Flexible electrochemical paper-based device for detection of breast cancer- derived exosome using nickel nanofoam 3D nanocomposite

Flexible electrochemical paper-based device for detection of breast cancer- derived exosome using nickel nanofoam 3D nanocomposite | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Flexible electrochemical paper-based device for detection of breast cancer- derived exosome using nickel nanofoam 3D nanocomposite Nafiseh Sahraei, Mohammad Mazloum-Ardakani, Alireza Moradi, Farzaneh Hoseynidokht This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3994610/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 2 You are reading this latest preprint version Abstract Exosomes as new biomarkers for cancer diagnosis have attracted attention because they are highly released by tumor cells in various biological fluids. In this study, an electrochemical paper-based immunosensor device (Exo-sensing paper) is introduced for the detection of exosome in the serum. The Exo-sensing paper is a three electrode system that is prepared using pattern paper and carbon and silver inks. The sensor part of this immunosensor contains a three-dimensional porous nanocomposite of nickel nanofoam coupled with graphene oxide and gold nanoparticles. The high specific surface area of this nanocomposite increases the antibody loading on the sensor surface significantly and consequently leads to obtaining a wide linear range of 500–1 × 10 7 Exospore/µL with a detection limit of 110 Exosome/µL. Due to some advantages of this constructed Exo-sensing paper such as easy storage, simple application, low cost and good selectivity in the real samples, this system has a good potential to be used as a point of care testing for in situ detection of the exosomes and is a promising strategy for minimally invasive liquid biopsy. Paper-based electrochemical biosensor Three-dimensional nickel nanofoam Point of care testing (POCT) Exosome. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Exosomes are nanometer extracellular vesicles containing two lipid layers and a large number of biological molecules such as proteins, lipids, carbohydrates and nucleic acids [ 1 ]. They are secreted by all types of cells and are widely existence in different fluids of human body and mainly play a key role in the intercellular communication and indicate a specific molecular profile related to the origin of the parent cells [ 2 ]. Notably, the existence and progression of some cancers to the types and levels of Exosome membrane proteins are strongly related. [ 3 , 4 ]. Therefore, exosomes containing specific proteins are a main source of new biomarkers in diagnosis and prognosis of disease. Some common detection methods of exosomal protein are mass spectrometry [ 5 ], enzyme-linked immunosorbent assay (ELISA) [ 6 ], surface plasmon resonance [ 7 ] and western blot (WB) analysis [ 8 ]. These methods are usually complex, boring and long pretreatment. In addition, the sensitivity of these methods is not sufficient for detection of small surfaces of exosomal proteins. Point of care testing (POCT) is a novel technology for the immediate analysis of clinical samples at the sampling situation without complex processing, which can possess a high potential for diagnosis and monitoring of cancer [ 9 , 10 ]. Based on the advantages of POCT technology, some methods have been expanded to determine exosomes, which have high feasibility but low sensitivity or require specialized equipment [ 11 – 13 ]. Therefore, improving a sensitive platform with a simple and low-cost POCT protocol for detection of exosomes is as a challenge. Nowadays, paper-based sensors have been developed in bioanalytical applications [ 14 , 15 ]. Due to this fact that the cellulose papers are cheap, light, available, biodegradable, and easily assembled, a series of POCT based on the paper-based devices have been developed for colorimetric [ 16 ], fluorescence [ 17 ], and electrochemiluminescence [ 18 ] techniques. Electrochemical methods have attractive properties of simple measurement protocol, high flexibility and short detection time, as well as miniaturization and portability, which make them very suited for the evolution of POCT technologies [ 19 – 21 ]. In addition, some modified electrodes can be used without complex equipment, which are particularly suitable for the development of portable POCT instruments. For example, Liu et al developed a paper POCT aptasensor for exosome quantification using Zr-MOFs and aptamer as detection system along with chain reaction hybridization for signal amplification[ 22 ]. Also, Su et al designed a smartphone-based biosensor for measurement of exosome in serum using polyenzymatic signal amplification based on double-antibody sandwich method [ 23 ]. To promote the performance of paper POCT electrochemical devices, the nanomaterials of carbon, metal oxides and metals have been modified on the paper surface [ 24 – 26 ]. In addition, the three-dimensional porous metal nanofoams are widely used in electrochemistry field as a conductive materials with high catalytic properties and high specific surface area [ 27 ]. The porous nanofoam of nickel contains the properties of metals such as electrical and thermal conductivity and catalytic activity along with the extraordinary properties of nanostructure materials such as low density and high specific surface area, which leads to an increase in its properties specially increasing the active sites and catalytic activity [ 28 , 29 ]. For example, Deng et al. designed a biosensor based on decorated nickel foam with ZIF-8/CuO- for glucose determination [ 30 ]. The electrochemical analysis revealed that the electrochemical active surface of the electrode increased twice while the electrical resistance decreased during one week. So, various metal nanofoams have been used in electrochemical sensors to immobilize various biomolecules for detection of cancer biomarkers. In this study, carbon and silver ink (constructed in the laboratory) were printed on the paper pattern of a three-electrode system to prepare a paper-based electrochemical immunosensor (exo-sensing paper). Then, Au@rGO/Ni nanofoam nanocomposite was placed on the electrode surface of carbon ink to improve its conductivity. For the synthesis of the modifier, first, three-dimensional nickel nanofoam was prepared as a substrate using an easy and cheap method without a pattern. The size of the porous and the diameter of the obtained nickel nanofoam are much smaller than the commercial samples, and therefore it can accelerate the electron and ion transfer. Then, graphene oxide was placed on the nickel nanofoam as an anti-corrosion of metal and a three-dimensional network of graphene oxide was created using nickel nanofoam as the sub layer substrate. Finally, gold nanoparticles were prepared on the rGO/Ninanofoam nanocomposite by in situ reduction using ascorbic acid and a displacement reaction with nickel without using a surfactant. This nanocomposite offers a good potential in biosensor and catalytic applications due to the strong structure of nickel foam and good recyclability. Next, Anti-CD9 antibody was immobilized on the electrode surface and exposed to different concentrations of exosome extracted from cancer cell and finally the human serum. The exo-sensing paper has some advantages like simplification for general uses, low cost and the competency of the biosensor. Exo-sensing paper is developed based on the use of paper which leads to the mass production with good repeatability, cheap and disposable properties. Also, the used carbon ink to make the electrodes has a simple synthesis and is very cheap. The obtained low detection limit and wide linear range confirm the high sensitivity of this immunosensor platform as POCT for using in real samples. 2. Experimental section 2.1. Materials and devices Graphite powder, hydroxyethyl cellulose (HEC), 2-methoxyethanol, cellulose acetate, ascorbic acid, poly (acrylic acid), silver nitrate, hydrogen tetrachloroauate (AuHCl 4 .3H2O), Nickel nitrate, potassium ferrocyanide (K 3 [Fe(CN) 6 , potassium ferricyanide (K 4 [Fe(CN) 6 , iron (III) chloride, potassium chloride (KCl), acetone, methanol, 2-methoxyethanol, cyclohexanone, nitric acid and sulfuric acid were purchased from Merck Co or Sigma Chemical Co. Mouse monoclonal α-human CD9 antibody (Ab) was obtained from R&D systems. The electrochemical tests were performed on the electrochemical workstation (Metrohm AutoLab PGSTAT-302, Netherlands). A paper-based three-electrode configuration was usage, containing the working and auxiliary electrodes (carbon ink) and a reference electrode (silver ink). The laser cutter was usage for the fabrication of the pattern of electrodes (Mini Laser BCL-MU, Jinan Bodor CNC Machine Co, China). Ultracentrifuging was done with a Beckman Coulter Optima XPN-90 with the SW40 Ti rotor at 4°C. Cyclic voltammetry measurements were done in the electrolyte solution containing a redox system (Fe(CN) 6 −3/−4 with a scan rate of 0.1 V/s and in the potential range of -0.6 to + 0.8 V. Electrochemical impedance spectroscopy (EIS) was experimented in the frequency range of 100 Hz to 10 5 Hz. The main measurements were performed in the same solution with the iron probe monitoring using the differential pulse voltammetry (DPV) technique in the potential range of -0.4V to + 0.4V. 2.2. Cell culture Exosomes were purified from serum-free culture media of MCF-7 cell as described in the previous work [ 31 ]. Briefly, MCF-7 cells were grown in the culture medium of DMEM with penicillin/streptomycin (1%) without bovine serum for 72 h at 37°C and wet atmosphere (CO 2 5%). Exosomes were isolated from the serum-free supernatant using an ultracentrifuge. First, the Exosomes were centrifuged for 10 min (1200g) and then 10000g for 30 min to remove the cell remains and large vesicles. Then the supernatants were centrifuged at 100,000g for 2 h to collect the Exosomes. The exosome plates were re suspended in 100 µl of PBS and stored at -80°C. 2.3. The synthesis of nickel nanofoams Nickel nanofoams were synthesized without pattern by combustion process. For synthesis, (NiNO 3 ) 2 .6H 2 O (0.05 M) was poured in 50 mL of 2-methoxyethanol (C 3 H 8 O 2 ) as a solvent and reducing agent and stirred for 30 min until it was dissolved completely. The obtained solution was stirred for 2 h at 80°C in an oil bath and then dried to obtain a dark colored colloidal precursor. Subsequently, the precursor colloid was heated to 180°C in an oven and it was burned vigorously in seconds to obtain the nickel foams. The combustion products of the solvent (C 3 H 8 O 2 ) in the atmosphere contain carbon dioxide and large amounts of carbon monoxide, which can convert the divalent nickel ions (Ni + 2 ) into nickel (Ni) and create the porous nickel foams of open and continuous cells. 2.4. rGO/Ninanofoam and AuNPs@rGO/Ninanofoam synthesis Graphene oxide was prepared by the modified Homer method [ 32 ]. The synthesized nickel nanofoam was placed in a centrifuge tube containing dispersed graphene oxide (4 mg/mL), and centrifuged at 50 rmp for 15 min to fill the pores with GO and the graphene oxide sheets cover the continuous network of nickel nanofoams. The wet nickel nanofoam dried at room temperature and this process was repeated several times to increase the loading of rGO. To prepare the AuNPs@rGO/Ninanofoam nanocomposite, 1 mL of ascorbic acid solution (1 mM) was added to rGo/Ninanofoam suspension (5 mg/mL) at 80°C. The solution of AuHCl 4 .3H 2 O (5 mM) was added drop by drop to this suspension while stirring to form gold nanoparticles outside and inside of the holes. Finally, the nanocomposite was purified by centrifugation and then washed with ethanol and water. 