Electro-Nano Diagnostic Platform Based on Antibody–Antigen Interaction: An Electrochemical Immunosensor for Influenza A Virus Detection

H1N1 is a kind of influenza A virus that causes serious health issues throughout the world. Its symptoms are more serious than seasonal flu and can sometimes be lethal. For this reason, rapid, accurate, and effective diagnostic tests are needed. In this study, an electrochemical immunosensor for the sensitive, selective, and practical detection of the H1N1 virus was developed. The sensor platform included multi-walled carbon nanotube gold-platinum (MWCNT-Au-Pt) hybrid nanomaterial and anti-hemagglutinin (anti-H1) monoclonal antibody. For the construction of this biosensor, a gold screen-printed electrode (AuSPE) was used as a transducer. Firstly, AuSPE was modified with MWCNT-Au-Pt hybrid nanomaterial via drop casting. Anti-H1 antibody was immobilized onto the electrode surface after the modification process with cysteamine was applied. Then, the effect of the interaction time with cysteamine for surface modification was investigated. Following that, the experimental parameters, such as the amount of hybrid nanomaterial and the concentration of anti-H1 were optimized. Under the optimized conditions, the analytical characteristics of the developed electrochemical immunosensor were investigated for the H1N1 virus by using electrochemical impedance spectroscopy. As a result, a linear range was obtained between 2.5–25.0 µg/mL with a limit of the detection value of 3.54 µg/mL. The relative standard deviation value for 20 µg/mL of the H1N1 virus was also calculated and found as 0.45% (n = 3). In order to determine the selectivity of the developed anti-H1-based electrochemical influenza A immunosensor, the response of this system towards the H3N2 virus was investigated. The matrix effect was also investigated by using synthetic saliva supplemented with H1N1 virus.


Introduction
Concerns about the reemergence of an influenza pandemic have been increased since low rates of COVID-19 were observed. It has been reported that the rate of seasonal influenza has dropped since April 2020 based on the COVID-19 precautions such as quarantine, travel restrictions, the closures of companies and schools, the usage of masks, etc. [1][2][3][4][5]. However, this long-term decrease in influenza virus infections might reduce herd immunity which could result in massive influenza spread [6,7].
The influenza virus, which belongs to the Orthomyxoviridae family [8], is a respiratory virus. Based on the variations of its matrix protein and nucleoprotein, the virus is classified as influenza virus A, influenza virus B, and influenza virus C [9,10]. Considering morbidity and harm, influenza A is recognized as the most dangerous type [11][12][13][14]. On the virus surface, there are two enclosed glycoproteins, namely, hemagglutinin (H) and neuraminidase (N), which play very important roles in the infection process of the virus. Until now, 18 H and 11 N subtypes have been verified [14][15][16].

Instruments and Procedures
EIS measurements were performed with a FRA-modeled µ-AUTOLAB potentiostatgalvanostat device that was supported by NOVA Software(version 1.10, Nova Software Inc., China), in the presence of 10 mM [Fe(CN) 6 ] −3/−4 in pH 7.4, 50 mM phosphate buffer. The EIS working frequency range was set between 0.1 Hz-10 kHz at a potential value of 0.10 V. Throughout the entire study, a commercial 220 AT model Methrohm branded screen-printed gold electrode (AuSPE) was used, which contained the gold working electrode (diameter = 4 mm), gold auxiliary electrode, and silver reference electrode on a single platform.
The morphological and structural characterizations of the MWCNT-Au-Pt hybrid nanomaterial were carried out with scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis via a ZEISS ULTRA PLUS.

Hybrid Nanomaterials Preparation 2.3.1. MWCNT-Based Hybrid Nanomaterials Preparation
For this purpose, activated MWCNTs were used. First of all, in order to perform the acid activation procedure, the proper amount of MWCNT powder was sonicated in 40 mL of acid mixture including H 2 SO 4 /HNO 3 (3:1 v/v) for 6 h. After that, the activated MWCNT powder was rinsed with pure water, centrifuged, and then dried in a vacuum oven at 50 • C for 14 h. Then, platinum nanoparticle (PtNP) and gold nanoparticle (AuNP) or only AuNP deposition procedure on MWCNT was started with the sonication of 20 mg activated MWCNT in the presence of 20 mL of the Au and Pt nanoparticle solutions (for MWCNT-Au-Pt) or only AuNp (for MWCNT-Au) solution, which were prepared according to previous studies [35,36]. After the sonication step for 4 h, the mixture was annealed at 150 • C for 4 h to increase functionality.

