Hemoglobin–Polyaniline Composite and Electrochemical Field Effective Transistors

: A composite of hemoglobin/polyaniline was prepared. The chemical structure of this obtained composite was conﬁrmed using infrared absorption spectroscopy measurement. The luminol reaction of the composite manifested chemical emissions from the composite. Furthermore, electrochemical transistors using the composite were created. The hemoglobin/polyaniline-based electrochemical transistor could switch to external current ﬂow via an electrochemical reaction. The color of the transistor surface changed from green to red upon applying electrochemical potential.


Introduction
Hemoglobin (Hb) is a protein found in the red blood corpuscle. This protein is essential for the bonding of oxygen molecules and transporting them throughout the body. Hb has high oxidation activity, redox properties, and stability [1][2][3][4]. Polyaniline (PANI) is a conductive polymer which can be prepared in water. PANI has good redox properties.
In this study, we synthesized a composite of Hb and PANI (Hb/PANI). The chemical structure of the composite was confirmed using infrared (IR) absorption spectroscopy measurement. Since PANI can be prepared in a water medium, PANI is biocompatible and can be expected to be used in vivo. We carried out direct deposition of Hb/PANI composite on an electrode during chemical preparation to create electrochemical transistors.
Moreover, acidic surfactants are used to tune the solubility and processability of PANI. Surfactants show liquid crystallinity. Applications of liquid crystals for reaction field can produce functional polymers [5][6][7][8][9][10]. Cholic acid was used as an acidic surfactant to prepare the composite to yield Hb/PANI/cholic acid. The chemical structure of the composite was characterized via IR spectroscopy.

Synthesis of Hb/PANI
Hemoglobin (1.023 g), aniline monomer (1.058 g), and sulfuric acid (1.046 g) were dissolved in water (200 mL) during cooling in an ice bath. Subsequently, ammonium peroxodisulfate (APS, 1.022 g) was added. A large volume of methanol was poured into the solution after 24 h to wash the polymer. The product was filtered, collected, and vacuumdried to produce 1.696 g of Hb/PANI composites. During the reaction, a comb-shaped electrode was immersed for creating an electrochemical transistor. A comb-shaped electrode for a rain drop sensor YL-83 (weight: 13 g, dimensions: 54 mm × 40 mm (L × W)), glass and iron), purchased from Aitendo (Tokyo, Japan), was employed. Following polymerization, the composite was deposited onto the electrode (Scheme 1). During cooling in an ice bath, Hb (1.017 g), aniline monomer (1.11 g), sulfuric acid (1.023 g), and sodium cholic acid (1.016 g) as a biosurfactant produced by the liver were dissolved in water. Subsequently, 1.109 g of APS was added. A large volume of methanol was poured into the solution after 48 h to wash the polymer. The product was filtered, collected, and vacuum-dried to produce 2.902 g of the Hb/PANI/cholic acid composite. Figure 1 shows the change in pH during the polymerization process. Addition of aniline as a monomer to water gradually increased the pH value. Next, addition of sulfuric acid decreased the pH value to ca. pH = 2. Aniline sulfate was produced in the reaction medium by means of the addition of sulfuric acid. Subsequent addition of APS increased the pH value in the initial stage, and decreased the pH value in the polymerization. The gradual decrease in the pH value indicated the progression of the polymerization reaction.

IR
The absorption of Hb/PANI and Hb/PANI/cholic acid was measured using Fourier transform infrared (FT-IR) spectroscopy. Figure 2a depicts the entire absorption spectrum of Hb/PANI. The out-of-plane bending vibration of C-H in the aromatic ring (820 cm −1 ), During cooling in an ice bath, Hb (1.017 g), aniline monomer (1.11 g), sulfuric acid (1.023 g), and sodium cholic acid (1.016 g) as a biosurfactant produced by the liver were dissolved in water. Subsequently, 1.109 g of APS was added. A large volume of methanol was poured into the solution after 48 h to wash the polymer. The product was filtered, collected, and vacuum-dried to produce 2.902 g of the Hb/PANI/cholic acid composite. Figure 1 shows the change in pH during the polymerization process. Addition of aniline as a monomer to water gradually increased the pH value. Next, addition of sulfuric acid decreased the pH value to ca. pH = 2. Aniline sulfate was produced in the reaction medium by means of the addition of sulfuric acid. Subsequent addition of APS increased the pH value in the initial stage, and decreased the pH value in the polymerization. The gradual decrease in the pH value indicated the progression of the polymerization reaction. During cooling in an ice bath, Hb (1.017 g), aniline monomer (1.11 g), sulfuric acid (1.023 g), and sodium cholic acid (1.016 g) as a biosurfactant produced by the liver were dissolved in water. Subsequently, 1.109 g of APS was added. A large volume of methanol was poured into the solution after 48 h to wash the polymer. The product was filtered, collected, and vacuum-dried to produce 2.902 g of the Hb/PANI/cholic acid composite. Figure 1 shows the change in pH during the polymerization process. Addition of aniline as a monomer to water gradually increased the pH value. Next, addition of sulfuric acid decreased the pH value to ca. pH = 2. Aniline sulfate was produced in the reaction medium by means of the addition of sulfuric acid. Subsequent addition of APS increased the pH value in the initial stage, and decreased the pH value in the polymerization. The gradual decrease in the pH value indicated the progression of the polymerization reaction.