2.5. Preparation of the Exo-sensing paper In this work, Exo-sensing papers were made by prototyping technique. First, the pattern of the paper-based device was designed, and then the patterns were prepared on a self-adhesive glass paper by laser cutting as a hydrophobic border. The patterns were pasted on a filter paper (Whatman number 1). For a three-electrode system on Exo-sensing papers, a working electrode (WE) with a standard deviation of 2.8% and area = 0.04 cm2), an auxiliary electrode (CE) and their connections using carbon ink and a reference electrode (RE) using silver ink was printed. The Exo-sensing papers were dried in an oven at 55°C. The carbon ink was made by mixing graphite powder (0.5 g) and 1.25 g of cellulose acetate solution (7.5% w in cyclohexane and acetone) [ 33 ]. Silver ink was synthesized by reduction of silver nitrate by poly acrylic acid (PAA) and diethanolamine (DEA) as a surface trapping agent in an aqueous solution in several steps. Briefly, 1 g of PAA was mixed with 20 g of DEA in 25 mL of water for 2 h. Then 10 ml of silver nitrate solution (0.06 mol) was slowly added to the above solution and stirred for 24 h. To increase the size of silver nanoparticles, it was ultra sonicated for 90 min. To coagulate the silver ink, the solution was titrated with ethanol. Finally, to purify and separate PAA, it was washed with water and then centrifuged at 9000 rpm [ 34 ]. The details of the synthesis of carbon and silver inks and the preparation of the Ag/AgCl reference electrode have been described in detail in our previous work [ 31 ]. Then, 4 µL of AuNPs@rGO/Ninanofoam porous nanocomposite (5 mg/mL) was casted on the surface of the carbon working electrode. Then 2 µL of CD9 antibody (5 ng /µL) was immobilized on the surface of the modified working electrode and the electrode was placed for 1 h in a wet atmosphere at 4°C to form the binding with the modified electrode surface. For each step, the electrode surface was washed with phosphate buffer solution to remove un-bounded species. Next, the electrode surface was exposed to 1 µL of BSA solution for 15 min, to block the remaining possible sites on the modified electrode surface to prevent non-specific binding. Eventually, 1.5 µL of different concentrations of the exosome solution was placed on the surface of the fabricated Exo-sensing paper for 30 min, and the differential pulse voltammetry curves in the presence of redox [Fe(CN) 6 ] −3/−4 solution with potassium chloride electrolyte (0.1 M) were recorded in the potential range of -0.4 V to + 0.4 V. The electrical repulsion between the modified surface and the probe, as well as the design of the modified electrode, prevents the transfer of electrons from the probe to the electrode surface and leads to decreasing the current. DPV currents were recorded before and after trapping the exosomes by antibody and this decrease in the analytical signal which is proportional to the exosome concentration can be act a confirmation of the good potential of this platform to detect exosomes. CV and EIS measurements were also performed at each steps of the surface modification, which confirms the successful modification of each step of the antibody and exosome binding. 3. Results and discussion 3.1. Characterization of the designed biosensor The processes of nickel nanofoam and AuNPs@rGO/Nickelnanofoam nanocomposite construction are schematically shown in Scheme 1. During the annealing of the colloidal precursor, due to the use of 2-methoxyethanol solvent, a large amount of carbon monoxide is produced which can cause reduction of the divalent nickel ion (Ni + 2 ) to nickel (Ni). As shown in this figure, the dark nickel nanofoams are very soft and light. Nickel nanofoams were fabricated by annealing Ni(NO 3 ) 2 colloid and C 3 H 8 O 2 in air by using an easy and template-free approach. This fabrication process is very low cost and can be used for large scale. Scheme 1. The processes of Nickel nanofoam construction Figure 1 indicates the morphology of the prepared nickel nanofoam. The FE-SEM images presented in Fig. 1 A and B confirms that the sample has a three-dimensional continuous network structure. The skeleton diameter is 200–300 nm and the pore size is 150–350 nm, which is much smaller than the pore size of commercial foams. Due to the homogeneous structure of the nanofoam, the prepared nickel nanofoam has a much lower density and a high specific surface area. This nickel nanofoam as a current collector can be useful for improving the electrochemical performance of the electrode. Figure 1 C displays the attached graphene layers on the nickel nanofoam surface. A large number of wrinkles and waves are observed on the graphene sheets indicating a good binding between graphene oxide and nickel nanofoam skeleton. Also, due to the difference in the thermal expansion coefficient between graphene and nickel nanofoam these wrinkles and waves may have been created. Figure 1 D shows the three-dimensional image of gold nanoparticles that are dispersed throughout the rGO/nickel nanofoam substrate, uniformly. Figure 2 A shows the X-ray diffraction (XRD) pattern of nickel nanofoam. The three diffraction peaks are (111) 44.5°, (200) 52° and (222) 76.5°, respectively, which are the characteristic of cubic nickel crystal plates (JCPDS card 04-0850). There is no sign of other crystalline phases indicating that the nickel nanofoam has a relatively high purity. The adsorption-desorption isotherm of N 2 was usage to investigate the structure of Ni nanofoam (Fig. 2 B). These isotherms show a type of mesopore hysteresis loop with absorption and desorption branches at high relative pressures. According to the IUPAC patterns, the structure of type IV is taken from the isotherm curves indicating a porous structure for synthetized nickel nanofoam [ 35 , 36 ]. High specific surface area and pores volume of (1975 m 2 /g and 3.5 cm 3 /g) were obtained from these isotherms, which are also related to BET data (Fig. 2 C). The pores diameter average of nickel nanofoams was acquired to be 5.3 nm by BJH analysis that is in good agreement with other analyzes including FE-SEM. The crystal structure of AuNPs@rGO/Ninanofoam nanocomposite was analyzed by X-ray diffraction. As can be seen in Fig. 2 D, in addition to the peaks related to the diffraction of the nickel crystal, the specific peaks at 38.5 2 θ have also appeared, which confirms the formation of gold nanostructured crystal. Also, a wide peak with low height is related to rGO deposited on the nickel nanofoam with an amorphous structure [ 37 ]. The diffraction pattern of AuNPs@rGO/Nickel nanofoam shows that the gold nanoparticles on the rGO/Nickel nanofoam have a relatively high crystallinity. To confirm the components of AuNPs@rGO/Nickel nanofoam nanocomposite and also to confirm the formation of AuNPs on rGO/Nickel nanofoam, the elemental analysis by X-ray energy diffraction spectroscopy (EDS) was also performed. Figure 3 A shows the related peaks to the nanocomposite elements. EDS analysis was obtained to be Ni 56%, C 30%, Au 12% and oxygen 3%. In addition, elemental mapping was done to discover the distribution of the constituent components of the nanocomposite. As seen in Fig. 3 B, a uniform distribution of all elements in the composition of the nanocomposite especially for gold nano particles can be seen. EDX mapping analysis as well as XRD confirms the successful synthesis of AuNPs@rGO/Nickel nanofoam nanocomposite. In the synthesis of this nanocomposite, the nickel nanofoam as a substrate acts as a support frame for rGO and AuNPs, and also is easily recyclable. This advantage is very important for paper-based electrodes. Also, it was used as a reducing agent in the synthesis of gold nanoparticles. AuNPs can be formed by the following substitution reaction: Ni + Au + 3 → Ni + 2 + Au Of course, some gold nanoparticles are also obtained during the reduction of GO. The high activity of AuNPs@rGO/Nickel nanofoam can be related to the synthesis method of gold nanoparticles prepared without surfactants and therefore they can expose their active sites, completely. Also, the nickel nanofoam along with graphene oxide create a good stability and conductivity and also an increase in the electrons transfer [ 38 , 39 ]. Electrodes with this kind of modified structures can provide multiple directions for electron transfer and lead to increasing the effective loading of materials (biomolecules such as antibodies, enzyme, aptamers, etc.) and consequently increase of the linear range, effectively. The nanocomposite of AuNPs@rGO/Nickel nanofoam was synthesized by a green, easy and efficient method. The prepared nickel nanofoam was light and flexible, and the fabrication method was easy and template-free, too. Gold nanoparticles are without chemical surfactants and distributed on the surface of rGO/Nickel nanofoam uniformly with a good performance. 3.2. The electrochemical behavior of electrodes An electrochemical immunosensor (three-electrode) paper-based device was made with a patterned sticker on a filter paper coated with paraffin (on one side). This patterned sticker creates hydrophobic limits around the electrodes. The thickness of the electrodes depends on the number of ink print on the paper, which can be controlled by the thickness of the stencil (100 µm). A very simple and cheap method that also can use for the mass production of paper-based devices. The working electrode of carbon ink was modified with AuNPs@rGO/Nickel nanofoam to prepare an activated three-electrode system. The electrochemical properties of the prepared Exo-sensing paper were investigated by cyclic voltammetry in the presence of [Fe(CN) 6 ] 3−/4− as probe in the range of -0.6 V to + 0.8 V. The obtained cyclic voltammogram of the carbon ink (working electrode) surface with a pair of redox peaks (ΔEp = 129 mV) suggests that the electrochemical behavior of probe on the carbon ink surface has a semi-reversible behavior. Also, the stability of the products of redox probe on the surface of carbon ink was studied by testing the ratio of anodic to cathodic peak currents. To obtain a good understanding of mass transfer on the working electrode surface, the electrochemical characteristics of Exo-sensing paper were investigated and compared using the cyclic voltammetry method at different scan rates before and after modification of the working electrode surface with AuNPs@rGO/Nickel nanofoam nanocomposite (Figure S1 and S2). The activated surface of the working electrode was analyzed according to the slope of the anodic peak currents diagram versus the square of the scan rate (inset in Figure S1 and S2) according to the Randles-Sevcik equation: I pa = (2.69 × 10 5 ) n 2/3 A eff D 1/2 ʋ 1/2 C 0 where n is the number of transferred electrons, A is the specific active surface, I is the anode peak currents at different scan rates of the working electrode, C is the concentration of K 4 Fe(CN) 6 , D is the diffusion coefficient of iron probe and ʋ is the scan rate [ 40 ]. The results showed that the effective surface of the working electrode was improved 42% after modification. A paper-based electrochemical immunosensor (Exo-sensing paper) based on AuNPs/rGO/Nickel nanofoam was developed for exosome with CD9 surface marker detection. CV was used for investigation of the preparation process of the Exo-sensing paper, and the experiments were done in a solution containing K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] (5 mM) and KCl (0.1 M) at a potential of -0.6 to + 0.8 V. The obtained CV curves of modified electrodes with different nanocomposits and biomolecules are displayed in Fig. 4 A. Compared to the bare electrode (carbon ink), the rGO/Nickel nanofoam electrode revealcs a higher current signal, and probably due to the higher conductivity of rGO. The electrode AuNPs@rGO/Nickel nanofoam, shows an increase of the current signal, which is due to the higher conductivity of nanomaterials of nanocomposite, especially gold nanoparticles. Due to low conductivity of immunoproteins, the current signal of Ab/AuNPs@rGO/Nikel nanofoam electrode is decreased. In addition, after incubation with exosome, the current signal of the exosome/Ab/AuNPs@rGO/Nickel nanofoam electrode is decreased more strongly due to the formation of immune complexes on the surface of the electrode by prevention of the electron transfer. Also, electrochemical impedance spectroscopy (ESI) was performed to investigate the surface characteristics of the modified electrodes at different stages. As shown in Fig. 4 B, the ESI spectra of different surface electrode modification are different. The electron transfer resistance (Rct) value of the bare electrode (carbon ink) has a large semicircle. After modifying the electrode surface with nanocomposite of rGO/Nickel nanofoam and AuNPs@rGO/Nickel nanofoam, the semicircles became smaller indicating less electron transfer resistance in the modified electrode surfaces. As shown in this figure the impedance values of the Exo-sensing paper are increased by layer-by-layer modification with antibody and Exosome. It can be related to this fact that by introduction of the macromolecular proteins to the surface of the electrodes, the capacity of transfer electrons on the electrode surfaces are decreased. The results of ESI analysis have a good agreement with the CV signals, which indicates the successful fabrication of this Exo-sensing paper. 