Graphene-Based Hybrid Nanomaterial Preparation
In order to prepare graphene-Au and graphene-Pt, the first step was the synthesis of graphene oxide (GO) by the Hummers-Offeman method [37]. For this purpose, by stirring at 25 • C for 24 h, graphite powder was dispersed in 23 mL of 98% H 2 SO 4 solution for the separation of the graphite layers. After 100 mg of NaNO 3 was put into the final solution and mixed for 30 min, 3 mg of KMnO 4 was added drop-by-drop to the medium, which the temperature was controlled by an ice bath to oxidize the graphite layers. Then, 46 mL of ultra-pure water was added to that solution, after the solution was heated up to 40 • C. As the final step of the Hummers method, 140 mL of ultrapure water and 10 mL of 30% H 2 O 2 solution were put into the solution, respectively to stop the oxidation reaction with KMnO 4 .
After the residues of the reaction were eliminated via centrifuge, water was removed from the obtained GO powder.
Moreover, the graphene-Au and graphene-Pt hybrid nanomaterials were synthesized according to the study of Xu et al. [38]. First, 10 mg GO powder was dispersed in 10 mL of pure water and sonicated for 1 h to obtain a stable GO colloid [10]. Next, 20 mL of EG solution (reducing reagent) and 0.5 mL of 0.01 M HAuCl 4 ·3H 2 O or H 2 PtCl 6 ·6H 2 O (metal source) were added into the GO colloidal solution and mixed for 30 min. Then, after 6 h of the stirring procedure at 100 • C, the graphene-Au and graphene-Pt hybrid nanomaterials were obtained. For the separation of obtained hybrid nanomaterials from an excess of EG, centrifugation and a subsequent washing procedure with pure water for five times were applied.
In the last step, the graphene-Au and graphene-Pt hybrid nanomaterials were dried in an oven at 60 • C for 12 h.
Before using electrode modification, all types of hybrid nanopowders were dispersed in pure water at a concentration of 10 mg mL −1 [10].

Fabrication of Anti-H1 Based Electrochemical Immunosensor
In this study, AuSPE was used as a transducer. The first step involved the modification of AuSPE with MWCNT-Au-Pt hybrid nanomaterial. For this purpose, 10 µL of MWCNT-Au-Pt suspension (10 mg/mL in pure water) was dropped onto the working electrode surface and left until the suspension dehydrated at 25 • C for 15 min. Next, MWCNT-Au-Pt hybrid nanomaterial-modified electrode surface was treated with 10µL, 100 mM of cysteamine for 1 h for the formation of -NH 2 functional groups on the surface. Then, 10 µL, 60 µg/mL anti-H3 antibodies in the presence of 10 µL, 5 mM of EDC, and 8 mM of NHS linker reagents mixture were immobilized on the electrode surface by waiting for 1 h in order to achieve a specific bioactive layer. After the antibody immobilization, 10 µL of 0.1% BSA was placed on the electrode surface as a blocking solution for the prevention of nonspecific bindings. After each step, the electrodes were washed with pH 7.4, 50 mM phosphate buffer. The fabricated anti-H1-based electrochemical immunosensors were prepared for single-use (Scheme 1).
in pure water at a concentration of 10 mg mL −1 [10].

Preparation of Hybrid Nanomaterial Modified AuSPE
MWCNT-Au, MWCNT-Au-Pt, graphene-Au, and graphene-Pt hybrid nanomaterials were used separately to modify AuSPE. For this purpose, 10 µL of hybrid nanomaterial suspension (10 mg/mL in pure water) was dropped onto the working electrode surface and left until the suspension dehydrated at 25 °C for 15 min. At the end of this period, AuSPE/Grafen-Au, AuSPE/MWCNT-Au, AuSPE/Grafen-Pt, and AuSPE/MWCNT-Au-Pt nanomaterials were obtained.

