregate clearance, and surprisingly, viral infection [1,2].For example, it has been reported that a low level of Plg was correlated with inflammatory markers such as c-reactive protein (CRP) and interleukin-6 (IL-6), coagulation markers such as international normalized ratio (INR) and activated partial thromboplastin time (APTT), and organ dysfunction indicators such as high level of fasting blood glucose level and reduced glomerular filtration rate [3].More interestingly, Plg has been found to play an important role in COVID-19 complications, with several reports on the correlation between low levels of Plg and increased mortality [3] and Plg's effectiveness in the treatment of associated lung lesions and hypoxemia [4].Thus, Plg is a very promising COVID-19 prognosis biomarker and therapeutic agent.
The interaction between proteins and nanoparticles (NPs) is a complex process that has huge implications for diagnostics and drug delivery.Typically, proteins are adsorbed on the surface of the nanoparticles forming a "protein corona" with the reaction kinetics and dynamics determined by the type of interacting protein and NPs.Such interaction leads to changes in the functionality of the nanoparticles and also the protein structure, which exhibits conformational changes and unfolding processes with a major impact on its properties [5].Protein-NP complexation has been studied in different systems and for different applications.For example, bovine serum albumin (BSA) has been used as a capping agent and stabilizer in the synthesis of Au [6] and Ag [7] NPs.Several types of NPs have been loaded with Plg activators for highly efficient and targeted delivery [8].Recently, Boehmler et al. studied the reaction kinetics between Ag NPs and BSA, reporting an increase in the Ag dissolution rate constant upon BSA adsorption on its surface, which was dependent on the size of the Ag NPs and the BSA concentration.They explained this observation by BSA-Ag + complex formation and displacement by excess BSA [9].
Plg-NP complexation has wide implications in Plg detection and hemostasis management and needs to be well studied.Bychkova et al. studied the interaction between Plg and iron oxide NPs through nonspecific binding and found ~70% decrease in Plg fibrinolytic activity upon conjugation [10].Several studies have reported an inherent thrombolytic activity of Ag NPs that could be mediated through its interaction with Plg, triggering its activation, in addition to antagonizing its activation inhibitors [11][12][13].Despite the evidence of major functionality changes upon Plg-NP interaction, studies on the dynamics and kinetics of such complexation, and its implications on conjugate complex properties and functionalities are very scarce.
In this work, we studied the interaction between Plg and Ag NPs, and characterized the Plg-Ag complexation at a wide range of Plg concentrations (0.942-18.2 nM), spectroscopically using UV-Vis and fluorimetric techniques, and electrochemically using square wave voltammetry (SWV).In addition, dynamic light scattering (DLS) was used to track complex size changes as the Plg concentration increased.Plg was found to impact the spectroscopic and electrochemical properties of Ag NPs significantly.Both UV-Vis spectroscopy and SWV showed a steady decrease in Ag NP response as the Plg concentration increased to 14.7 nM and 7.43 nM, respectively, followed by a slower decrease at higher concentrations.Fluorimetry showed a relatively stable Ag emission till 4.57 nM Plg, beyond which there was a sudden emission increase that decreased gradually upon adding more Plg.The steady and linear change of Ag NPs response as a function of Plg resulted in calibration curves with a wide linear range and low limits of detection (LOD) and quantification (LOQ).SWV showed the highest sensitivity with LOD and LOQ of 0.740 nM and 2.47 nM, respectively.Dynamic light scattering showed the appearance of larger particles as Plg concentration increased, which indicated Plg-induced Ag NP agglomeration that could have contributed to the change in Ag NP properties.The significant change in the optical, electrochemical, and structural properties of Ag NPs upon complexation with Plg shed light on the nature of their conjugation and demonstrated a robust methodology to develop a highly sensitive Plg sensor that could help in the development of Plg as a biomarker.

Materials and Chemicals
Plasminogen purified from human plasma using affinity column with Lysine-Sepharose beads, silver nitrate, sodium citrate dihydrate, and citric acid monohydrate were purchased from Sigma-Aldrich.Noctiluca scintillans algae were collected from the eastern coast of the United Arab Emirates.Graphite pencil lead (GPE) with a diameter of 0.5 mm (Pentel, HB) was used as the working electrode.Ultrapure water with a resistivity of 10-15 MΩ cm (Milli-Q Elix Essential® 5 ) was used in all experiments.

Synthesis of Ag NPs
Ag nanoparticles were synthesized using Noctiluca scintillans algae according to our earlier report [14] using 2.0% w/v algae extract, 0.1 M AgNO3 at 80 °C, pH 7.2 for 2.5 h with an Ag:algal extract of 3:7 v/v.