3.3. The optimization of effective parameters for exosome detection To obtain a high performance for the Exo-sensing paper, some effective parameters such as the concentration of nanocomposite, antibody concentration, and the binding time of antibody to the electrode surface were optimized. The concentration of the modifier on the electrode surface significantly affects the detection performance. Because the different concentrations of modifier can affect the catalytic activity as well as the conductivity of the electrode. The electrochemical current signal increases by increasing the concentration of the modifier suspension up to 10 mg/mL, as shown in Figure S3. The incubation time of anti-CD9 antibody on the AuNPs@rGO/Nickel nanofoam was tested at different times. As shown in Figure S4, the DPV currents are decreased by increasing the antibody incubation time until the peak current be constant value at 60 min, and therefore, 60 min was chosen as an optimum value for the construction of the Exo-sensing paper. The results of the effect of antibody concentration on DPV signal rate are displayed in Figure S5. As the antibody concentration increased from 500 pg/mL to 100 ng/mL, the currents are decreased and reached a constant value at 5 ng/mL. As a result, the antibody concentration was optimized and the optimum concentration was considered to be 5 ng/mL. However, the incubation time of the exosome was evaluated for the complete formation of the antibody-exosome complex. The peak currents of DPVs are decreased by increasing the incubation time and becomes constant at 30 min. Therefore, 30 min was chosen as the optimum incubation time. 3.4. The analytical performance of Exo-sensing paper for exosome detection Under optimum conditions, the electrochemical performance of the Exo-sensing paper was analyzed by incubation of the exosome with different concentrations on the Ab/AuNPs@rGO/Nickel nanofoam electrode surface. The responses of DPV currents were decreased by increasing the exosome concentration (Fig. 5 A). The obtained ΔI values displayed a good linear relationship with the logarithm of the exosome concentration in the range of 500 to 1 × 10 7 exosome/µL, with a correlation coefficient (R 2 ) of 0.98 (Fig. 5 B). The calculated detection limit was obtained to be 110 exosome/µL. The performance of the constructed Exo-sensing paper was compared with the previous methods in Table 1 . This table shows that the proposed method has shown a good performance. This better performance can be related to the specific and excellent characteristics of nanocomposite and the synergistic effect of these three components in the nanocomposite structure. Porous nickel nanofoam has good conductivity, high stability and large specific surface area and therefore it is a very suitable substrate for electrocatalytic activities in combination with graphene oxide and gold nanoparticles. In addition, the process of an Exo-sensing paper fabrication is low-cost and relatively simple. Also, this Exo-sensing paper based on disposable paper is biodegradable and easy to use and has a short analysis time. Therefore, the use of this POCT in clinical diagnosis is suggested. 3.5. Repeatability, selectivity and stability of the Exo-sensing paper To investigate the selectivity of the designed Exo-sensing paper, the derived exosomes from MCF-7 cell were detected with interfering substances such as microvesicles, BSA, AFP and glucose with the same concentration (5ng/mL) using the same method. As seen in Fig. 6 A, it is clear that the DPV response to exosomes is greater than the response to interferences, which can be due to exosome-antibody interaction. The detection performance in the biological environment was also evaluated. As shown in Figure S6, the obtained results from human serum without exosome are almost similar to phosphate buffer solution, which indicates that the proposed biosensor can work well in complex physiological samples. Table 1 The comparison of performance of the Exo-sensing paper with other exosome detection biosensors Detection Methods Linear Range (Exosome/µL) Detection Limit (Exosome/µL) Reference Chronoamprometry 500 − 10 5 285 [ 41 ] Differential pulse voltammetry 10 2 – 10 7 70 [ 42 ] Electrochemiluminescence 500–5×10 5 229 [ 43 ] Chronoamprometry 10 2 – 10 6 200 [ 44 ] Differential pulse voltammetry 1.7×10 5 - 3.4×10 8 5×10 3 [ 22 ] Differential pulse voltammetry 500 − 10 7 110 This work Also the repeatability of the Exo-sensing paper was investigated by producing five identical Exo-sensing paper and incubating them with the same concentration of Exosome (1 × 10 3 exosome/µL). There was no significant difference in the current responses among these fabricated electrodes and the RSD was less than 7%. Another the important factor for the detection of exosomes is stability of electrode so, a continuous CV scans were used to study the stability of the Exo-sensing paper. After 10 CV scans, 97% of the initial current values were still detected. Furthermore, Exo-sensing paper retained 89% of the initial current value after 14 days at 4°C, indicating its long-term stability (Figure S7). 3.6. The clinical test of the constructed Exo-sensing paper To evaluate the efficiency of the clinical performance of the proposed method in real samples, the Exo-sensing paper was utilized to determine the exosomes in the serum samples of breast cancer patients and healthy. Human serum from two healthy human and four patients were collected for analysis using the fabricated Exo-sensing paper. As shown in Fig. 6 B, the DPV responses of patient's serums were higher than healthy subjects. The obtained RSD of the samples was less than 8.5%. The obtained results from the constructed Exo-sensing paper were compared with the ELISA method. The t-test (0.64, p < 0.05) did not show any significant difference between the obtained results by Exo-sensing paper and ELISA. Table 2 shows the results obtained from both methods. The developed Exo-sensing paper may be useful in clinical diagnosis and even have better diagnostic efficiency due to its wider linear range and lower detection limit than ELISA. Table 2 The obtained results of ELAISA and Exo-sensing paper sample ELAISA (exosome/µL) Exo-sensing paper (exosome/µL) H1 1248 1427 ± 0.78 H2 2055 2190 ± 0.85 P1 55863 56792 ± 0.94 P2 239764 241466 ± 0.75 P3 310374 314154 ± 0.65 P4 397259 408722 ± 0.72 4. Conclusions In summary, an electrochemical immunosensor based on a coupled paper with disposable nickel nanofoam for the selective and sensitive detection of exosomes derived from cancer cells was developed. A three-electrode system was prepared using a pattern paper on the filter paper along with carbon and silver inks. AuNPs@rGO/ckel nanofoam nanocomposite was placed on the electrode surface to achieve signal amplification and effective binding of antibody to the electrode surface. The flexible nickel nanofoam was prepared by a pattern-free and very simple method. Graphene oxide and 3D network of very light nickel nanofoam were deeply integrated without any additives and using a simple strategy, and finally AuNPs@rGO/Nickle nanofoam nanocomposite was prepared by an easy and efficient method without using chemical surfactants. The high specific surface area of the nanocomposite effectively increases the binding of antibody to the electrode surface and clearly increases the electric signal of the electrode and then leads to increasing the sensitivity and selectivity of the exosome detection. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Credit author statementN. Sahraei: Idea, synthesize experiment and writing - original draft.F. Hoseynidokht and A. Moradi: Experiment and calculation.M. Mazloum-Ardakani: Idea, editing and writing - original draft. Acknowledgements: The authors appreciate the financial assistance of the Iran National Science Foundation (INSF), Yazd University Research Council for this study. References Luo S, Wu Y, Pan W et al (2023) An integrated magneto-fluorescent nanosensor for rapid and sensitive detection of tumor-derived exosomes. Sens Actuators B 374:132792. https://doi.org/10.1021/acsami.3c01607 Lim J, Kang B, Son HY et al (2022) Microfluidic device for one-step detection of breast cancer-derived exosomal mRNA in blood using signal-amplifiable 3D nanostructure. Biosens Bioelectron 197:113753. https://doi.org/10.1016/j.bios.2021.113753 Wu H, Cai X, Zhang Q et al (2021) A Design of Precise Protection System for Endangered Species Based on Beidou + 4G. In: 2021 International Conference on Electronic Information Technology and Smart Agriculture (ICEITSA). IEEE, pp 71–75 10.1109/ICEITSA54226.2021.00023 LeBleu VS, Kalluri R (2020) Exosomes as a multicomponent biomarker platform in cancer. Trends cancer 6:767–774. https://doi.org/10.1016/j.trecan.2020.03.007 Zhang X-W, Liu M-X, He M-Q et al (2021) Integral multielement signals by DNA-programmed UCNP–AuNP nanosatellite assemblies for ultrasensitive ICP–MS detection of exosomal proteins and cancer identification. Anal Chem 93:6437–6445. https://doi.org/10.1021/acs.analchem.1c00152 Chen G, Huang AC, Zhang W et al (2018) Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature 560:382–386. https://doi.org/10.1038/s41586-018-0392-8 Wu W, Yu X, Wu J et al (2021) Surface plasmon resonance imaging-based biosensor for multiplex and ultrasensitive detection of NSCLC-associated exosomal miRNAs using DNA programmed heterostructure of Au-on-Ag. Biosens Bioelectron 175:112835. https://doi.org/10.1016/j.bios.2020.112835 Shao H, Im H, Castro CM et al (2018) New technologies for analysis of extracellular vesicles. Chem Rev 118:1917–1950. https://doi.org/10.1021/acs.chemrev.7b00534 Rosa BG, Akingbade OE, Guo X et al (2022) Multiplexed immunosensors for point-of-care diagnostic applications. Biosens Bioelectron 203:114050. https://doi.org/10.1016/j.bios.2022.114050 Sahraei N, Mazloum-Ardakani M, Hoseynidokht F (2022) Electrochemical paper-based biosensors for point-of-care diagnostics: Detection methods and applications. J Electrochem Sci Eng. https://doi.org/10.5599/jese.1104 Chattopadhyay S, Ram R, Sarkar A, Chakraborty S (2022) Smartphone-based automated estimation of plasma creatinine from finger-pricked blood on a paper strip via single-user step sample-to-result integration. Measurement 199:111492. https://doi.org/10.1021/acs.analchem.1c04910 Oliveira-Rodríguez M, Serrano-Pertierra E, García AC et al (2017) Point-of-care detection of extracellular vesicles: sensitivity optimization and multiple-target detection. Biosens Bioelectron 87:38–45. https://doi.org/10.1016/j.bios.2016.08.001 Park J, Park JS, Huang C-H et al (2021) An integrated magneto-electrochemical device for the rapid profiling of tumour extracellular vesicles from blood plasma. Nat biomedical Eng 5:678–689. https://doi.org/10.1038/s41551-021-00752-7 da Silva VAOP, de Freitas RC, de Oliveira PR et al (2020) Microfluidic paper-based device integrated with smartphone for point-of-use colorimetric monitoring of water quality index. Measurement 164:108085. https://doi.org/10.1016/j.measurement.2020.108085 Shu Y, Su T, Lu Q et al (2022) Paper-based electrochemical immunosensor device via Ni-Co MOF nanosheet as a peroxidase mimic for the label-free detection of alpha-fetoprotein. Sens Actuators B 373:132736. https://doi.org/https://doi.org/10.1016/j.snb.2022.132736 Liu D, Zhang Y, Zhu M et al (2020) Microfluidic-Integrated Multicolor Immunosensor for Visual Detection of HIV-1 p24 Antigen with the Naked Eye. Anal Chem 92:11826–11833. https://doi.org/10.1021/acs.analchem.0c02091 Wang J, Jiang C, Jin J et al (2021) Ratiometric Fluorescent Lateral Flow Immunoassay for Point-of‐Care Testing of Acute Myocardial Infarction. Angew Chem 133:13152–13159. https://doi.org/10.1002/ange.202103458 Adhikari J, Rizwan M, Dennany L, Ahmed MU (2021) Electrochemiluminescence nanoimmunosensor for CD63 protein using a carbon nanochips/iron oxide/nafion-nanocomposite modified mesoporous carbon interface. Measurement 170:108755. https://doi.org/https://doi.org/10.1016/j.measurement.2020.108755 Xu L, Shoaie N, Jahanpeyma F et al (2020) Optical, electrochemical and electrical (nano)biosensors for detection of exosomes: A comprehensive overview. Biosens Bioelectron 161:112222. https://doi.org/https://doi.org/10.1016/j.bios.2020.112222 Boonkaew S, Chaiyo S, Jampasa S et al (2019) An origami paper-based electrochemical immunoassay for the C-reactive protein using a screen-printed carbon electrode modified with graphene and gold nanoparticles. Microchim Acta 186:1–10. https://doi.org/10.1007/s00604-019-3245-8 Wang L, Shan T, Pu L et al (2022) Glucometer-based electrochemical biosensor for determination of microRNA (let-7a) using magnetic-assisted extraction and supersandwich signal amplification. Microchim Acta 189:444. https://doi.org/10.1007/s00604-022-05544-7 Liu X, Gao X, Yang L et al (2021) Metal–Organic Framework-Functionalized Paper-Based Electrochemical Biosensor for Ultrasensitive Exosome Assay. Anal Chem 93:11792–11799. https://doi.org/10.1021/acs.analchem.1c02286 Su J, Chen S, Dou Y et al (2022) Smartphone-Based Electrochemical Biosensors for Directly Detecting Serum-Derived Exosomes and Monitoring Their Secretion. Anal Chem 7:3235–3244. https://doi.org/10.1021/acs.analchem.1c04910 Yang H, Zhao J, Dong J et al (2022) Simultaneous detection of exosomal microRNAs by nucleic acid functionalized disposable paper-based sensors. Chem Eng J 438:135594. https://doi.org/10.1016/j.cej.2022.135594 Khaksari S, Abnous K, Hadizadeh F et al (2022) Signal amplification strategies in biosensing of extracellular vesicles (EVs). Talanta 124244. https://doi.org/10.1016/j.talanta.2022.124244 Mazloum-Ardakani M, Tavakolian-Ardakani Z, Sahraei N, Moshtaghioun SM (2019) Fabrication of an ultrasensitive and selective electrochemical aptasensor to detect carcinoembryonic antigen by using a new nanocomposite. Biosens Bioelectron 129:1–6. https://doi.org/10.1016/j.bios.2018.12.047 Tappan BC, Steiner SA III, Luther EP (2010) Nanoporous metal foams. Angew Chem Int Ed 49:4544–4565. https://doi.org/10.1002/anie.200902994 Beer M, Rybár R, Kaľavský M (2019) Experimental heat transfer analysis of open cell hollow ligament metal foam at low Reynolds number. Measurement 133:214–221. https://doi.org/10.1016/j.measurement.2018.10.025 Ni W, Wu H, Bin, Wang B et al (2012) One-Pot Synthesis of Ultra‐Light Nickel Nanofoams Composed of Nanowires and Their Transformation into Various Functional Nanofoams. Small 8:3432–3437 Deng L, Fan S, Chen Y et al (2022) In situ growing CuO/ZIF-8 into nickel foam to fabricate a binder-free self-supported glucose biosensor. Ind Eng Chem Res 61:7312–7321. https://doi.org/10.1021/acs.iecr.2c01298 Sahraei N, Mazloum-Ardakani M, Mohiti J et al (2022) Flexible Paper-Based Immunosensor for the Detection of Specific Cancer-Derived Exosomes. J Electrochem Soc 169:127514. 