Fabrication of Anti-H1 Based Electrochemical Immunosensor
In this study, AuSPE was used as a transducer. The first step involved the modification of AuSPE with MWCNT-Au-Pt hybrid nanomaterial. For this purpose, 10 µL of MWCNT-Au-Pt suspension (10 mg/mL in pure water) was dropped onto the working electrode surface and left until the suspension dehydrated at 25 °C for 15 min. Next, MWCNT-Au-Pt hybrid nanomaterial-modified electrode surface was treated with 10µL, 100 mM of cysteamine for 1 h for the formation of -NH2 functional groups on the surface. Then, 10 µL, 60 µg/mL anti-H3 antibodies in the presence of 10 µL, 5 mM of EDC, and 8 mM of NHS linker reagents mixture were immobilized on the electrode surface by waiting for 1 h in order to achieve a specific bioactive layer. After the antibody immobilization, 10 µL of 0.1% BSA was placed on the electrode surface as a blocking solution for the prevention of nonspecific bindings. After each step, the electrodes were washed with pH 7.4, 50 mM phosphate buffer. The fabricated anti-H1-based electrochemical immunosensors were prepared for single-use (Scheme 1). Scheme 1. Schematic illustration of the fabrication of developed anti-H1based MWCNTAuPt hybrid nanomaterialmodified electrochemical influenza A biosensor.

Electrochemical H1N1 Virus Detection Procedure
Different concentrations of 10 µL of H1N1 virus solutions were incubated with developed influenza A immunosensor at 25 °C for 1 h. For the investigation of interactions between H1N1 virus and anti-H1 antigen which was immobilized onto the working electrode, EIS measurements were carried out in 50 mM pH 7.4 phosphate buffer including 10 mM [Fe(CN)6] 4− /[Fe(CN)6] 3− as a redox probe indicator in the frequency range of 0.1 Hz-10 kHz and with a potential of 0.10 V. Each experiment was carried out in triplicate, and the results were presented with error bars.

Electrochemical H1N1 Virus Detection Procedure
Different concentrations of 10 µL of H1N1 virus solutions were incubated with developed influenza A immunosensor at 25 • C for 1 h. For the investigation of interactions between H1N1 virus and anti-H1 antigen which was immobilized onto the working electrode, EIS measurements were carried out in 50 mM pH 7.4 phosphate buffer including 10 mM [Fe(CN) 6 ] 4− /[Fe(CN) 6 ] 3− as a redox probe indicator in the frequency range of 0.1 Hz-10 kHz and with a potential of 0.10 V. Each experiment was carried out in triplicate, and the results were presented with error bars.

Sample Application
In total, 10 µL of 5 µg/mL H1N1 virus solution was put in synthetic saliva solution and incubated with developed anti-H1-based electrochemical influenza A immunosensor. A synthetic saliva solution was prepared by incorporating the reagents into pH 7.3 phosphate buffer including 0.05 mol/L NaHCO 3 , 8.55 × 10 −3 mol/L NaCl and 2.68 × 10 −3 mol/L KCl [39]. Next, electrochemical measurements were performed by using EIS in triplicate.

Selectivity Study
In order to appraise the selectivity of developed anti-H1-based electrochemical influenza A immunosensor, EIS measurements were performed to monitor the resistance changes on the electrode surface after incubating developed immunosensor with 10 µL, 5 µg/mL of the H1N1 and H3N2 influenza A viruses which were model and control viruses, respectively.