IR
The absorption of Hb/PANI and Hb/PANI/cholic acid was measured using Fourier transform infrared (FT-IR) spectroscopy. Figure

IR
The absorption of Hb/PANI and Hb/PANI/cholic acid was measured using Fourier transform infrared (FT-IR) spectroscopy. Figure 2a depicts the entire absorption spectrum of Hb/PANI. The out-of-plane bending vibration of C-H in the aromatic ring (820 cm −1 ), the in-plane bending vibration of C-H in the aromatic ring (1130 cm −1 ), the stretching vibration of the N atom adjacent to the benzene ring (1515 cm −1 ), and the stretching vibration of the N atom adjacent to the quinoid ring (1650 cm −1 ) were observed as characteristic peaks in polyaniline (Figure 2b,c). The entire absorption spectrum of Hb/PANI/cholic acid is shown in Figure 3. Furthermore, the out-of-plane bending vibration of C-H in aromatic ring (770 cm −1 ), the in-plane bending vibration of C-H in aromatic ring (1100 cm −1 ), the stretching vibration of two N atoms adjacent to the benzene ring (1500 cm −1 ), and the stretching vibration of two N atoms adjacent to the quinoid ring (1610 cm −1 ) were observed. In both cases, the presence of signals at 1300-1700 cm −1 in the spectrum was indicative of the absorption of the amide bonds. Therefore, the FT-IR spectra confirmed that the composites contain both hemoglobin and polyaniline as components.
J. Compos. Sci. 2021, 5, x FOR PEER REVIEW 3 of 7 the in-plane bending vibration of C-H in the aromatic ring (1130 cm −1 ), the stretching vibration of the N atom adjacent to the benzene ring (1515 cm −1 ), and the stretching vibration of the N atom adjacent to the quinoid ring (1650 cm −1 ) were observed as characteristic peaks in polyaniline (Figure 2b,c). The entire absorption spectrum of Hb/PANI/cholic acid is shown in Figure 3. Furthermore, the out-of-plane bending vibration of C-H in aromatic ring (770 cm −1 ), the in-plane bending vibration of C-H in aromatic ring (1100 cm −1 ), the stretching vibration of two N atoms adjacent to the benzene ring (1500 cm −1 ), and the stretching vibration of two N atoms adjacent to the quinoid ring (1610 cm −1 ) were observed. In both cases, the presence of signals at 1300-1700 cm −1 in the spectrum was indicative of the absorption of the amide bonds. Therefore, the FT-IR spectra confirmed that the composites contain both hemoglobin and polyaniline as components.

Luminol Emission
NaOH (0.6 g), luminol (0.02 g), and 3% hydrogen peroxide solution (30 mL) were added to distilled water (60 mL) to prepare a luminol solution. Hb/PANI and approximately ten milligrams of Hb/PANI/cholic acid were dissolved in luminol solution (5 mL). Light emission upon the irradiation of a UV light was observed. Photoluminescence (PL) spectroscopy measurements for the composite confirmed the emission, as shown in Figure 4. The PL spectra for the composites are shown in Figure 5. Spectral forms were replotted using the least-squares method from the original data. The luminol reaction allows the Hb/PANI to show intense blue emission and Hb/PANI/cholic acid a weak emission, as shown in Figure 4a

Luminol Emission
NaOH (0.6 g), luminol (0.02 g), and 3% hydrogen peroxide solution (30 mL) were added to distilled water (60 mL) to prepare a luminol solution. Hb/PANI and approximately ten milligrams of Hb/PANI/cholic acid were dissolved in luminol solution (5 mL). Light emission upon the irradiation of a UV light was observed. Photoluminescence (PL) spectroscopy measurements for the composite confirmed the emission, as shown in Figure  4. The PL spectra for the composites are shown in Figure 5. Spectral forms were replotted using the least-squares method from the original data. The luminol reaction allows the Hb/PANI to show intense blue emission and Hb/PANI/cholic acid a weak emission, as shown in Figure 4a

Transistor
The setup of a Hb/PANI-based electrochemical transistor was conducted. Hb/PANI was deposited on the comb-shaped electrode for the experiment. During the polymerization process of aniline, the reaction was carried out in the solution, and deposition of the resultant PANI on the wall of the reaction vessel simultaneously occurred. Therefore, immersion of the comb-shaped electrode in the polymerization solution allows the Hb/PANI to be deposited on the electrode surface. We refer to this method as direct polymerizationdeposition, as a convenient method compared with the cast method, or the spray deposition method.
This circuit diagram is shown in Figure 6. First, 200 mL of 0.1 M sulfuric acid was prepared as an electrolytic solution in a beaker. Subsequently, a stainless-steel spring (5 cm in diameter and 20 cm in length) was placed in the beaker as a counter electrode. The comb type electrode was set in the center position of the spring.