10.1149/1945–7111/aca836 Marcano DC, Kosynkin DV, Berlin JM et al (2010) Improved synthesis of graphene oxide. ACS Nano 4:4806–4814. https://doi.org/10.1021/nn1006368 Rungsawang T, Punrat E, Adkins J et al (2016) Development of Electrochemical Paper-based Glucose Sensor Using Cellulose‐4‐aminophenylboronic Acid‐modified Screen‐printed Carbon Electrode. Electroanalysis 28:462–468. https://doi.org/10.1002/elan.201500406 Russo A, Ahn BY, Adams JJ et al (2011) Pen-on‐paper flexible electronics. Adv Mater 23:3426–3430. https://doi.org/10.1002/adma.201101328 Abebe B, Murthy HCA, Amare E (2018) Summary on adsorption and photocatalysis for pollutant remediation: Mini review. J Encapsulation Adsorpt Sci 8:225–255. 10.4236/jeas.2018.84012 Ravikovitch PI, Neimark AV (2001) Characterization of nanoporous materials from adsorption and desorption isotherms. Colloids Surf A 187:11–21. https://doi.org/10.1016/S0927-7757(01)00614-8 Ye S, Feng J, Wu P (2013) Deposition of three-dimensional graphene aerogel on nickel foam as a binder-free supercapacitor electrode. ACS Appl Mater Interfaces 5:7122–7129. https://doi.org/10.1021/am401458x Fu Y, Yang Z, Li X et al (2013) Template-free synthesized Ni nanofoams as nanostructured current collectors for high-performance electrodes in lithium ion batteries. J Mater Chem A 1:10002–10007. https://doi.org/10.1039/C3TA11753G Liu YY, Guo XL, Zhao L et al (2018) Facile preparation of surfactant-free Au NPs/RGO/Ni foam for degradation of 4-nitrophenol and detection of hydrogen peroxide. Nanotechnology 29:235706. 10.1088/1361–6528/aab936 Bard AJ, Faulkner LR, White HS (2022) Electrochemical methods: fundamentals and applications. Wiley Wang L, Zeng L, Wang Y et al (2021) Electrochemical aptasensor based on multidirectional hybridization chain reaction for detection of tumorous exosomes. Sens Actuators B 332:129471 Sahraei N, Mazloum-Ardakani M, Khoshroo A et al (2022) Electrochemical system designed on a paper platform as a label-free immunosensor for cancer derived exosomes based on a mesoporous carbon foam-ternary nanocomposite. J Electroanal Chem 920:116590. https://doi.org/10.1016/j.jelechem.2022.116590 Zhang H, Wang Z, Wang F et al (2021) Ti3C2 MXene mediated Prussian blue in situ hybridization and electrochemical signal amplification for the detection of exosomes. Talanta 224:121879. https://doi.org/10.1016/j.talanta.2020.121879 Doldán X, Fagúndez P, Cayota A et al (2016) Electrochemical sandwich immunosensor for determination of exosomes based on surface marker-mediated signal amplification. Anal Chem 88:10466–10473. https://doi.org/10.1021/acs.analchem.6b02421 Scheme Scheme 1 is available in Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Onlinefloatimage1.png Scheme 1. The processes of Nickel nanofoam construction SupplementaryFile.docx Cite Share Download PDF Status: Under Review Version 1 posted Submission checks completed at journal 27 Feb, 2024 First submitted to journal 27 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3994610","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275299058,"identity":"2d404909-59a4-4337-8286-99c2c03cf9fe","order_by":0,"name":"Nafiseh Sahraei","email":"","orcid":"","institution":"Yazd University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nafiseh","middleName":"","lastName":"Sahraei","suffix":""},{"id":275299059,"identity":"e9357ff7-449a-4ba6-b5c9-a9e70f20b0ce","order_by":1,"name":"Mohammad Mazloum-Ardakani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYHACxgNAQg7EkgARbMToAWkxJl1LYgNMC0Gg2378wYGPbdvSNxw//vAGQ40dA5/0AfxazM4kJByc2XY7d8OZhGQLhmPJDGx8CQS0HEg4cJgXpOVAwjEJBrYDDGw8BBxmdv5hA0hLusH5h20SDP+I0XIjmQGkJcHgRjKbBGMbUVqeMRycce624cwbz5gtEvuSeYhwWPrDBx/KbsvzARk3Pnyzk5PvIaAFDhQOAIkEBgZCdiAB+Qbi1Y6CUTAKRsEIAwDvpUcEQiuKpgAAAABJRU5ErkJggg==","orcid":"","institution":"Yazd University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Mazloum-Ardakani","suffix":""},{"id":275299060,"identity":"5ee89057-9809-474e-99f8-9106ca15d250","order_by":2,"name":"Alireza Moradi","email":"","orcid":"","institution":"Shahid Sadoughi University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alireza","middleName":"","lastName":"Moradi","suffix":""},{"id":275299061,"identity":"22ed264f-7899-496a-a060-be3bea0dee2b","order_by":3,"name":"Farzaneh Hoseynidokht","email":"","orcid":"","institution":"Yazd University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Farzaneh","middleName":"","lastName":"Hoseynidokht","suffix":""}],"badges":[],"createdAt":"2024-02-27 18:21:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3994610/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3994610/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51824634,"identity":"36047a81-e082-408a-99ae-33bfab7ffcdd","added_by":"auto","created_at":"2024-02-29 16:39:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":343561,"visible":true,"origin":"","legend":"\u003cp\u003eFESEM images of (A and B) Nickel nanofoam, (C) rGO/Nickel nanofoam, (D) AuNPs@ rGO/Nickel nanofoam\u003c/p\u003e","description":"","filename":"Onlinefloatimage212.png","url":"https://assets-eu.researchsquare.com/files/rs-3994610/v1/545fc495b94bc746234a85b3.png"},{"id":51824633,"identity":"8a7aa045-7914-4c7a-88f8-ccc82e4144ce","added_by":"auto","created_at":"2024-02-29 16:39:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":32572,"visible":true,"origin":"","legend":"\u003cp\u003e(A) XRD pattern of Nickel nanofoam, (B) The N\u003csub\u003e2\u003c/sub\u003e adsorption-desorption isotherm of Nickel nanofoam, (C) BJH analysis of Nickel nanofoam, (D) XRD pattern of AuNPs@ rGO/Nickel nanofoam\u003c/p\u003e","description":"","filename":"Onlinefloatimage312.png","url":"https://assets-eu.researchsquare.com/files/rs-3994610/v1/ce99cba3463ec9671a1b8e94.png"},{"id":51825046,"identity":"286ec34f-658f-4f50-897e-6b839fac15ff","added_by":"auto","created_at":"2024-02-29 16:47:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":231073,"visible":true,"origin":"","legend":"\u003cp\u003e(A) EDS spectra of AuNPs@ rGO/Nickel nanofoam, (B) SEM-mapping of AuNPs@ rGO/Nickel nanofoam\u003c/p\u003e","description":"","filename":"Onlinefloatimage414.png","url":"https://assets-eu.researchsquare.com/files/rs-3994610/v1/beabe75be50d08d7c885d54c.png"},{"id":51824636,"identity":"3951abc7-1004-42ae-84e6-d962ff8d4607","added_by":"auto","created_at":"2024-02-29 16:39:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20762,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Cyclic voltammograms at scan rate 0.1 V/s (B) Nyquist diagram in a frequency range of 100 - 10\u003csup\u003e5\u003c/sup\u003e Hz, of (a) bare electrode (carbon ink), (b) rGO/Nickel nanofoam electrode, (c) AuNPs@ rGO/Nickel nanofoam electrode, (d) Ab/AuNPs@ rGO/Nickel nanofoam electrode, (e) exosome/Ab/AuNPs@rGO/Nickelnanofoam\u003c/p\u003e","description":"","filename":"Onlinefloatimage512.png","url":"https://assets-eu.researchsquare.com/files/rs-3994610/v1/4bffee0021de80514fb3dad9.png"},{"id":51824640,"identity":"e267972a-1593-41a2-8298-b34fb06727dc","added_by":"auto","created_at":"2024-02-29 16:39:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":12993,"visible":true,"origin":"","legend":"\u003cp\u003e(A) DPV responses of the Exo-sensing paper to different concentration of exosome (B) curve of current change vs. the logarithmic value of exosome concentration (Y= 26.44 ± 0.29 -3.50 ± 0.068 Log [exosome], R\u003csup\u003e2\u003c/sup\u003e =0.995). The electrochemical tests were performed at 5 mM [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3-/4- \u003c/sup\u003eand 0.1 M KCl\u003c/p\u003e","description":"","filename":"Onlinefloatimage66.png","url":"https://assets-eu.researchsquare.com/files/rs-3994610/v1/9d54c3a52e15638dcf1dffa2.png"},{"id":51824635,"identity":"812854c1-b3c0-4c67-8b37-00f6e5823020","added_by":"auto","created_at":"2024-02-29 16:39:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8710,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Peak current of DPV exosome and other interferences. (B) Detection of exosome in the clinical samples (H1-2\u003csub\u003e\u0026nbsp; \u003c/sub\u003ehealthy and P1-4 patient individuals, n=3)\u003c/p\u003e","description":"","filename":"Onlinefloatimage72.png","url":"https://assets-eu.researchsquare.com/files/rs-3994610/v1/31e2db27cadc5f6fbd54a54e.png"},{"id":51825231,"identity":"6edff6d5-eb60-4b97-b1f6-cb884dad8e2b","added_by":"auto","created_at":"2024-02-29 16:55:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1064904,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3994610/v1/4901f0ec-05fc-4c7f-a05c-296862f8d04d.pdf"},{"id":51824637,"identity":"c2b78d53-47b1-4790-8909-06963851215d","added_by":"auto","created_at":"2024-02-29 16:39:19","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":50110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e The processes of Nickel nanofoam construction\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3994610/v1/5c4e81c6645a9c2fe1b6cbaa.png"},{"id":51824638,"identity":"bb762f19-c4b9-4f15-bc27-a6f3a99f6b12","added_by":"auto","created_at":"2024-02-29 16:39:19","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":287673,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-3994610/v1/ee9eebb00c0e47133732fd2d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Flexible electrochemical paper-based device for detection of breast cancer- derived exosome using nickel nanofoam 3D nanocomposite","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eExosomes are nanometer extracellular vesicles containing two lipid layers and a large number of biological molecules such as proteins, lipids, carbohydrates and nucleic acids [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They are secreted by all types of cells and are widely existence in different fluids of human body and mainly play a key role in the intercellular communication and indicate a specific molecular profile related to the origin of the parent cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Notably, the existence and progression of some cancers to the types and levels of Exosome membrane proteins are strongly related. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, exosomes containing specific proteins are a main source of new biomarkers in diagnosis and prognosis of disease. Some common detection methods of exosomal protein are mass spectrometry [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], enzyme-linked immunosorbent assay (ELISA) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], surface plasmon resonance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and western blot (WB) analysis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These methods are usually complex, boring and long pretreatment. In addition, the sensitivity of these methods is not sufficient for detection of small surfaces of exosomal proteins. Point of care testing (POCT) is a novel technology for the immediate analysis of clinical samples at the sampling situation without complex processing, which can possess a high potential for diagnosis and monitoring of cancer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Based on the advantages of POCT technology, some methods have been expanded to determine exosomes, which have high feasibility but low sensitivity or require specialized equipment [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, improving a sensitive platform with a simple and low-cost POCT protocol for detection of exosomes is as a challenge.\u003c/p\u003e \u003cp\u003eNowadays, paper-based sensors have been developed in bioanalytical applications [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Due to this fact that the cellulose papers are cheap, light, available, biodegradable, and easily assembled, a series of POCT based on the paper-based devices have been developed for colorimetric [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], fluorescence [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and electrochemiluminescence [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] techniques. Electrochemical methods have attractive properties of simple measurement protocol, high flexibility and short detection time, as well as miniaturization and portability, which make them very suited for the evolution of POCT technologies [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition, some modified electrodes can be used without complex equipment, which are particularly suitable for the development of portable POCT instruments. For example, Liu et al developed a paper POCT aptasensor for exosome quantification using Zr-MOFs and aptamer as detection system along with chain reaction hybridization for signal amplification[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Also, Su et al designed a smartphone-based biosensor for measurement of exosome in serum using polyenzymatic signal amplification based on double-antibody sandwich method [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. To promote the performance of paper POCT electrochemical devices, the nanomaterials of carbon, metal oxides and metals have been modified on the paper surface [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, the three-dimensional porous metal nanofoams are widely used in electrochemistry field as a conductive materials with high catalytic properties and high specific surface area [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The porous nanofoam of nickel contains the properties of metals such as electrical and thermal conductivity and catalytic activity along with the extraordinary properties of nanostructure materials such as low density and high specific surface area, which leads to an increase in its properties specially increasing the active sites and catalytic activity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For example, Deng et al. designed a biosensor based on decorated nickel foam with ZIF-8/CuO- for glucose determination [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The electrochemical analysis revealed that the electrochemical active surface of the electrode increased twice while the electrical resistance decreased during one week. So, various metal nanofoams have been used in electrochemical sensors to immobilize various biomolecules for detection of cancer biomarkers.