Selection of Type of Hybrid Nanomaterial
In order to select the most convenient hybrid nanomaterial, experiments by using plain AuSPE, AuSPE/Grafen-Au, AuSPE/MWCNT-Au, AuSPE/Grafen-Pt, and AuSPE/ MWCNT-Au-Pt, were conducted via EIS in 50 mM pH 7.4 phosphate buffer including 10 mM [Fe(CN) 6 ] 4− /[Fe(CN) 6 ] 3− as a redox probe in the frequency range of 0.1 Hz-10 kHz and with a potential of 0.10 V. Figure 1 shows the electrochemical performances of different types of hybrid nanomaterials. According to the resistance values obtained from the Nyquist plots, it is possible to say that MWCNT-Au-Pt/AuSPE has the lowest resistance value due to the high conductivity of the MWCNT-Au-Pt hybrid nanomaterial. For this reason, in order to increase the efficacy of the developed immunosensor, this hybrid nanomaterial was selected and used for further studies.
KCl [39]. Next, electrochemical measurements were performed by using EIS in trip

Selectivity Study
In order to appraise the selectivity of developed anti-H1-based electrochemica enza A immunosensor, EIS measurements were performed to monitor the res changes on the electrode surface after incubating developed immunosensor with 5µg/mL of the H1N1 and H3N2 influenza A viruses which were model and con ruses, respectively.

Selection of Type of Hybrid Nanomaterial
In order to select the most convenient hybrid nanomaterial, experiments by plain AuSPE, AuSPE/Grafen-Au, AuSPE/MWCNT-Au, AuSPE/Grafen-Pt AuSPE/MWCNT-Au-Pt, were conducted via EIS in 50 mM pH 7.4 phosphate bu cluding 10 mM [Fe(CN)6] 4− /[Fe(CN)6] 3− as a redox probe in the frequency range of 0 10 kHz and with a potential of 0.10 V. Figure 1 shows the electrochemical perform of different types of hybrid nanomaterials. According to the resistance values ob from the Nyquist plots, it is possible to say that MWCNT-Au-Pt/AuSPE has the resistance value due to the high conductivity of the MWCNT-Au-Pt hybrid nanom For this reason, in order to increase the efficacy of the developed immunosensor, t brid nanomaterial was selected and used for further studies.

MWCNT-Au-Pt Hybrid Nanomaterial Characterization
SEM and EDX analyses were used to provide information regarding the morp ical and structural composition of the synthesized MWCNT-Au-Pt hybrid nanom Figure 2A shows the typical CNT shape in the form of sticks with a thickness of 4 The deposited AuNPs and PtNPs on the surface of CNT were observed as roundand homogeneously dispersed bright dots in Figure 2B. From SEM images, it is p to say that AuNPs and PtNPs were accumulated together on the CNT structure tionally, the sizes of these metal nanomaterials on the MWCNT were obtained in th of 45 to 75 nm.

MWCNT-Au-Pt Hybrid Nanomaterial Characterization
SEM and EDX analyses were used to provide information regarding the morphological and structural composition of the synthesized MWCNT-Au-Pt hybrid nanomaterial. Figure 2A shows the typical CNT shape in the form of sticks with a thickness of 400 nm. The deposited AuNPs and PtNPs on the surface of CNT were observed as round-shaped and homogeneously dispersed bright dots in Figure 2B. From SEM images, it is possible to say that AuNPs and PtNPs were accumulated together on the CNT structure. Additionally, the sizes of these metal nanomaterials on the MWCNT were obtained in the range of 45 to 75 nm.
In Figure 2C, EDX results confirmed that Au and Pt nanoparticles were successfu deposited on MWCNT surface. The weight and atomic percentages of the elements in prepared hybrid nanomaterial structure were determined as 69.19% C, 12.57% Pt, a 18.24% Au, and 97.35% C, 1.09% Pt, and 1.56% Au, respectively.