Instrumentation
UV-Vis spectroscopy was carried out using Jenway 6850 spectrophotometer at a wavelength range of 300-800 nm and a resolution of 1 nm -1 .Transmission electron microscopy (TEM) was conducted using Titan Themis G2 Probe Cs Corrected Scanning Transmission Electron Microscope operating at 300 kV.Fluorimetry was conducted using a Shimadzu RF-6000 fluorimeter with an excitation wavelength of 434 nm and an emission wavelength range of 444-900 nm, a scan speed of 6000 nm/min, and a resolution of 1 nm -1 .Electrochemical measurements were conducted using CHI 1232c electrochemical workstation in a three-electrode cell using a graphite pencil working electrode with an immersion depth of 1 cm, Pt wire counter electrode, and Ag/AgCl (1 M KCl) reference electrode in a 0.1 M citrate buffer (pH 4) electrolyte.Dynamic light scattering was performed using Microtrac Nanotrac Wave II.

Results and Discussion
Metal NPs including Ag NPs show a unique optical phenomenon that arises from their nanoscale dimensions, which confine surface electrons.They can absorb electromagnetic radiation at a specific frequency that resonates with that of their surface electrons, which is mainly a function of the nanoparticle size.This phenomenon is known as surface plasmon resonance (SPR).The SPR absorbance band position and intensity are significantly affected by changes on the surface of the NPs, thus, SPR is widely applied for analytical applications [15,16].We synthesized Ag nanoparticles using an algal extract according to our previous work [14].The Ag NP absorbance peak was detected at 434 nm (Figure 1a), which indicated a size of ~60-80 nm [17].This size range was confirmed using TEM, which showed Ag nanoparticles within the size range of 50-80 nm (Figure 1c).Plg was mixed with the Ag nanoparticles at increasing concentrations, and the effect on the Ag NPs SPR peak was monitored.It was observed that the peak intensity decreased steadily as the concentration of Plg increased from 0.942 nM to 14.7 nM, and then further decreased at a slower rate or almost stabilized at higher concentrations till 18.2 nM (Figure 1a-b).Both ranges were linear and we used the former as our main calibration curve from which we could calculate the LOD and LOQ to be 1.07 nM and 3.56 nM, respectively according to Equations (1-2) [18].
Where  is the standard error of the intercept of the calibration curve and s is the slope of the linear range of 0.942-14.7 nM.The effect of Plg on the SPR peak of Ag nanoparticles could be explained by its adsorption on the surface of the NPs [5], which disrupted the electron cloud oscillation and resulted in a change in SPR peak intensity.We were able to demonstrate that such change was concentration dependant and linear, and thus could be used for the quantitative detection of Plg.
Fluorescence is another optical phenomenon of metal nanoparticles that arises due to the quantum confinement effect that results in discrete energy levels [19].Such discrete levels allow for the absorbance and emission of electromagnetic radiation, with several reported mechanisms, including the transition between the conduction band and holes in the d band, the presence of oligomeric metallic clusters, and electron interband transitions [20,21].As with absorbance, metal NPs emissions are sensitive to surface changes that could form hybrid states at the interface of the two interacting moieties [19], and thus could be applied in electrochemical systems, especially given the inherently high sensitivity of fluorimetric analytical systems [22].To this end, the Ag nanoparticles were excited at 434.0 nm and the emission peak could be detected at 868.5 nm (Figure 2a), which was close to the values reported earlier [19].The fluorimetry study revealed a more complex interaction between Ag NPs and Plg, where the emission peak intensity was stable upon Plg addition till 4.67 nm, which was a threshold Plg concentration upon which the emission intensity increased by ~32% and then decreased steadily thereafter (Figure 2a).A linear calibration curve could be established at the Plg concentration range of 5.60-18.2nM, from which LOD and LOQ could be calculated to be 2.65 nM and 8.82 nM (Figure 2b).The sudden fluorescence intensity change is an intriguing phenomenon that could be explained by either Plg adopting certain conformational changes or Ag NPs structural rearrangement at the threshold concentration of 4.67 nM.This phenomenon is very interesting and will be further investigated in our future work.Although fluorimetry is inherently a very sensitive technique and showed a linear response after the threshold concentration, the system was not as sensitive as UV-Vis spectroscopy, which could be due to the nature of Plg effect on SPR and emission, which was more significant in the former than the latter.
Metal nanoparticles have excellent electrochemical properties due to their high conductivity and electrocatalytic activity, which is especially enhanced due to the large surface area of NPs [23].The interaction of Ag NPs with BSA has been studied using an electrochemical voltammetric approach, where BSA was found to enhance the dissolution rate of Ag NPs [9].In addition, electrochemical techniques are known to be among the most facile, cost-effective, and sensitive analytical systems, and thus could be very promising for point-of-care Plg detection [18,24].We studied the interaction between Ag NPs and Plg on a graphite working electrode in a citrate buffer at pH 4 using SWV, recoding the oxidation peak of Ag NPs at ~0.4 V (Figure 3a).Upon the addition of Plg, the Ag oxidation peak intensity started to decrease at a relatively high rate till 7.43 nM, after which the rate of change decreased but was still linear (Figure 3a).Two linear ranges could be derived from such calibration experiment, at 0.942-7.43nM and 7.43-18.2nM.LOD and LOQ were calculated using the former range to be 0.740 nM and 2.47 nM, respectively (Figure 3b).The decreasing Ag oxidation peak could be the result of two factors, the first is a Plg protective layer formed as a surface "protein corona" [5] and the second is an enhanced surface etching of Ag NPs as a result of Plg-Ag(I) complex formation, as reported earlier in the Ag-BSA system [9].However, conclusive results would need further study of the system.The electrochemical approach achieved lower LOD and LOQ, as compared to spectroscopic approaches (Table 1), indicating its very high sensitivity and providing a guide for future studies on protein-metal NP interaction.The application of Ag-Plg complexation in Plg analytical detection using different techniques is summarized in Scheme 1.  DLS or photon correlation spectroscopy is a cost-effective and facile approach for nanoparticle sizing and monitoring size change over time or upon interaction with a specific reagent.It is based on the determination of diffusion coefficient based on the light scattering of particles, and then correlating the diffusion coefficient to their hydrodynamic radius using the Stokes-Einstein equation [25][26][27].We used DLS to study the interaction between Ag nanoparticles and Plg to try to get an insight into the morphological changes taking place with increasing Plg concentration (Figure 4).The hydrodynamic size of Ag nanoparticles was in the range of 100-120 nm, which was not changed upon Plg addition till a concentration of 4.67 nM.The relatively larger size of Ag nanoparticles obtained using DLS, as compared to TEM, could be explained by the enclosure of Ag nanoparticles within residues of the algal extract (Figure 1c), which resulted in a larger hydrodynamic size.However, at higher Plg concentrations, a second peak appeared indicating the formation of larger size particles of ~240 nm and ~470 nm at Plg concentrations of 8.