\u003c/p\u003e \u003cp\u003eIn this study, carbon and silver ink (constructed in the laboratory) were printed on the paper pattern of a three-electrode system to prepare a paper-based electrochemical immunosensor (exo-sensing paper). Then, Au@rGO/Ni nanofoam nanocomposite was placed on the electrode surface of carbon ink to improve its conductivity. For the synthesis of the modifier, first, three-dimensional nickel nanofoam was prepared as a substrate using an easy and cheap method without a pattern. The size of the porous and the diameter of the obtained nickel nanofoam are much smaller than the commercial samples, and therefore it can accelerate the electron and ion transfer. Then, graphene oxide was placed on the nickel nanofoam as an anti-corrosion of metal and a three-dimensional network of graphene oxide was created using nickel nanofoam as the sub layer substrate. Finally, gold nanoparticles were prepared on the rGO/Ninanofoam nanocomposite by in situ reduction using ascorbic acid and a displacement reaction with nickel without using a surfactant. This nanocomposite offers a good potential in biosensor and catalytic applications due to the strong structure of nickel foam and good recyclability. Next, Anti-CD9 antibody was immobilized on the electrode surface and exposed to different concentrations of exosome extracted from cancer cell and finally the human serum. The exo-sensing paper has some advantages like simplification for general uses, low cost and the competency of the biosensor. Exo-sensing paper is developed based on the use of paper which leads to the mass production with good repeatability, cheap and disposable properties. Also, the used carbon ink to make the electrodes has a simple synthesis and is very cheap. The obtained low detection limit and wide linear range confirm the high sensitivity of this immunosensor platform as POCT for using in real samples.\u003c/p\u003e"},{"header":"2. Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials and devices\u003c/h2\u003e \u003cp\u003eGraphite powder, hydroxyethyl cellulose (HEC), 2-methoxyethanol, cellulose acetate, ascorbic acid, poly (acrylic acid), silver nitrate, hydrogen tetrachloroauate (AuHCl\u003csub\u003e4\u003c/sub\u003e.3H2O), Nickel nitrate, potassium ferrocyanide (K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e, potassium ferricyanide (K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e, iron (III) chloride, potassium chloride (KCl), acetone, methanol, 2-methoxyethanol, cyclohexanone, nitric acid and sulfuric acid were purchased from Merck Co or Sigma Chemical Co. Mouse monoclonal α-human CD9 antibody (Ab) was obtained from R\u0026amp;D systems.\u003c/p\u003e \u003cp\u003eThe electrochemical tests were performed on the electrochemical workstation (Metrohm AutoLab PGSTAT-302, Netherlands). A paper-based three-electrode configuration was usage, containing the working and auxiliary electrodes (carbon ink) and a reference electrode (silver ink). The laser cutter was usage for the fabrication of the pattern of electrodes (Mini Laser BCL-MU, Jinan Bodor CNC Machine Co, China). Ultracentrifuging was done with a Beckman Coulter Optima XPN-90 with the SW40 Ti rotor at 4\u0026deg;C. Cyclic voltammetry measurements were done in the electrolyte solution containing a redox system (Fe(CN)\u003csub\u003e6\u003c/sub\u003e \u003csup\u003e\u0026minus;3/\u0026minus;4\u003c/sup\u003e with a scan rate of 0.1 V/s and in the potential range of -0.6 to +\u0026thinsp;0.8 V. Electrochemical impedance spectroscopy (EIS) was experimented in the frequency range of 100 Hz to 10\u003csup\u003e5\u003c/sup\u003e Hz. The main measurements were performed in the same solution with the iron probe monitoring using the differential pulse voltammetry (DPV) technique in the potential range of -0.4V to +\u0026thinsp;0.4V.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell culture\u003c/h2\u003e \u003cp\u003eExosomes were purified from serum-free culture media of MCF-7 cell as described in the previous work [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Briefly, MCF-7 cells were grown in the culture medium of DMEM with penicillin/streptomycin (1%) without bovine serum for 72 h at 37\u0026deg;C and wet atmosphere (CO\u003csub\u003e2\u003c/sub\u003e 5%). Exosomes were isolated from the serum-free supernatant using an ultracentrifuge. First, the Exosomes were centrifuged for 10 min (1200g) and then 10000g for 30 min to remove the cell remains and large vesicles. Then the supernatants were centrifuged at 100,000g for 2 h to collect the Exosomes. The exosome plates were re suspended in 100 \u0026micro;l of PBS and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. The synthesis of nickel nanofoams\u003c/h2\u003e \u003cp\u003eNickel nanofoams were synthesized without pattern by combustion process. For synthesis, (NiNO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO (0.05 M) was poured in 50 mL of 2-methoxyethanol (C\u003csub\u003e3\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) as a solvent and reducing agent and stirred for 30 min until it was dissolved completely. The obtained solution was stirred for 2 h at 80\u0026deg;C in an oil bath and then dried to obtain a dark colored colloidal precursor. Subsequently, the precursor colloid was heated to 180\u0026deg;C in an oven and it was burned vigorously in seconds to obtain the nickel foams. The combustion products of the solvent (C\u003csub\u003e3\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) in the atmosphere contain carbon dioxide and large amounts of carbon monoxide, which can convert the divalent nickel ions (Ni\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e) into nickel (Ni) and create the porous nickel foams of open and continuous cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. rGO/Ninanofoam and AuNPs@rGO/Ninanofoam synthesis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eGraphene oxide was prepared by the modified Homer method [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The synthesized nickel nanofoam was placed in a centrifuge tube containing dispersed graphene oxide (4 mg/mL), and centrifuged at 50 rmp for 15 min to fill the pores with GO and the graphene oxide sheets cover the continuous network of nickel nanofoams. The wet nickel nanofoam dried at room temperature and this process was repeated several times to increase the loading of rGO. To prepare the AuNPs@rGO/Ninanofoam nanocomposite, 1 mL of ascorbic acid solution (1 mM) was added to rGo/Ninanofoam suspension (5 mg/mL) at 80\u0026deg;C. The solution of AuHCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO (5 mM) was added drop by drop to this suspension while stirring to form gold nanoparticles outside and inside of the holes. Finally, the nanocomposite was purified by centrifugation and then washed with ethanol and water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Preparation of the Exo-sensing paper\u003c/h2\u003e \u003cp\u003eIn this work, Exo-sensing papers were made by prototyping technique. First, the pattern of the paper-based device was designed, and then the patterns were prepared on a self-adhesive glass paper by laser cutting as a hydrophobic border. The patterns were pasted on a filter paper (Whatman number 1). For a three-electrode system on Exo-sensing papers, a working electrode (WE) with a standard deviation of 2.8% and area\u0026thinsp;=\u0026thinsp;0.04 cm2), an auxiliary electrode (CE) and their connections using carbon ink and a reference electrode (RE) using silver ink was printed. The Exo-sensing papers were dried in an oven at 55\u0026deg;C. The carbon ink was made by mixing graphite powder (0.5 g) and 1.25 g of cellulose acetate solution (7.5% w in cyclohexane and acetone) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Silver ink was synthesized by reduction of silver nitrate by poly acrylic acid (PAA) and diethanolamine (DEA) as a surface trapping agent in an aqueous solution in several steps. Briefly, 1 g of PAA was mixed with 20 g of DEA in 25 mL of water for 2 h. Then 10 ml of silver nitrate solution (0.06 mol) was slowly added to the above solution and stirred for 24 h. To increase the size of silver nanoparticles, it was ultra sonicated for 90 min. To coagulate the silver ink, the solution was titrated with ethanol. Finally, to purify and separate PAA, it was washed with water and then centrifuged at 9000 rpm [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The details of the synthesis of carbon and silver inks and the preparation of the Ag/AgCl reference electrode have been described in detail in our previous work [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Then, 4 \u0026micro;L of AuNPs@rGO/Ninanofoam porous nanocomposite (5 mg/mL) was casted on the surface of the carbon working electrode. Then 2 \u0026micro;L of CD9 antibody (5 ng /\u0026micro;L) was immobilized on the surface of the modified working electrode and the electrode was placed for 1 h in a wet atmosphere at 4\u0026deg;C to form the binding with the modified electrode surface. For each step, the electrode surface was washed with phosphate buffer solution to remove un-bounded species. Next, the electrode surface was exposed to 1 \u0026micro;L of BSA solution for 15 min, to block the remaining possible sites on the modified electrode surface to prevent non-specific binding. Eventually, 1.5 \u0026micro;L of different concentrations of the exosome solution was placed on the surface of the fabricated Exo-sensing paper for 30 min, and the differential pulse voltammetry curves in the presence of redox [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e\u0026minus;3/\u0026minus;4\u003c/sup\u003e solution with potassium chloride electrolyte (0.1 M) were recorded in the potential range of -0.4 V to +\u0026thinsp;0.4 V. The electrical repulsion between the modified surface and the probe, as well as the design of the modified electrode, prevents the transfer of electrons from the probe to the electrode surface and leads to decreasing the current. DPV currents were recorded before and after trapping the exosomes by antibody and this decrease in the analytical signal which is proportional to the exosome concentration can be act a confirmation of the good potential of this platform to detect exosomes. CV and EIS measurements were also performed at each steps of the surface modification, which confirms the successful modification of each step of the antibody and exosome binding.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of the designed biosensor\u003c/h2\u003e \u003cp\u003eThe processes of nickel nanofoam and AuNPs@rGO/Nickelnanofoam nanocomposite construction are schematically shown in Scheme 1. During the annealing of the colloidal precursor, due to the use of 2-methoxyethanol solvent, a large amount of carbon monoxide is produced which can cause reduction of the divalent nickel ion (Ni\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e) to nickel (Ni). As shown in this figure, the dark nickel nanofoams are very soft and light. Nickel nanofoams were fabricated by annealing Ni(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e colloid and C\u003csub\u003e3\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in air by using an easy and template-free approach. This fabrication process is very low cost and can be used for large scale.\u003c/p\u003e \u003cp\u003e \u003cb\u003eScheme 1.\u003c/b\u003e The processes of Nickel nanofoam construction\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicates the morphology of the prepared nickel nanofoam. The FE-SEM images presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B confirms that the sample has a three-dimensional continuous network structure. The skeleton diameter is 200\u0026ndash;300 nm and the pore size is 150\u0026ndash;350 nm, which is much smaller than the pore size of commercial foams. Due to the homogeneous structure of the nanofoam, the prepared nickel nanofoam has a much lower density and a high specific surface area. This nickel nanofoam as a current collector can be useful for improving the electrochemical performance of the electrode. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC displays the attached graphene layers on the nickel nanofoam surface. A large number of wrinkles and waves are observed on the graphene sheets indicating a good binding between graphene oxide and nickel nanofoam skeleton. Also, due to the difference in the thermal expansion coefficient between graphene and nickel nanofoam these wrinkles and waves may have been created. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD shows the three-dimensional image of gold nanoparticles that are dispersed throughout the rGO/nickel nanofoam substrate, uniformly.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows the X-ray diffraction (XRD) pattern of nickel nanofoam. The three diffraction peaks are (111) 44.5\u0026deg;, (200) 52\u0026deg; and (222) 76.5\u0026deg;, respectively, which are the characteristic of cubic nickel crystal plates (JCPDS card 04-0850). There is no sign of other crystalline phases indicating that the nickel nanofoam has a relatively high purity. The adsorption-desorption isotherm of N\u003csub\u003e2\u003c/sub\u003e was usage to investigate the structure of Ni nanofoam (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These isotherms show a type of mesopore hysteresis loop with absorption and desorption branches at high relative pressures. According to the IUPAC patterns, the structure of type IV is taken from the isotherm curves indicating a porous structure for synthetized nickel nanofoam [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. High specific surface area and pores volume of (1975 m\u003csup\u003e2\u003c/sup\u003e/g and 3.5 cm\u003csup\u003e3\u003c/sup\u003e/g) were obtained from these isotherms, which are also related to BET data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The pores diameter average of nickel nanofoams was acquired to be 5.3 nm by BJH analysis that is in good agreement with other analyzes including FE-SEM. The crystal structure of AuNPs@rGO/Ninanofoam nanocomposite was analyzed by X-ray diffraction. As can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, in addition to the peaks related to the diffraction of the nickel crystal, the specific peaks at 38.5 2 θ have also appeared, which confirms the formation of gold nanostructured crystal.\u003c/p\u003e \u003cp\u003eAlso, a wide peak with low height is related to rGO deposited on the nickel nanofoam with an amorphous structure [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The diffraction pattern of AuNPs@rGO/Nickel nanofoam shows that the gold nanoparticles on the rGO/Nickel nanofoam have a relatively high crystallinity. To confirm the components of AuNPs@rGO/Nickel nanofoam nanocomposite and also to confirm the formation of AuNPs on rGO/Nickel nanofoam, the elemental analysis by X-ray energy diffraction spectroscopy (EDS) was also performed. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows the related peaks to the nanocomposite elements. EDS analysis was obtained to be Ni 56%, C 30%, Au 12% and oxygen 3%. In addition, elemental mapping was done to discover the distribution of the constituent components of the nanocomposite. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, a uniform distribution of all elements in the composition of the nanocomposite especially for gold nano particles can be seen. EDX mapping analysis as well as XRD confirms the successful synthesis of AuNPs@rGO/Nickel nanofoam nanocomposite.\u003c/p\u003e \u003cp\u003eIn the synthesis of this nanocomposite, the nickel nanofoam as a substrate acts as a support frame for rGO and AuNPs, and also is easily recyclable. This advantage is very important for paper-based electrodes. Also, it was used as a reducing agent in the synthesis of gold nanoparticles. AuNPs can be formed by the following substitution reaction:\u003c/p\u003e \u003cp\u003eNi\u0026thinsp;+\u0026thinsp;Au\u003csup\u003e+\u0026thinsp;3\u003c/sup\u003e \u0026rarr; Ni\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e+ Au\u003c/p\u003e \u003cp\u003eOf course, some gold nanoparticles are also obtained during the reduction of GO. The high activity of AuNPs@rGO/Nickel nanofoam can be related to the synthesis method of gold nanoparticles prepared without surfactants and therefore they can expose their active sites, completely. Also, the nickel nanofoam along with graphene oxide create a good stability and conductivity and also an increase in the electrons transfer [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Electrodes with this kind of modified structures can provide multiple directions for electron transfer and lead to increasing the effective loading of materials (biomolecules such as antibodies, enzyme, aptamers, etc.) and consequently increase of the linear range, effectively. The nanocomposite of AuNPs@rGO/Nickel nanofoam was synthesized by a green, easy and efficient method. The prepared nickel nanofoam was light and flexible, and the fabrication method was easy and template-free, too. Gold nanoparticles are without chemical surfactants and distributed on the surface of rGO/Nickel nanofoam uniformly with a good performance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. The electrochemical behavior of electrodes\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAn electrochemical immunosensor (three-electrode) paper-based device was made with a patterned sticker on a filter paper coated with paraffin (on one side). This patterned sticker creates hydrophobic limits around the electrodes. The thickness of the electrodes depends on the number of ink print on the paper, which can be controlled by the thickness of the stencil (100 \u0026micro;m). A very simple and cheap method that also can use for the mass production of paper-based devices. The working electrode of carbon ink was modified with AuNPs@rGO/Nickel nanofoam to prepare an activated three-electrode system. The electrochemical properties of the prepared Exo-sensing paper were investigated by cyclic voltammetry in the presence of [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e as probe in the range of -0.6 V to +\u0026thinsp;0.8 V. The obtained cyclic voltammogram of the carbon ink (working electrode) surface with a pair of redox peaks (ΔEp\u0026thinsp;=\u0026thinsp;129 mV) suggests that the electrochemical behavior of probe on the carbon ink surface has a semi-reversible behavior. Also, the stability of the products of redox probe on the surface of carbon ink was studied by testing the ratio of anodic to cathodic peak currents. To obtain a good understanding of mass transfer on the working electrode surface, the electrochemical characteristics of Exo-sensing paper were investigated and compared using the cyclic voltammetry method at different scan rates before and after modification of the working electrode surface with AuNPs@rGO/Nickel nanofoam nanocomposite (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). The activated surface of the working electrode was analyzed according to the slope of the anodic peak currents diagram versus the square of the scan rate (inset in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2) according to the Randles-Sevcik equation:\u003c/p\u003e \u003cp\u003eI\u003csub\u003epa\u003c/sub\u003e = (2.69 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) n\u003csup\u003e2/3\u003c/sup\u003e A\u003csub\u003eeff\u003c/sub\u003e D\u003csup\u003e1/2\u003c/sup\u003e ʋ\u003csup\u003e1/2\u003c/sup\u003e C\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003cp\u003ewhere n is the number of transferred electrons, A is the specific active surface, I is the anode peak currents at different scan rates of the working electrode, C is the concentration of K\u003csub\u003e4\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e, D is the diffusion coefficient of iron probe and ʋ is the scan rate [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The results showed that the effective surface of the working electrode was improved 42% after modification.\u003c/p\u003e \u003cp\u003eA paper-based electrochemical immunosensor (Exo-sensing paper) based on AuNPs/rGO/Nickel nanofoam was developed for exosome with CD9 surface marker detection. CV was used for investigation of the preparation process of the Exo-sensing paper, and the experiments were done in a solution containing K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]/K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e] (5 mM) and KCl (0.1 M) at a potential of -0.6 to +\u0026thinsp;0.8 V. The obtained CV curves of modified electrodes with different nanocomposits and biomolecules are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Compared to the bare electrode (carbon ink), the rGO/Nickel nanofoam electrode revealcs a higher current signal, and probably due to the higher conductivity of rGO. The electrode AuNPs@rGO/Nickel nanofoam, shows an increase of the current signal, which is due to the higher conductivity of nanomaterials of nanocomposite, especially gold nanoparticles. Due to low conductivity of immunoproteins, the current signal of Ab/AuNPs@rGO/Nikel nanofoam electrode is decreased. In addition, after incubation with exosome, the current signal of the exosome/Ab/AuNPs@rGO/Nickel nanofoam electrode is decreased more strongly due to the formation of immune complexes on the surface of the electrode by prevention of the electron transfer.\u003c/p\u003e \u003cp\u003eAlso, electrochemical impedance spectroscopy (ESI) was performed to investigate the surface characteristics of the modified electrodes at different stages. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, the ESI spectra of different surface electrode modification are different. The electron transfer resistance (Rct) value of the bare electrode (carbon ink) has a large semicircle. After modifying the electrode surface with nanocomposite of rGO/Nickel nanofoam and AuNPs@rGO/Nickel nanofoam, the semicircles became smaller indicating less electron transfer resistance in the modified electrode surfaces. As shown in this figure the impedance values of the Exo-sensing paper are increased by layer-by-layer modification with antibody and Exosome. It can be related to this fact that by introduction of the macromolecular proteins to the surface of the electrodes, the capacity of transfer electrons on the electrode surfaces are decreased. The results of ESI analysis have a good agreement with the CV signals, which indicates the successful fabrication of this Exo-sensing paper.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3. The optimization of effective parameters for exosome detection\u003c/h2\u003e \u003cp\u003eTo obtain a high performance for the Exo-sensing paper, some effective parameters such as the concentration of nanocomposite, antibody concentration, and the binding time of antibody to the electrode surface were optimized. The concentration of the modifier on the electrode surface significantly affects the detection performance. Because the different concentrations of modifier can affect the catalytic activity as well as the conductivity of the electrode. The electrochemical current signal increases by increasing the concentration of the modifier suspension up to 10 mg/mL, as shown in Figure S3. The incubation time of anti-CD9 antibody on the AuNPs@rGO/Nickel nanofoam was tested at different times. As shown in Figure S4, the DPV currents are decreased by increasing the antibody incubation time until the peak current be constant value at 60 min, and therefore, 60 min was chosen as an optimum value for the construction of the Exo-sensing paper. The results of the effect of antibody concentration on DPV signal rate are displayed in Figure S5. As the antibody concentration increased from 500 pg/mL to 100 ng/mL, the currents are decreased and reached a constant value at 5 ng/mL. As a result, the antibody concentration was optimized and the optimum concentration was considered to be 5 ng/mL. However, the incubation time of the exosome was evaluated for the complete formation of the antibody-exosome complex. The peak currents of DPVs are decreased by increasing the incubation time and becomes constant at 30 min. Therefore, 30 min was chosen as the optimum incubation time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4. The analytical performance of Exo-sensing paper for exosome detection\u003c/h2\u003e \u003cp\u003eUnder optimum conditions, the electrochemical performance of the Exo-sensing paper was analyzed by incubation of the exosome with different concentrations on the Ab/AuNPs@rGO/Nickel nanofoam electrode surface. The responses of DPV currents were decreased by increasing the exosome concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The obtained ΔI values displayed a good linear relationship with the logarithm of the exosome concentration in the range of 500 to 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e exosome/\u0026micro;L, with a correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e) of 0.98 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The calculated detection limit was obtained to be 110 exosome/\u0026micro;L. The performance of the constructed Exo-sensing paper was compared with the previous methods in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This table shows that the proposed method has shown a good performance. This better performance can be related to the specific and excellent characteristics of nanocomposite and the synergistic effect of these three components in the nanocomposite structure. Porous nickel nanofoam has good conductivity, high stability and large specific surface area and therefore it is a very suitable substrate for electrocatalytic activities in combination with graphene oxide and gold nanoparticles. In addition, the process of an Exo-sensing paper fabrication is low-cost and relatively simple. Also, this Exo-sensing paper based on disposable paper is biodegradable and easy to use and has a short analysis time. Therefore, the use of this POCT in clinical diagnosis is suggested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Repeatability, selectivity and stability of the Exo-sensing paper\u003c/h2\u003e \u003cp\u003eTo investigate the selectivity of the designed Exo-sensing paper, the derived exosomes from MCF-7 cell were detected with interfering substances such as microvesicles, BSA, AFP and glucose with the same concentration (5ng/mL) using the same method. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, it is clear that the DPV response to exosomes is greater than the response to interferences, which can be due to exosome-antibody interaction. The detection performance in the biological environment was also evaluated. As shown in Figure S6, the obtained results from human serum without exosome are almost similar to phosphate buffer solution, which indicates that the proposed biosensor can work well in complex physiological samples.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe comparison of performance of the Exo-sensing paper with other exosome detection biosensors\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetection Methods\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLinear Range\u003c/p\u003e \u003cp\u003e(Exosome/\u0026micro;L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDetection Limit\u003c/p\u003e \u003cp\u003e(Exosome/\u0026micro;L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChronoamprometry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u0026thinsp;\u0026minus;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDifferential pulse voltammetry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e2\u003c/sup\u003e \u0026ndash; 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectrochemiluminescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u0026ndash;5\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChronoamprometry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e2\u003c/sup\u003e \u0026ndash; 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDifferential pulse voltammetry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.7\u0026times;10\u003csup\u003e5\u003c/sup\u003e- 3.4\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026times;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDifferential pulse voltammetry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u0026thinsp;\u0026minus;\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAlso the repeatability of the Exo-sensing paper was investigated by producing five identical Exo-sensing paper and incubating them with the same concentration of Exosome (1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e exosome/\u0026micro;L). There was no significant difference in the current responses among these fabricated electrodes and the RSD was less than 7%. Another the important factor for the detection of exosomes is stability of electrode so, a continuous CV scans were used to study the stability of the Exo-sensing paper. After 10 CV scans, 97% of the initial current values were still detected. Furthermore, Exo-sensing paper retained 89% of the initial current value after 14 days at 4\u0026deg;C, indicating its long-term stability (Figure S7).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.6. The clinical test of the constructed Exo-sensing paper\u003c/h2\u003e \u003cp\u003eTo evaluate the efficiency of the clinical performance of the proposed method in real samples, the Exo-sensing paper was utilized to determine the exosomes in the serum samples of breast cancer patients and healthy. Human serum from two healthy human and four patients were collected for analysis using the fabricated Exo-sensing paper. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, the DPV responses of patient's serums were higher than healthy subjects. The obtained RSD of the samples was less than 8.5%. The obtained results from the constructed Exo-sensing paper were compared with the ELISA method. The t-test (0.64, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) did not show any significant difference between the obtained results by Exo-sensing paper and ELISA. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the results obtained from both methods. The developed Exo-sensing paper may be useful in clinical diagnosis and even have better diagnostic efficiency due to its wider linear range and lower detection limit than ELISA.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe obtained results of ELAISA and Exo-sensing paper\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003esample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eELAISA (exosome/\u0026micro;L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExo-sensing paper (exosome/\u0026micro;L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1427\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2190\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55863\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e56792\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e239764\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e241466\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e310374\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e314154\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e397259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e408722\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn summary, an electrochemical immunosensor based on a coupled paper with disposable nickel nanofoam for the selective and sensitive detection of exosomes derived from cancer cells was developed. A three-electrode system was prepared using a pattern paper on the filter paper along with carbon and silver inks. AuNPs@rGO/ckel nanofoam nanocomposite was placed on the electrode surface to achieve signal amplification and effective binding of antibody to the electrode surface. The flexible nickel nanofoam was prepared by a pattern-free and very simple method. Graphene oxide and 3D network of very light nickel nanofoam were deeply integrated without any additives and using a simple strategy, and finally AuNPs@rGO/Nickle nanofoam nanocomposite was prepared by an easy and efficient method without using chemical surfactants. The high specific surface area of the nanocomposite effectively increases the binding of antibody to the electrode surface and clearly increases the electric signal of the electrode and then leads to increasing the sensitivity and selectivity of the exosome detection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCredit author statementN. Sahraei: Idea, synthesize experiment and writing - original draft.F. Hoseynidokht and A. Moradi: Experiment and calculation.M. Mazloum-Ardakani: Idea, editing and writing - original draft.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThe authors appreciate the financial assistance of the Iran National Science Foundation (INSF), Yazd University Research Council for this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLuo S, Wu Y, Pan W et al (2023) An integrated magneto-fluorescent nanosensor for rapid and sensitive detection of tumor-derived exosomes. Sens Actuators B 374:132792. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acsami.3c01607\u003c/span\u003e\u003cspan address=\"10.1021/acsami.3c01607\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim J, Kang B, Son HY et al (2022) Microfluidic device for one-step detection of breast cancer-derived exosomal mRNA in blood using signal-amplifiable 3D nanostructure. Biosens Bioelectron 197:113753. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bios.2021.113753\u003c/span\u003e\u003cspan address=\"10.1016/j.bios.2021.113753\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu H, Cai X, Zhang Q et al (2021) A Design of Precise Protection System for Endangered Species Based on Beidou\u0026thinsp;+\u0026thinsp;4G. In: 2021 International Conference on Electronic Information Technology and Smart Agriculture (ICEITSA). IEEE, pp 71\u0026ndash;75 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1109/ICEITSA54226.2021.00023\u003c/span\u003e\u003cspan address=\"10.1109/ICEITSA54226.2021.00023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeBleu VS, Kalluri R (2020) Exosomes as a multicomponent biomarker platform in cancer. Trends cancer 6:767\u0026ndash;774. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.trecan.2020.03.007\u003c/span\u003e\u003cspan address=\"10.1016/j.trecan.2020.03.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X-W, Liu M-X, He M-Q et al (2021) Integral multielement signals by DNA-programmed UCNP\u0026ndash;AuNP nanosatellite assemblies for ultrasensitive ICP\u0026ndash;MS detection of exosomal proteins and cancer identification. Anal Chem 93:6437\u0026ndash;6445. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.1c00152\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.1c00152\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen G, Huang AC, Zhang W et al (2018) Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature 560:382\u0026ndash;386. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-018-0392-8\u003c/span\u003e\u003cspan address=\"10.1038/s41586-018-0392-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu W, Yu X, Wu J et al (2021) Surface plasmon resonance imaging-based biosensor for multiplex and ultrasensitive detection of NSCLC-associated exosomal miRNAs using DNA programmed heterostructure of Au-on-Ag. Biosens Bioelectron 175:112835. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bios.2020.112835\u003c/span\u003e\u003cspan address=\"10.1016/j.bios.2020.112835\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao H, Im H, Castro CM et al (2018) New technologies for analysis of extracellular vesicles. Chem Rev 118:1917\u0026ndash;1950. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.chemrev.7b00534\u003c/span\u003e\u003cspan address=\"10.1021/acs.chemrev.7b00534\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosa BG, Akingbade OE, Guo X et al (2022) Multiplexed immunosensors for point-of-care diagnostic applications. Biosens Bioelectron 203:114050. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bios.2022.114050\u003c/span\u003e\u003cspan address=\"10.1016/j.bios.2022.114050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahraei N, Mazloum-Ardakani M, Hoseynidokht F (2022) Electrochemical paper-based biosensors for point-of-care diagnostics: Detection methods and applications. J Electrochem Sci Eng. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5599/jese.1104\u003c/span\u003e\u003cspan address=\"10.5599/jese.1104\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChattopadhyay S, Ram R, Sarkar A, Chakraborty S (2022) Smartphone-based automated estimation of plasma creatinine from finger-pricked blood on a paper strip via single-user step sample-to-result integration. Measurement 199:111492. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.1c04910\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.1c04910\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira-Rodr\u0026iacute;guez M, Serrano-Pertierra E, Garc\u0026iacute;a AC et al (2017) Point-of-care detection of extracellular vesicles: sensitivity optimization and multiple-target detection. Biosens Bioelectron 87:38\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bios.2016.08.001\u003c/span\u003e\u003cspan address=\"10.1016/j.bios.2016.08.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark J, Park JS, Huang C-H et al (2021) An integrated magneto-electrochemical device for the rapid profiling of tumour extracellular vesicles from blood plasma. Nat biomedical Eng 5:678\u0026ndash;689. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41551-021-00752-7\u003c/span\u003e\u003cspan address=\"10.1038/s41551-021-00752-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eda Silva VAOP, de Freitas RC, de Oliveira PR et al (2020) Microfluidic paper-based device integrated with smartphone for point-of-use colorimetric monitoring of water quality index. Measurement 164:108085. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.measurement.2020.108085\u003c/span\u003e\u003cspan address=\"10.1016/j.measurement.2020.108085\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShu Y, Su T, Lu Q et al (2022) Paper-based electrochemical immunosensor device via Ni-Co MOF nanosheet as a peroxidase mimic for the label-free detection of alpha-fetoprotein. Sens Actuators B 373:132736. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.snb.2022.132736\u003c/span\u003e\u003cspan address=\"10.1016/j.snb.2022.132736\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu D, Zhang Y, Zhu M et al (2020) Microfluidic-Integrated Multicolor Immunosensor for Visual Detection of HIV-1 p24 Antigen with the Naked Eye. Anal Chem 92:11826\u0026ndash;11833. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.0c02091\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.0c02091\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Jiang C, Jin J et al (2021) Ratiometric Fluorescent Lateral Flow Immunoassay for Point-of‐Care Testing of Acute Myocardial Infarction. Angew Chem 133:13152\u0026ndash;13159. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ange.202103458\u003c/span\u003e\u003cspan address=\"10.1002/ange.202103458\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdhikari J, Rizwan M, Dennany L, Ahmed MU (2021) Electrochemiluminescence nanoimmunosensor for CD63 protein using a carbon nanochips/iron oxide/nafion-nanocomposite modified mesoporous carbon interface. Measurement 170:108755. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.measurement.2020.108755\u003c/span\u003e\u003cspan address=\"10.1016/j.measurement.2020.108755\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu L, Shoaie N, Jahanpeyma F et al (2020) Optical, electrochemical and electrical (nano)biosensors for detection of exosomes: A comprehensive overview. Biosens Bioelectron 161:112222. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.bios.2020.112222\u003c/span\u003e\u003cspan address=\"10.1016/j.bios.2020.112222\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoonkaew S, Chaiyo S, Jampasa S et al (2019) An origami paper-based electrochemical immunoassay for the C-reactive protein using a screen-printed carbon electrode modified with graphene and gold nanoparticles. Microchim Acta 186:1\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00604-019-3245-8\u003c/span\u003e\u003cspan address=\"10.1007/s00604-019-3245-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Shan T, Pu L et al (2022) Glucometer-based electrochemical biosensor for determination of microRNA (let-7a) using magnetic-assisted extraction and supersandwich signal amplification. Microchim Acta 189:444. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00604-022-05544-7\u003c/span\u003e\u003cspan address=\"10.1007/s00604-022-05544-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Gao X, Yang L et al (2021) Metal\u0026ndash;Organic Framework-Functionalized Paper-Based Electrochemical Biosensor for Ultrasensitive Exosome Assay. Anal Chem 93:11792\u0026ndash;11799. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.1c02286\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.1c02286\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu J, Chen S, Dou Y et al (2022) Smartphone-Based Electrochemical Biosensors for Directly Detecting Serum-Derived Exosomes and Monitoring Their Secretion. Anal Chem 7:3235\u0026ndash;3244. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.1c04910\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.1c04910\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Zhao J, Dong J et al (2022) Simultaneous detection of exosomal microRNAs by nucleic acid functionalized disposable paper-based sensors. Chem Eng J 438:135594. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cej.2022.135594\u003c/span\u003e\u003cspan address=\"10.1016/j.cej.2022.135594\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhaksari S, Abnous K, Hadizadeh F et al (2022) Signal amplification strategies in biosensing of extracellular vesicles (EVs). Talanta 124244. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.talanta.2022.124244\u003c/span\u003e\u003cspan address=\"10.1016/j.talanta.2022.124244\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazloum-Ardakani M, Tavakolian-Ardakani Z, Sahraei N, Moshtaghioun SM (2019) Fabrication of an ultrasensitive and selective electrochemical aptasensor to detect carcinoembryonic antigen by using a new nanocomposite. Biosens Bioelectron 129:1\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bios.2018.12.047\u003c/span\u003e\u003cspan address=\"10.1016/j.bios.2018.12.047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTappan BC, Steiner SA III, Luther EP (2010) Nanoporous metal foams. Angew Chem Int Ed 49:4544\u0026ndash;4565. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/anie.200902994\u003c/span\u003e\u003cspan address=\"10.1002/anie.200902994\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeer M, Ryb\u0026aacute;r R, Kaľavsk\u0026yacute; M (2019) Experimental heat transfer analysis of open cell hollow ligament metal foam at low Reynolds number. Measurement 133:214\u0026ndash;221. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.measurement.2018.10.025\u003c/span\u003e\u003cspan address=\"10.1016/j.measurement.2018.10.025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi W, Wu H, Bin, Wang B et al (2012) One-Pot Synthesis of Ultra‐Light Nickel Nanofoams Composed of Nanowires and Their Transformation into Various Functional Nanofoams. Small 8:3432\u0026ndash;3437\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng L, Fan S, Chen Y et al (2022) In situ growing CuO/ZIF-8 into nickel foam to fabricate a binder-free self-supported glucose biosensor. Ind Eng Chem Res 61:7312\u0026ndash;7321. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.iecr.2c01298\u003c/span\u003e\u003cspan address=\"10.1021/acs.iecr.2c01298\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahraei N, Mazloum-Ardakani M, Mohiti J et al (2022) Flexible Paper-Based Immunosensor for the Detection of Specific Cancer-Derived Exosomes. J Electrochem Soc 169:127514. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1149/1945\u0026ndash;7111/aca836\u003c/span\u003e\u003cspan address=\"10.1149/1945\u0026ndash;7111/aca836\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarcano DC, Kosynkin DV, Berlin JM et al (2010) Improved synthesis of graphene oxide. ACS Nano 4:4806\u0026ndash;4814. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/nn1006368\u003c/span\u003e\u003cspan address=\"10.1021/nn1006368\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRungsawang T, Punrat E, Adkins J et al (2016) Development of Electrochemical Paper-based Glucose Sensor Using Cellulose‐4‐aminophenylboronic Acid‐modified Screen‐printed Carbon Electrode. Electroanalysis 28:462\u0026ndash;468. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/elan.201500406\u003c/span\u003e\u003cspan address=\"10.1002/elan.201500406\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRusso A, Ahn BY, Adams JJ et al (2011) Pen-on‐paper flexible electronics. Adv Mater 23:3426\u0026ndash;3430. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.201101328\u003c/span\u003e\u003cspan address=\"10.1002/adma.201101328\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbebe B, Murthy HCA, Amare E (2018) Summary on adsorption and photocatalysis for pollutant remediation: Mini review. J Encapsulation Adsorpt Sci 8:225\u0026ndash;255. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4236/jeas.2018.84012\u003c/span\u003e\u003cspan address=\"10.4236/jeas.2018.84012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRavikovitch PI, Neimark AV (2001) Characterization of nanoporous materials from adsorption and desorption isotherms. Colloids Surf A 187:11\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0927-7757(01)00614-8\u003c/span\u003e\u003cspan address=\"10.1016/S0927-7757(01)00614-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe S, Feng J, Wu P (2013) Deposition of three-dimensional graphene aerogel on nickel foam as a binder-free supercapacitor electrode. ACS Appl Mater Interfaces 5:7122\u0026ndash;7129. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/am401458x\u003c/span\u003e\u003cspan address=\"10.1021/am401458x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu Y, Yang Z, Li X et al (2013) Template-free synthesized Ni nanofoams as nanostructured current collectors for high-performance electrodes in lithium ion batteries. J Mater Chem A 1:10002\u0026ndash;10007. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C3TA11753G\u003c/span\u003e\u003cspan address=\"10.1039/C3TA11753G\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu YY, Guo XL, Zhao L et al (2018) Facile preparation of surfactant-free Au NPs/RGO/Ni foam for degradation of 4-nitrophenol and detection of hydrogen peroxide. Nanotechnology 29:235706. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1088/1361\u0026ndash;6528/aab936\u003c/span\u003e\u003cspan address=\"10.1088/1361\u0026ndash;6528/aab936\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBard AJ, Faulkner LR, White HS (2022) Electrochemical methods: fundamentals and applications. Wiley\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Zeng L, Wang Y et al (2021) Electrochemical aptasensor based on multidirectional hybridization chain reaction for detection of tumorous exosomes. Sens Actuators B 332:129471\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahraei N, Mazloum-Ardakani M, Khoshroo A et al (2022) Electrochemical system designed on a paper platform as a label-free immunosensor for cancer derived exosomes based on a mesoporous carbon foam-ternary nanocomposite. J Electroanal Chem 920:116590. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jelechem.2022.116590\u003c/span\u003e\u003cspan address=\"10.1016/j.jelechem.2022.116590\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Wang Z, Wang F et al (2021) Ti3C2 MXene mediated Prussian blue in situ hybridization and electrochemical signal amplification for the detection of exosomes. Talanta 224:121879. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.talanta.2020.121879\u003c/span\u003e\u003cspan address=\"10.1016/j.talanta.2020.121879\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDold\u0026aacute;n X, Fag\u0026uacute;ndez P, Cayota A et al (2016) Electrochemical sandwich immunosensor for determination of exosomes based on surface marker-mediated signal amplification. Anal Chem 88:10466\u0026ndash;10473. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.6b02421\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.6b02421\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-electrochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jach","sideBox":"Learn more about [Journal of Applied Electrochemistry](http://link.springer.com/journal/10800)","snPcode":"10800","submissionUrl":"https://submission.nature.com/new-submission/10800/3","title":"Journal of Applied Electrochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Paper-based electrochemical biosensor, Three-dimensional nickel nanofoam, Point of care testing (POCT), Exosome.","lastPublishedDoi":"10.21203/rs.3.rs-3994610/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3994610/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExosomes as new biomarkers for cancer diagnosis have attracted attention because they are highly released by tumor cells in various biological fluids. In this study, an electrochemical paper-based immunosensor device (Exo-sensing paper) is introduced for the detection of exosome in the serum. The Exo-sensing paper is a three electrode system that is prepared using pattern paper and carbon and silver inks. The sensor part of this immunosensor contains a three-dimensional porous nanocomposite of nickel nanofoam coupled with graphene oxide and gold nanoparticles. The high specific surface area of this nanocomposite increases the antibody loading on the sensor surface significantly and consequently leads to obtaining a wide linear range of 500\u0026ndash;1 \u0026times; 10 \u003csup\u003e7\u003c/sup\u003e Exospore/\u0026micro;L with a detection limit of 110 Exosome/\u0026micro;L. Due to some advantages of this constructed Exo-sensing paper such as easy storage, simple application, low cost and good selectivity in the real samples, this system has a good potential to be used as a point of care testing for in situ detection of the exosomes and is a promising strategy for minimally invasive liquid biopsy.\u003c/p\u003e","manuscriptTitle":"Flexible electrochemical paper-based device for detection of breast cancer- derived exosome using nickel nanofoam 3D nanocomposite","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-29 16:39:15","doi":"10.21203/rs.3.rs-3994610/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"checksComplete","content":"","date":"2024-02-28T02:49:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Electrochemistry","date":"2024-02-27T18:18:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-electrochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jach","sideBox":"Learn more about [Journal of Applied Electrochemistry](http://link.springer.com/journal/10800)","snPcode":"10800","submissionUrl":"https://submission.nature.com/new-submission/10800/3","title":"Journal of Applied Electrochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e5120140-d680-4e6c-ad3a-291d5087aaa4","owner":[],"postedDate":"February 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-12T11:53:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-29 16:39:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3994610","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3994610","identity":"rs-3994610","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}