Electrochemical Characterization of Developed Biosensor
EIS was used for the layer-by-layer electrochemical characterization of developed fluenza A immunosensor. The Nyquist plots consist of two parts which are a semicir and a straight line. Whereas the straight line demonstrates the Warburg impedance, wh is due to diffusion at low frequencies, the semicircle diameter represents the charge tra fer resistance on the electrode surface. As shown in Figure 2D, compared to the bare g working electrode surface resistance, MWCNT-Au-Pt hybrid nanoparticle-modif AuSPE has a smaller semicircle signal due to the facile electron transfer process becau of the conductivity that was provided by AuNP structure (a,b). Then, when anti-H1 w immobilized onto the electrode surface after modification with cysteamine (c), the sem circle diameter became bigger owing to harder electron transfer (d). At this stage, the el trode surface was saturated with BSA to prevent non-specific binding, and for this reas the radius of the semicircle became bigger (e). Next, based on the specific binding of proteins on H1N1 virus to anti-H1 antibodies on developed immunosensor surface, blocking layer originating from the bound H1N1 virus was formed on the electrode s face. Therefore, as the electron transfer became harder because of that layer, the bigg semicircle was obtained (f). In Figure 2C, EDX results confirmed that Au and Pt nanoparticles were successfully deposited on MWCNT surface. The weight and atomic percentages of the elements in the prepared hybrid nanomaterial structure were determined as 69.19% C, 12.57% Pt, and 18.24% Au, and 97.35% C, 1.09% Pt, and 1.56% Au, respectively.

Electrochemical Characterization of Developed Biosensor
EIS was used for the layer-by-layer electrochemical characterization of developed influenza A immunosensor. The Nyquist plots consist of two parts which are a semicircle and a straight line. Whereas the straight line demonstrates the Warburg impedance, which is due to diffusion at low frequencies, the semicircle diameter represents the charge transfer resistance on the electrode surface. As shown in Figure 2D, compared to the bare gold working electrode surface resistance, MWCNT-Au-Pt hybrid nanoparticle-modified AuSPE has a smaller semicircle signal due to the facile electron transfer process because of the conductivity that was provided by AuNP structure (a,b). Then, when anti-H1 was immobilized onto the electrode surface after modification with cysteamine (c), the semicircle diameter became bigger owing to harder electron transfer (d). At this stage, the electrode surface was saturated with BSA to prevent non-specific binding, and for this reason, the radius of the semicircle became bigger (e). Next, based on the specific binding of H1 proteins on H1N1 virus to anti-H1 antibodies on developed immunosensor surface, the blocking layer originating from the bound H1N1 virus was formed on the electrode surface. Therefore, as the electron transfer became harder because of that layer, the biggest semicircle was obtained (f).

Experimental Parameters Optimization
Because of the presence of -SH and -NH 2 functional groups in its structure, cysteamine plays a significant role in binding the biological materials onto the gold surface. Without any pre-thiolation, cysteamine can bind AuNPs from -SH ends and biological materials from -NH 2 ends [40,41]. As mentioned in the experimental part, the electrode surface was activated with cysteamine so that the antibodies could bind effectively onto the electrode surface. For this reason, a series of experiments were carried out to determine the optimum time required for the modification of the electrode surface. According to Nyquist plots that were obtained before (0 min) and after (15,30,60, 120 min) the interaction with cysteamine (Figure 3), it was seen that the optimum modification time is 60 min. When the modification time was increased, the formation of a double semicircle curve was observed. This situation may be explained as the oxidation of -SH ends of the cysteamine into -SO 2 and -SO 3 in a long waiting time. In addition, there is a possibility that -COOH ends in the MWCNT-Au-Pt structure on the electrode surface may have an additional interaction with cysteamine [42,43].

