Modern Physics Letters B
2150476 (9 pages)
© World Scientific Publishing Company
DOI: 10.1142/s0217984921504765
Features of electrochemical reduction of silk fibroin in thepresence of phosphate tricalcium in the form of nanocating
KhakkulovJakhongir Mardonovich∗, Kholmuminov Abdufatto Akhatovich and Temirov ZokirShukurulloevich
NationalUniversity of Uzbekistan, Tashkent 100174, Uzbekistan ∗jmkhakkulov@mail.ru
Received 21June 2021
Revised 31July 2021
Accepted 2August 2021
Published 16September 2021
The possibilities of movement andelectrochemical reduction of fibroin macroions in the presence of tricalciumphosphate ions in the form of a nanocoating during electrolysis have beenstudied. The manifestation of a non-Newtonian flow of a mixture of macroionsand ions in a shear flow, the conditions for their electrochemical reduction inthe form of a nanocoating with uniform morphology, and thickness on theelectrode surface are revealed. It was found that the excess ions in themixture and the uneven relief of the electrode surface contribute to theformation of a nanocoating with an inhomogeneous and uneven thickness.
Keywords: Electrolysis; macroion; fibroin;tricalcium phosphate; nanocoating; implant.
1. Introduction
The production of polymernanostructured materials, in particular, nanocoatings with functional activeproperties, is one of the urgent scientific problems of modern nanotechnology.1,3In such cases, ionic polymers behave like macroions and the reduction ofwhich is accompanied by the formation of chemical bonds of various kinds.
In the case of choosing medical implants as anelectrode, it is important to have nanocoatings with the property ofbiocompatibility, bioactivity, non-toxicity, etc. This was the subject of sometargeted studies, and the possibility of forming such coatings based on thebiopolymer chitosan (ChZ) on the surface of a titanium dental implant wasrevealed.6 ChZ has its own bioactivity, thanks to the NH2-amine
∗Corresponding author.
group in the elementary units. Thelarge-scale use of ChZ is to a certain extent difficult due to its productionby using a high-tech method for deacetylation of chitin (biopolymer) releasedfrom the protective coating of various small creatures, such as crabs,crayfish, fish, pupae, etc.7,8
In principle, ChZ can be replaced by anotherbiopolymer, in particular, fibroin (FB) of natural silk, which is moreaccessible and does not require preliminary chemical modification for use inelectrolysis.9,10 The content of relatively bulky amino acidresidues such as aspartic and glutamic acids in FBs does not fully allow theformation of the α-helixof this protein molecule in solution, as well as the β-form in silk fiber.11 The choice ofsuch solvents for FB is very limited, and the most suitable is dilute solutionsof formic acid (HCOOH) for electrolysis.12
In Ref. 13, the possibility of obtaining microcoatings andnanocoatings of FB in a semi-saturated solution of octacalcium phosphate (Ca8 (HPO4)2 (PO4)6) by electrolysis wasstudied. The presence of a large amount of octacalcium phosphate in a solutionof FB in 50% HCOOH:H2Opromotes noticeable precipitation of the protein as a result of the formationof a salt complex. This makes it difficult to carry out electrolysis, which ispossible when the temperature rises above 60◦C,when weak salt complexes are destroyed and conditions for an electrochemicalreduction reaction are created for ions and macroions on the electrode surface.However, in the region above 60◦C,denaturation of proteins is inevitable, which can be avoided with a decrease intemperature below 50◦C.One of the ways to solve this issue is to replace octacalcium phosphate withorthocalcium (tricalcium) phosphate (Ca3(PO4)2).14 When tricalcium phosphate(TCP) is used, even in the form of a saturated solution, protein is not saltedout, and effective electrolysis of macroions and ions is realized in the rangeof 30–50◦C.
Such an oxidation electrode can be carbon, forexample, graphite rods.15 As a recovery electrode, it is of interestto use electrically conductive materials and targeted products, for example,dental and orthopedic implants.16
In general, it is possible to obtain nanocoatingsbased on PB and TCP with a certain characteristic by carrying out in-depthstudies to find the optimal conditions for the movement and recovery ofmacroions in the presence of ions on the surface, for example, a titaniumelectrode of various shapes and reliefs. Such studies were carried out withinthe framework of this work using a specially assembled electrolysis unit.
