key: cord-0019268-biqmbxrm
authors: Yoo, Jhongryul; Jeong, Hongin; Park, Seo Kyung; Park, Sungho; Lee, Je Seung
title: Interdigitated Electrode Biosensor Based on Plasma-Deposited TiO(2) Nanoparticles for Detecting DNA
date: 2021-06-29
journal: Biosensors (Basel)
DOI: 10.3390/bios11070212
sha: 374b3066f1ade5091af7c43faae15689f32b6962
doc_id: 19268
cord_uid: biqmbxrm

Bioelectrodes mediated by metal oxide nanoparticles have facilitated the development of new sensors in medical diagnosis. High-purity TiO(2) nanoparticles (NPs) were synthesized through thermal plasma and deposited directly on an interdigitated electrode. The surface of the TiO(2)-deposited electrode was activated with (3-aminopropyl) triethoxysilane (APTES) followed by fixing the single-stranded probe deoxyribonucleic acid (DNA) to fabricate the DNA biosensor. The structural properties of the deposited TiO(2) nanoparticles were analyzed using a transmission electron microscope (TEM), X-ray diffraction (XRD), and a dynamic light scattering (DLS) system. The chemical composition and structural properties of the TiO(2) nanoparticle layer and the fixed layer were analyzed by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). E. coli O157:H7, a well-known pernicious pathogenic bacterial species, was detected as a target DNA of the prepared DNA biosensor, and the characteristics of DNA detection were determined by the current change using a picoammeter. The degree of binding between the probe DNA and the target DNA was converted into an electrical signal using the picoammeter method to quantitatively analyze the concentration of the target DNA. With the specificity experiment, it was confirmed that the biosensor was able to discriminate between nucleotides with mismatched, non-complementary, or complementary sequences.

Due to the food poisoning epidemic that is spreading worldwide, more and more attention is being paid to public health issues. The most well-known causes of food poisoning are pathogenic bacteria including Escherichia coli (E. coli) O157:H7, Clostridium perfringens, Salmonella, Campylobacter, Listeria monocytogenes, etc. [1] [2] [3] [4] . E. coli O157:H7 was first discovered in 1982 and classified as an enterohemorrhagic E. coli (EHEC), which is a serotype bacterial species and can produce Shiga-like toxins [5] [6] [7] . It is the causative agent of food-borne diseases caused by contaminated raw food, including unsterilized milk or raw ground beef [8, 9] . As the incidence of bacteria-related diseases and the number of deaths continue to increase worldwide, there is an urgent need to develop a biosensor capable of quickly and inexpensively detecting pathogenic bacteria such as E. coli O157:H7 with high sensitivity and specificity.

Several traditional tests, such as microbial culture and microscopic counting methods, are commonly used as pathogen detection and counting methods, but have the disadvantage of being time consuming and having low accuracy. Therefore, various analytical methods have been developed including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) probe [10] , micro-electromechanical systems (MEMS) [11] , flow cytometry, immunomagnetic separation (IMS) [12] , quartz crystal microbalance (QCM) [13] , polymerase chain reaction (PCR) [14] , and surface plasmon resonance (SPR) [15] to detect these

Glass substrates were cleaned with organic solvents prior to the fabrication of the interdigitated electrode (IDE) followed by drying under nitrogen atmosphere. To pattern finger electrodes in the active area, the lift-off lithography method was used. On the glass substrate, a negative photoresist (ma-N 1405) was spin-coated with a spin speed of 2000 rpm followed by heating at 150 • C for 60 s to harden the spin-coated resist layer. After developing the resist by exposing RD-6 to UV light under a photomask for 240 s, the residual solvent in the resist was removed, and the film was affixed to the substrate by heating at 100 • C for 60 s. The Pt film was deposited by a DC magnetron sputtering system (RSP 5004, SNTEK, Suwon, Korea). The photoresist was stripped using acetone followed by rinsing with distilled water until solid IDEs were exposed.

To deposit the crystalline TiO 2 NPs, self-developed equipment capable of depositing the precursor (TiCl 4 ) using thermal plasma directly on a Pt IDE patterned glass substrate was used [33] . The IDE electrode fabricated glass substrate was immobilized on the substrate holder of the nanoparticle manufacturing and deposition equipment. