Oxidative Stripping of Starch-AgNPs for Nanomolar Indirect Electrochemical Sensing of BSA Using an L-Cysteine Modified Glassy Carbon Electrode

In this study, an L-cystine-modified glassy carbon electrode (GCE) was developed as an electrochemical platform based on AgNP oxidative stripping for nanomolar indirect detection of bovine serum albumin (BSA) biomacromolecules. The Poly-L-Cys was formed by L-cysteine (L-Cys) monomers electro-polymerization on the GCE surface. L-Cys pending groups interacted with AgNPs through Ag-S chemical bonds while it interacted with BSA through van der Waals forces and hydrogen bonding. To characterize the bare GCE and GCE/L-Cys, cyclic voltammetry and electrochemical impedance spectroscopy were used. The oxidative stripping of AgNPs by the addition of BSA was monitored using linear sweep stripping analysis.


Introduction
Genetic information encoded in nucleic acids has enormously fascinated researchers and caught their interest in unraveling its mysteries.Proteins, which are the molecular expression of genetic information, are the major constituent of biological activities.As a vital component of the cell, proteins also play an important role in biological processes.These can be released in pathological conditions, which can be termed protein biomarkers.The sensing of these proteins can be vital in disease diagnosis and treatment.Quantitative analysis of proteins has real applications in pharmacology, clinical medicine, biotechnology, and food nutrition [1,2].In vertebrate blood, serum albumin is the most abundant protein.It has a major role in the circulation system mainly in transporting and deporting many proteins and different compounds.They maintain plasma osmotic pressure while also exerting a great role in scavenging free oxygen radicals [2,3].
Bovine serum albumin (BSA) is a typical serum protein, which has many physiological functions such as binding, transportation, and delivery of different molecules like fatty acids, bilirubin, porphyrins, and steroids [4].It is one of the dietary proteins, which can be absorbed from food.By consuming bovine milk and meat, humans also get BSA.It is reported that exposure to pathogenic BSA can result in diseases such as membranous nephropathy [5], mad cow disease [6], insulin-dependent diabetes mellitus [7], and Creutzfeldt-Jakob disease [8].As a result, more attention is being paid to the detection of BSA in immunological and bio-analytical studies, such as fluorimetric determination [9,10], quartz crystal microbalance [11], electrochemical polarography [12], and infrared spectroscopy [13].The major drawbacks of these analytical methods are that they necessitate costly equipment and complicated procedures and are associated with limited selectivity.Electrochemical methods, on the other hand, have benefits over conventional techniques as electrochemical biosensors facilitate simple and rapid sensing at low cost [14].Metal nanoparticles such as Pt, Ag, and Au were extensively employed for fabricating electrochemical sensors due to the intrinsic characteristics existing in these nanoparticles, mainly the electrocatalytic properties, small size, mass transfer, electrochemical conductivity, in addition to their compatibility with living organisms [15,16].
Modified electrodes fabricated by electro-polymerization of amino acids have been of interest recently due to the simplicity of their preparation, displaying thin films at the modified surface with pending selective functional groups originating from the R-side chains of the amino acids, which can help in forming multilayers functionalized with very selective arm groups [17].In addition to the amine (-NH2) and carboxyl (-COOH) terminal functional groups existing in all amino acids, L-Cys has the advantageous thiol functional group (-SH), which makes it a special monomer for modifying electrochemical sensors either using chemical or electrochemical modifications [18].Benefiting from the above-mentioned properties, the naturally occurring zwitterion (L-Cys) is a very promising linker that could be used to stabilize the noble metal nanoparticles, such as AgNPs at the surface of an electrochemical platform and help therefore in indirect electrochemical detection of biomolecules using oxidative stripping voltammetry [19][20][21].
Electrochemical sensing of BSA was achieved in this work by altering the surface of GCE with L-cysteine while using Starch-AgNPs (SAgNPs) as the electroactive species.In this study, we focused on the electrochemical detection of BSA, with a particular emphasis on the complexation of BSA with SAgNPs.The electrochemical detection protocol is based on the stripping of Ag 0 , which is present in nanoparticles and bound to BSA at the surface of an L-Cysteine-modified glassy carbon electrode (GCE/L-Cys).

Instrumentation
A three-electrode electrochemical cell was used in all experiments, employing bare GCE and GCE/L-Cys working electrodes, KCl-saturated Ag/AgCl as reference electrode, and a Pt wire as counter electrode.A CHI1232 electrochemical workstation was used to run all voltammetry techniques and record the electrochemical measurements, while a Zahner Zennium Pro electrochemical workstation was employed to run electrochemical impedance spectroscopy (EIS).

Working electrode preparation (GCE/L-Cys)
5.0 mM of L-cystine was dissolved in 0.3 M phosphate buffer solution (PBS) at pH = 9.0, and its electrochemical polymerization on the surface of GCE was done using cyclic voltammetry (CV) with three successive cycles at the potential range of -0.8 V to +1.8 V and a scan rate of 20 mV/s.To remove the residual physically adsorbed L-Cys, the GCE/L-Cys electrode surface was rinsed several times with deionized water.CV and EIS were used to characterize both sensors, the bare GCE and GCE/L-Cys modified sensor, using 5.0 mM equimolar solution of ferro/ferricyanide [Fe(CN)6] 3-/4-in 0.1 M KCl.
Linear sweep voltammetry (LSV) was used to evaluate the electrocatalytic activity of bare GCE and GCE/L-Cys electrodes towards the sensing of BSA, where different concentrations of BSA were spiked into a 2.0 mL cell containing 1.0 mL of SAgNPs and 1.0 mL of citrate buffer (pH=4), based on which a calibration curve was then established.

Preparation of BSA/SAgNPs
SAgNPs were synthesized according to an experimental procedure reported in our earlier work [22].The obtained SAgNPs were centrifuged for 10 min at 4000 rpm before use.The absorbance spectra of SAgNPs and BSA/SAgNPs were obtained using UV-vis spectrometry and were used to determine the stoichiometry of complexation between BSA and SAgNPs.All analyses were performed in a microplate reader at a wavelength range of 200-800 nm.BSA concentrations of 0.0, 45.5, 83.3, 115.4,142.9, 333.3, 375.0, 444.4,and 500.0nM were analyzed by spiking a constant volume of 100 μL of SAgNP with increasing volumes of BSA.Δλ/Δλmax was plotted against the concentration of BSA to find the complexation stoichiometry of BSA and SAgNPs, where Δ is the shift in the peak maximum and Δmax is the maximum absorbance shift.

Results and discussion
Figure 1a depicts a plot of Δ/Δmax vs. various BSA concentrations in nM.When the BSA concentration was increased, the Δ/Δmax began to rise until it reached a constant value of 1.0.The optimal BSA/SAgNPs stoichiometry was 83.3 nM, after which the value of Δ/Δmax stabilized at 1.0.Following that, a stock working solution of BSA/SAgNPs was prepared by mixing 1000 L of SAgNPs with 200 L of BSA and ultrasonicating the solution for 5 minutes.The obtained BSA/SAgNPs solution was then used for constructing an LSV calibration curve.
The electrochemical activities of unmodified GCE and GCE/L-Cys electrodes were studied using CV in PBS (pH=7) as the blank and 5.0 mM K3Fe(CN)6/K4Fe(CN)6 in 0.1 M KCl.The CVs of both sensors, the unmodified GCE and GCE/L-Cys, were carried out at different scan rates ranging from 5.0 mV/s to 200 mV/s, as shown in Figures 1b,c.Both Figures show CVs of bare GCE and GCE/L-Cys electrodes with reversible anodic and cathodic processes taking place at the surface of the sensor for the redox couple K3Fe(CN)6/K4Fe(CN)6.The CVs of the electrodes in the PBS buffer (pH=7) revealed no electrochemical activity.The electrochemical surface areas of the sensors were determined using the Randles-Sevcik equation ( 1), given below.
Where A is the electrochemical active surface in cm 2 , D is the diffusion coefficient of the [Fe(CN)6] 4-which is subjected to oxidation at the surface of the electrode taken as 6.31×10 −6 cm 2 s -1 , C is the concentration of the same ion, taken as 5.0 mM, n represents the number of electrons involved in the ferro/ferricyanide redox reaction, taken as 1 e -, and ν is the scan rate of the reaction and is represented in V/s.A voltammogram overlay for GCE and GCE/L-Cys electrodes is depicted in Figures 1b and 1c.The electroactive surface areas were calculated using the CVs shown in Figures 1b-c, and are shown in Figure 1d as a bar graph.The GCE active electrochemical area was determined to be 6.6610 -3 cm 2 while a surface area of 4.4410 -3 cm 2 was found for GCE/L-Cys.The non-conductive nature of L-Cys caused the decrease in surface area.The oxidative stripping of SAgNPs in the presence of BSA was monitored using LSV. Figure 2a depicts LSVs of various solutions at the surface of GCE/L-Cys in citrate buffer (pH=4.0)over a potential range of -0.50-0.50V.In the absence of SAgNPs, no anodic peak was observed for GCE/L-Cys in both the blank and BSA solutions, as shown in Figure 2a.When AgNPs were added to the buffer solution, a clearly defined oxidation peak was observed at +0.16 V, confirming the stripping of SAgNPs on the electrode's surface.The addition of BSA to the Ag-containing solution improved the current response.This improvement supported the formation of a BSA/SAgNPs complex on the surface of the GCE electrode.
The influence of the concentration of BSA was studied and a calibration curve was established (Figure 2b-c).Different concentrations of BSA were spiked into a 2.0 mL cell containing 1.0 ml of SAgNPs and 1.0 ml of citrate buffer (pH=4.0).The concentration of BSA was varied from 0.0 to 45.5 nM, and the relative peak current was measured by linear sweep anodic stripping voltammetry (LSASV) using GCE/L-Cys electrode as shown in Figure 2b.There was a linear increase in the peak current with BSA concentration from 1.4 A to 23 A, with a limit of detection of 4.0 nM and a limit of quantification of 13.5 nM, as shown in Figure 2c.

Conclusion
For the first time, the mechanism of BSA sensing with the assistance of SAgNPs stripped on a GCE modified by L-Cys was studied.The sensors were electrochemically characterized using CV and EIS, and LSASV was used for the BSA detection process.Under optimized conditions, the electropolymerization of the selected monomer (L-Cys) yields a polymer with a high affinity for Ag nanoparticles and BSA.This phenomenon was used to fabricate a voltammetric sensor for BSA detection.This was done by using Starch-AgNPs as an electroactive species to modify a GCE by immobilizing L-Cys on its surface.L-Cys interacted with BSA via van der Waals weak interactions as well as strong hydrogen bonding leading to a reduction in the interaction with competitive water molecules.Moreover, the thiol group of L-Cys may have interacted strongly with AgNPs via Ag-S covalent bond leading to the production of more sensitive analytical results.LSV was used to detect BSA on the surface of GCE/L-Cys indirectly by stripping the Ag metal in its nanoparticle form (SAgNPs).The results revealed that an enhancement in the stripping oxidation peak of Ag upon the addition of BSA was obtained only in the case of LSV, due to the ability to obtain relevant current responses within a few milliseconds after the electrode was stimulated.

Declaration of Competing Interest
No known conflict or competing interest

Figure 1 .
Figure 1.(a) Δ/Δmax plot vs. BSA concentrations ranging from 0 to 500 nM.(b, c) Overlay of CV at various scan rates ranging from 5.0 mVs -1 to 200 mVs -1 , used for the determination of the electroactive surface area for (b) unmodified GCE and (c) GCE/L-Cys.(d) Surface area calculated for bare GCE and GCE/L-Cys.