2. ExperimentalPart
2.1. Preparationof a solution and a mixture of FB
An initial sample of FB fibers wasisolated from a natural silk cocoon by washing with the following naturalcomponents: (1) sericin in a 5% aqueous solution of 0.2 M CaCO3 for 100 min at 80◦C and (2) fat wax andminerals in acetone on the Soxhlet apparatus.17 The washed FB fiberswere dried to a constant weight, and the yield of which was 72% of the initialcocoon weight, which is in good agreement with the literature data.18
Silk FB fiber is characterized by anamorphous-crystalline structure, and dissolution is possible as a result of thedisintegration of crystalline regions, which, in the case of HCOOH, isaccompanied by noticeable acidic hydrolysis of the protein. Therefore,solutions of FB for electrolysis were prepared from amorphous FB in 50% HCOOH:H2O. Amorphous FB was obtainedby dissolving silk fibers in a solvent that does not cause protein degradation,namely, in an aqueous solution of 50% CaCl2at 90◦Cand precipitation by dialysis of Ca and Cl ions from the solution through asemipermeable membrane of cellulose xanthate. The precipitated mass was driedby freeze drying to obtain powdered FB. Polarization-optical observation showedthat the powdery sample is almost completely characterized by the amorphousstate of the chains and dissolves in HCOOH.
The average molecular weight (M) of FB was determinedby the Ubbelohde viscometry method by measuring the specific viscosity (ηsp) at various concentrations (C) of asolution of this biopolymer in 2.5 M LiCl-DMF. Based on the measurements, thedependence of the reduced viscosity (ηsp/C) on C was constructed according to the Huggins formula:ηsp/C = [η] + k[η]2C(here k is aconstant). The intrinsic viscosity value [η] = 118 ml/g was found byextratsolation C → 0.According to the Mark–Kuhn–Houwink equation M≈ ([η]/1.23 × 10−5)1/0.91,the value of the molecular weight of FB, M= 295,000,was calculated.
2.2. 
Improvementof the electrolysis plant
Electrochemical reduction of FBmacroions from solutions and mixtures was carried out on a specially assembledelectrolysis unit, which is schematically shown in Fig. 1.
It consists of three main parts, namely, a cylindricalglass tank (R), an electrical system (E), and an optical (O) system. Thereservoir (volume 1 l) has a thermostatically controlled jacket (1) withfittings (8, 8*) and optical windows (2, 2*), installed on an electric mixer(3).
Fig. 1. (Coloronline) Schematic diagram of the installation for electrolysis of macroions.
The reservoir contains a solution (4), into which theoxidation (5) and reduction (6) electrodes are lowered at the level of thequartz glass optical window. Rotation of the magnet (7) by means of an electricmixer ensures intensive movement of the solution. The solution temperature ismaintained by means of a water thermostat connected to the reservoir throughthe fittings (8) and (8*). The solution temperature is monitored with athermometer (9). The optical system consists of a light source (10) and amicroscope (11). The supply of the light beam from the top to the bottom alongthe axis of the cylindrical tank and the observation along the horizontal axisallows monitoring according to the principle of ultramicroscopy. This approachmakes it possible to record the appearance of nanoscale structures at a levelof 10−7 m. From aconstant voltage source (E), a current is supplied to the electrodes, which ismonitored using a voltmeter (V) and a microammeter (A).
The experiments were carriedout using a titanium plate (40 × 10 ×2 mm3) as a reduction electrode and acarbon rod as an oxidation electrode. The experiments were carried out using atitanium plate as the electrode into which the oxidation and reductionelectrodes were lowered. Here, the size of the oxidation electrode, i.e. carbonrod, is as follows: length is l = 40 mm and its diameter is d =5. With the help of these electrodes, the restoration of macroions onnon-surface surfaces during electrolysis is partially hampered due to thesmoothness of the surface, but the formation of coatings in the range of 10−7÷10−3 m of the electrode surface thicknessallows the formation of hard coatings. Since the electrolysis process iskinetic in nature, this made it possible to conduct exploratory studies on theformation of a coating with a certain thickness by choosing the composition ofthe solution and the mixture of FBs with ions, temperature, and time of theexperiment. The thickness and elemental composition of the coating weredetermined using a scanning electron microscope ZEISS SIGMA SEM 500 Aztec. Theadhesion strength of the coating was evaluated by washing in water and thefunctional activity by interaction with salt ions.