Conclusion
We have studied the interaction between Ag nanoparticles and Plg using four different techniques; UV-Vis spectroscopy, fluorimetry, voltammetry, and dynamic light scattering, thus probing the effect of Plg on the SPR, emission, electrochemical, and size properties of Ag NPs.It was observed that the absorbance and electrochemical oxidation response of Ag NPs decreased steadily as Plg concentration increased, while that of fluorescence exhibited a more complex behavior, increasing after a threshold Plg concentration and then decreasing steadily at higher concentrations.The hydrodynamic size of Ag NPs showed a dual range upon Plg addition at concentrations higher than 4.67 nM indicating Plginduced agglomeration.Calibration curves were established based on the spectroscopic and voltammetric studies, achieving very low LOD and LOQ of 0.740 nM and 2.47 nM, respectively, which opens the door for Plg detection as a biomarker in different biomedical applications.

Figure 1 .
Figure 1.(a) UV-Vis absorbance spectra of Ag NPs at increasing Plg concentrations and (b) the corresponding calibration curve.(c) TEM image of the Ag nanoparticles.

Figure 2 .
Figure 2. (a) Emission spectra of Ag NPs at increasing Plg concentrations and (b) the corresponding calibration curve.

Figure 3 .
Figure 3. (a) SWV of Ag NPs at increasing Plg concentrations and (b) the corresponding calibration curve.
35 nM and 12.0/18.2nM, respectively.The larger particles could have originated from Plg-induced Ag NP agglomeration, which could have increased the hydrodynamic size of the interacting particles resulting in the dual size range.This behavior could help to explain the reduction in absorbance, fluorescence, and electrochemical oxidation signals of Ag NPs as Plg concentration increased, where the NP agglomeration would have compromised the optical and electrochemical behavior of Ag NPs.

Figure 4 .
Figure 4. DLS size plots of Ag nanoparticles at increasing Plg concentrations.

Table 1 .
Comparison between the Plg analytical parameters using spectroscopic and voltammetric techniques.Schematic displaying the application of Ag-Plg complexation in the analytical detection of Plg using different techniques.