Transistor
The setup of a Hb/PANI-based electrochemical transistor was conducted. Hb/PANI was deposited on the comb-shaped electrode for the experiment. During the polymerization process of aniline, the reaction was carried out in the solution, and deposition of the resultant PANI on the wall of the reaction vessel simultaneously occurred. Therefore, immersion of the comb-shaped electrode in the polymerization solution allows the Hb/PANI to be deposited on the electrode surface. We refer to this method as direct polymerization-deposition, as a convenient method compared with the cast method, or the spray deposition method.
This circuit diagram is shown in Figure 6. First, 200 mL of 0.1 M sulfuric acid was prepared as an electrolytic solution in a beaker. Subsequently, a stainless-steel spring (5 cm in diameter and 20 cm in length) was placed in the beaker as a counter electrode. The comb type electrode was set in the center position of the spring.
The setup of a Hb/PANI-based electrochemical transistor was conducted. Hb/PANI was deposited on the comb-shaped electrode for the experiment. During the polymerization process of aniline, the reaction was carried out in the solution, and deposition of the resultant PANI on the wall of the reaction vessel simultaneously occurred. Therefore, immersion of the comb-shaped electrode in the polymerization solution allows the Hb/PANI to be deposited on the electrode surface. We refer to this method as direct polymerizationdeposition, as a convenient method compared with the cast method, or the spray deposition method.
This circuit diagram is shown in Figure 6. First, 200 mL of 0.1 M sulfuric acid was prepared as an electrolytic solution in a beaker. Subsequently, a stainless-steel spring (5 cm in diameter and 20 cm in length) was placed in the beaker as a counter electrode. The comb type electrode was set in the center position of the spring.    A change in the color of the surface of the comb-shaped electrode before and after applying the voltage was visually observed. Figure 8a presents an image of the Hb/PANI deposited on the interdigitated array electrode before the electrochemical application of voltage. Figure 8b presents an image of Hb/PANI after the application of voltage. The surface color changed from green to red upon the application of voltage accompanied by doping-dedoping. No such color change was observed for pure PANI with the electrochemical doping-dedoping. Hb/PANI in the dedoped (neutral) state and the PANI component reflect a purple (complete dedope) or green (mild dedope) color, mainly due to the reflection of light from the PANI component in the composite (Figure 8a). Conversely, the PANI component in the doped state of the Hb/PANI manifested a pale color, inducing the reflection of a red color from the Hb component (Figure 8b). In this case, PANI is almost transparent, and Hb with a red color reflects red light. A change in the color of the surface of the comb-shaped electrode before and after applying the voltage was visually observed. Figure 8a presents an image of the Hb/PANI deposited on the interdigitated array electrode before the electrochemical application of voltage. Figure 8b presents an image of Hb/PANI after the application of voltage. The surface color changed from green to red upon the application of voltage accompanied by doping-dedoping. No such color change was observed for pure PANI with the electrochemical doping-dedoping. Hb/PANI in the dedoped (neutral) state and the PANI component reflect a purple (complete dedope) or green (mild dedope) color, mainly due to the reflection of light from the PANI component in the composite (Figure 8a). Conversely, the PANI component in the doped state of the Hb/PANI manifested a pale color, inducing the reflection of a red color from the Hb component (Figure 8b). In this case, PANI is almost transparent, and Hb with a red color reflects red light.
A change in the color of the surface of the comb-shaped electrode before and after applying the voltage was visually observed. Figure 8a presents an image of the Hb/PANI deposited on the interdigitated array electrode before the electrochemical application of voltage. Figure 8b presents an image of Hb/PANI after the application of voltage. The surface color changed from green to red upon the application of voltage accompanied by doping-dedoping. No such color change was observed for pure PANI with the electrochemical doping-dedoping. Hb/PANI in the dedoped (neutral) state and the PANI component reflect a purple (complete dedope) or green (mild dedope) color, mainly due to the reflection of light from the PANI component in the composite (Figure 8a). Conversely, the PANI component in the doped state of the Hb/PANI manifested a pale color, inducing the reflection of a red color from the Hb component (Figure 8b). In this case, PANI is almost transparent, and Hb with a red color reflects red light.

Conclusions
We successfully synthesized a composite of hemoglobin and polyaniline. FT-IR measurements confirmed the chemical structure of polyaniline. Furthermore, the Hb/PANI composite demonstrated luminol activity. An e-FET based on the Hb/PANI

Conclusions
We successfully synthesized a composite of hemoglobin and polyaniline. FT-IR measurements confirmed the chemical structure of polyaniline. Furthermore, the Hb/PANI composite demonstrated luminol activity. An e-FET based on the Hb/PANI composite was built. The combination of the oxygen capture function and the good redox properties of the composite may be of use for oxygen storage plastics or sensors.