3. Resultsand Discussion
Experiments have shown the possibilityof reduction of isolated (C [η] ≤ 1) macroions of FB in HCOOH during electrolysis underthe action of direct current (I) in the range of 2–8 mA at room temperature. Itwas revealed that for isolated FB macroions, the most suitable solvent is amixture of formic acid and water (HCOOH:H2O)at a volume ratio of 50:50 (or 50% HCOOH). The addition of Ca3(PO4)2 to the solution leads to a noticeableincrease in viscosity, i.e. slowing down the movement of macroions due tointeraction with the ions of this salt.
It has been determined that an increase intemperature up to 50◦Cis accompanied by a decrease in viscosity; hence, there is an acceleration inthe movement of macroions and ions to the electrode. Above 60◦C, the restoration ofFB macroions becomes more complicated due to conformational changes; α −β transitions characteristic of protein molecules under theinfluence of heat are not excluded. This determines the identification of thefeatures of the movement of macroions in the presence of ions and their jointreduction on the electrode surface at different temperatures.
3.1. Thepeculiarity of the movement of macroions
To reveal the features of themovement of FB macroions surrounded by Ca and P ions, comparative rheologicalstudies in a shear flow were carried out, and the results obtained are shown inFig. 2 as a dependence of the shear rate (γ) on the shear stress (σ)at temperatures of 25◦Cand 50◦C.
Figure 2 shows the dependence of the velocity gradient (γ) on the shear stress (σ). As can be seen from thegraph, at the minimum (γ → 0) valueof the velocity gradient, the value of the effective viscosity decreases, andthe change in the effective viscosity γ> 400c−1 inthe field is minimized, i.e. the non-Newtonian flow persists even when itapproaches the Newtonian flow.
Fig. 2. Dependence of the velocity gradient (γ) on the shear stress (σ) for 1% solution of FB in HCOOH:H2O (50:50) at 25◦C (1) and 50◦C (1*) and for a mixture of PB:Ca2(PO4)3 (1:10) in HCOOH:H2O (50:50) at 25◦C (2) and 50◦C (2*).
It was noticed that with an increase in temperature,the yield stress (σγ) shifts toward the region of smallvalues of shear stress, that is, the fluidity of the solution and the mixtureof FB and ChZ increases. This is due to the rise in temperature, i.e. anincrease in thermal motion and a decrease in interactions between macroions andions. As a result, favorable conditions are created for the efficient movementof macroions and ions to the electrodes, which is important for electrolysis.The values of the limiting stresses determined at different temperatures aregiven in Table 1.
Table 1. Quantitative boundary leakage stressesof chitosan and FB solutions at different temperatures.
Yield stress (σγ) (Pa)
|
Polymer |
Solvent |
Concentration (g/dl) |
25◦C |
50◦C |
|
Chitosan |
2% CH3COOH:H2O |
0.25 |
15 Представленная информация была полезной? ДА 61.18% НЕТ 38.82% Проголосовало: 1512 |
10 |
|
FB |
HCOOH:H2O (1:1) |
0.25 |
30 |
20 |
3.2. Electrochemicalreduction of macroions in the presence of ions
Experiments carried out on a speciallyassembled electrolysis installation system showed that the recovery of isolatedmacroions of FB in HCOOH:H2O(50:50) in the form of microcoatings and nanocoatings is possible under theaction of a direct current of 2–8 mA in the temperature range of 25–50◦C for 1–10 h. In thecase of adding TCP, a joint reduction of macroions and ions occurs, which ismore efficiently realized when the ratio of FB:Ca2(PO4)3 is about 1 ≥ 10, and when the ratio is 1 < 10, areas appear thatcharacterize mainly the reduction of salt ions. This feature of recovery isseen from the comparative SEM images shown in Fig. 3 for FB:Ca2(PO4)3 in different ratios.