Experimental parameters optimization
Because of the presence of -SH and -NH2 functional groups in its structure, cy ine plays a significant role in binding the biological materials onto the gold surface out any pre-thiolation, cysteamine can bind AuNPs from -SH ends and biological als from -NH2 ends [40,41]. As mentioned in the experimental part, the electrode was activated with cysteamine so that the antibodies could bind effectively onto th trode surface. For this reason, a series of experiments were carried out to determ optimum time required for the modification of the electrode surface. Accord Nyquist plots that were obtained before (0 min) and after (15,30,60, 120 min) the i tion with cysteamine (Figure 3), it was seen that the optimum modification time is When the modification time was increased, the formation of a double semicircle was observed. This situation may be explained as the oxidation of -SH ends of t teamine into -SO2 and -SO3 in a long waiting time. In addition, there is a possibil -COOH ends in the MWCNT-Au-Pt structure on the electrode surface may have a tional interaction with cysteamine [42,43]. Moreover, MWCNT-Au-Pt hybrid nanomaterial amount in the develop munosensor structure was also optimized. For this purpose, five electrochemica enza A immunosensors that contain different amounts of MWCNT-Au-Pt hybrid materials (2,4,6,8, and 10 µL from 10 mg/mL suspension) and 60 µg/mL of anti-H body were prepared, and then the responses against 7.5 µg/mL of H1N1 virus de were investigated ( Figure 4). As can be seen in Figure 4f., the best result was ob with 4 µL of the MWCNT-Au-Pt hybrid nanomaterial-included immunosensor more than 4 µL of MWCNT-Au-Pt hybrid nanomaterial in the immunosensor st resulted in a decrease in the resistance value (Figure 4f). This may be related to the active surface area due to the excessive and irregular immobilization of MWCNT hybrid nanomaterial on the electrode surface [44,45]. Moreover, MWCNT-Au-Pt hybrid nanomaterial amount in the developed immunosensor structure was also optimized. For this purpose, five electrochemical influenza A immunosensors that contain different amounts of MWCNT-Au-Pt hybrid nanomaterials (2,4,6,8, and 10 µL from 10 mg/mL suspension) and 60 µg/mL of anti-H1 antibody were prepared, and then the responses against 7.5 µg/mL of H1N1 virus detection were investigated ( Figure 4). As can be seen in Figure 4f, the best result was obtained with 4 µL of the MWCNT-Au-Pt hybrid nanomaterial-included immunosensor. Using more than 4 µL of MWCNT-Au-Pt hybrid nanomaterial in the immunosensor structure resulted in a decrease in the resistance value (Figure 4f). This may be related to the limited active surface area due to the excessive and irregular immobilization of MWCNT-Au-Pt hybrid nanomaterial on the electrode surface [44,45].  The detection ability of the electrochemical biosensor depends on bioactive la preparation procedure and interaction conditions between that layer and the biologi analyte [43,46,47]. Therefore, in order to determine the electrode surface saturation c centration of anti-H1 antibody, EIS measurements were conducted with the develop immunosensor containing different concentrations of anti-H1 (20, 40, 60, 80, and 1 µg/mL) and 4 µL of MWCNT-Au-Pt hybrid nanomaterial after the interaction with µg/mL of H1N1 virus ( Figure 5). As can be seen from Figure 5f, the maximum resistan difference was obtained with 60 µg/mL anti-H1-included influenza A immunosens When more concentrated anti-H1 antibody was used in the developed immunosens the obtained resistance value decreased. This can be explained by the oversaturation the bioactive layer when using anti-H1 antibodies more concentrated than 60 µg/mL [4 The detection ability of the electrochemical biosensor depends on bioactive layer preparation procedure and interaction conditions between that layer and the biological analyte [43,46,47]. Therefore, in order to determine the electrode surface saturation concentration of anti-H1 antibody, EIS measurements were conducted with the developed immunosensor containing different concentrations of anti-H1 (20, 40, 60, 80, and 100 µg/mL) and 4 µL of MWCNT-Au-Pt hybrid nanomaterial after the interaction with 7.5 µg/mL of H1N1 virus ( Figure 5). As can be seen from Figure 5f, the maximum resistance difference was obtained with 60 µg/mL anti-H1-included influenza A immunosensor. When more concentrated anti-H1 antibody was used in the developed immunosensor, the obtained resistance value decreased. This can be explained by the oversaturation of the bioactive layer when using anti-H1 antibodies more concentrated than 60 µg/mL [48].

Analytical Characteristics
After the experimental parameters were optimized, analytical characteristics of the developed system towards different concentrations (2.5, 7.5, 10, 15, 20, 25 µg/mL) of H1N1 were examined with EIS technique (Figure 6). According to the obtained calibration curve, as shown in Figure 6, the developed electrochemical influenza A immunosensor shows a linear response over H1N1 virus concentration range of 2.5-25.0 µg/mL (R 2 = 0.99) with the linear regression equation of y = 20.424x + 159.01. In addition, limit of detection (LOD; 3 s/m; s is the standard deviation of blank solution for 3 points, m is the slope of the calibration plot) and limit of quantification (LOQ; 10 s/m) values were calculated as 3.54 µg/mL and 11.80 µg/mL, respectively. Relative standard deviation (RSD) value was found as 0.45% for 20 µg/mL concentration of the H1N1 virus (n = 3). µg/mL) and 4 µL of MWCNT-Au-Pt hybrid nanomaterial after the interaction with 7.5 µg/mL of H1N1 virus ( Figure 5). As can be seen from Figure 5f, the maximum resistance difference was obtained with 60 µg/mL anti-H1-included influenza A immunosensor. When more concentrated anti-H1 antibody was used in the developed immunosensor, the obtained resistance value decreased. This can be explained by the oversaturation of the bioactive layer when using anti-H1 antibodies more concentrated than 60 µg/mL [48]. Biosensors 2023, 13, x FOR PEER REVIEW 9

Analytical Characteristics
After the experimental parameters were optimized, analytical characteristics of developed system towards different concentrations (2.5, 7.5, 10, 15, 20, 25 µg/mL) of H were examined with EIS technique (Figure 6). According to the obtained calibration cu as shown in Figure 6, the developed electrochemical influenza A immunosensor show linear response over H1N1 virus concentration range of 2.5-25.0 µg/mL (R 2 = 0.99) w the linear regression equation of y = 20.424x + 159.01. In addition, limit of detection (L 3 s/m; s is the standard deviation of blank solution for 3 points, m is the slope of the bration plot) and limit of quantification (LOQ; 10 s/m) values were calculated as µg/mL and 11.80 µg/mL, respectively. Relative standard deviation (RSD) value was fo as 0.45% for 20 µg/mL concentration of the H1N1 virus (n = 3).

Selectivity Study
Selectivity study was performed by using 10 µL of 5 µg/mL H1N1 and H3N2 viru When the developed MWCNT-Au-Pt hybrid nanomaterial-modified anti-H1 antibo

Selectivity Study
Selectivity study was performed by using 10 µL of 5 µg/mL H1N1 and H3N2 viruses. When the developed MWCNT-Au-Pt hybrid nanomaterial-modified anti-H1 antibodybased electrochemical immunosensor for influenza A detection was incubated with H3N2 virus for 1 h, no change was observed in the resistance value, whereas a remarkable increase in the resistance value was determined after the interaction with H1N1 virus. The obtained results demonstrates specific binding of H1 protein on H1N1 virus to anti-H1 antibody. Moreover, as expected, since H3 proteins on H3N2 virus do not react with anti-H1 antibody, no response was observed for this type of virus (Figure 7).

Sample Application
The matrix effect was examined using synthetic saliva supplemented with H1N1 virus. For this purpose, EIS measurements were performed with developed influenza A immunosensor after the incubation with a synthetic saliva solution containing 5 µg/mL of H1N1 influenza A virus for 1 h. According to the obtained resistance values, recovery value was calculated as 99.18% ± 0.17. Table 1 shows the comparison of developed influenza A immunosensor with recent similar studies. As can be seen from Table 1, we can say that this study is at a good level in terms of linear range and LOD values.

Sample Application
The matrix effect was examined using synthetic saliva supplemented with H1N1 virus. For this purpose, EIS measurements were performed with developed influenza A immunosensor after the incubation with a synthetic saliva solution containing 5 µg/mL of H1N1 influenza A virus for 1 h. According to the obtained resistance values, recovery value was calculated as 99.18% ± 0.17. Table 1 shows the comparison of developed influenza A immunosensor with recent similar studies. As can be seen from Table 1, we can say that this study is at a good level in terms of linear range and LOD values.

Conclusions
In conclusion, an MWCNT-Au-Pt hybrid nanomaterial-modified anti-H1-based electrochemical immunosensor was produced for influenza A detection. Throughout the entire study, inactive H1N1 influenza A virus was used as a model virus. This developed platform has a potential application to diagnose different types of influenza viruses that contain the H1 protein on their surfaces. Besides being selective, sensitive, and practical, due to SPE usage, very small volumes of samples were analyzed with the developed immunosensor. In addition, we believe that developed system is of great importance because it can be adapted to all kinds of influenza virus determinations for the future just by changing the antibody type. Data Availability Statement: Data will be made available on request.