According to Faraday’s law, the thickness (or mass) of thecoating at certain values of the direct current can be regulated by choosingthe electrolysis time. Experiments have shown that in the case of the formationof a nanocoating from a mixture of macroions with ions, the surface relief ofthe electrode has a certain effect, especially when the surface ischaracterized with various micrometer and millimeter depressions and roughness.In such cases, it is important to select the optimal electrolysis conditionsfor obtaining a nanocoating with uniform thickness. In the case of usingimplants of various shapes as an electrode, for example, threaded dentures(pins), it is possible to obtain a nanocoating from a mixture of FB with Ca2(PO4)3 on their surface. The formation ofnanocoatings was recorded by ultramicroscopy in a specially assembled electrolysisunit (Fig. 1).
Fig. 3. SEM images of coatings of FB:Ca2(PO4)3 at a ratio of 1:10 (a) and 1:20 (b):(1) electrode; (2) coating based on the reduction of macroions with ions; (3)area of coverage containing a large number of reduced ions of TCP.
Fig.4. SEM image of the areas of the dental post covered with PB:Ca2(PO4)3.
Table 2. Elemental composition ofnanopocritium based on FB and Ca2(PO4)3.
ΦB:Ca2(PO4)3 (1:10) ΦB:Ca2(PO4)3 (1:20)
|
Elements |
Elements in the coating (%) |
|
C O N P Ca Ti |
|
Analysis of SEM imagesobtained from the end of the pin showed that the coating thickness ranges from10 s of nanometers to microns, depending on the shape of the electrode. Figure4 shows a SEM image of a portion of a dental post coated with FB:Ca2(PO4)3 (1:10 wt.%) during electrolysis under theaction of a direct current of 4 mA and a temperature of 40◦C for 4 h.
Here, 1 and 1* threads (height= 0.5 mm, pitch = 1.0mm) of the pin are covered with a layer of FB and Ca2(PO4)3 with a thickness of 50–150 nm; 2 refersto the area of the coating in the recess between the threads 100 nm and 250 nmthickness; 3 and 3* refer to the areas of the coating near the thread 300–500nm thickness; 4 and 4* are the coating areas between threads > 900 nm thickness.
The results of the elemental analysis were obtainedwith the special SEM program; the nanopocritium formed by the electrolysismethod can be seen in the following results (see Table 2).
In this case, the coating obtained from a mixture ofFB:Ca2(PO4)3 (1:20) contains 1.5 times more Ca and Pthan the coating obtained from a mixture of FB:Ca2(PO4)3 (1:10), as well as N-and C-elements of FB was 2.6 times less. The presence of differences in thecomposition of the coating due to the elements is due to the fact that duringelectrolysis, ions reach the electrode faster than macroions andelectrochemical reduction takes place. This can be done on the basis ofchanging the composition of the mixture to reduce or eliminate the differencesto achieve maximum electrochemical reduction of the macronions and ionstogether.
An important characteristic of the nanocoating isthe strength of its anchorage on the surface of the implant and themanifestation of functional activity upon contact with various substances,especially ions and ionic compounds. The adhesion strength of nanocoatings wasevaluated in a very simple way, namely, by washing them with distilled water.It was found that when washing in the range of 15–60◦C for 1 h, there are no noticeablemicroscopic changes on the electrode surface, and the viscosity and pHneutrality of water remain practically unchanged. We also studied the stabilityof PB:Ca2(PO4)3 nanocoatings to the action of FBsolvents, namely, aqueous solutions of 50% CaCl2 and 50% HCOOH, as well as 2.5 M LiCl-DMF.
4. Conclusion
Thus, the results of thestudies carried out have shown that mixtures of FB macroions and TCP ionsexhibit non-Newtonian flows caused by the conformational change of macroions inthe presence of ions in the flow. Such a conformational change inmacromolecules takes place during electrolysis; moreover, during the jointreduction of macroions and ions, ions have a certain advantage if they arecontained in an excessive amount in the mixture. This affects the uniformityand thickness of the nanocoating. It was found that the surface relief of theelectrode has a definite effect on the formation of a nanocoating. This isclearly seen in the case of joint restoration of macroions and ions in thethreaded parts of a titanium implant.
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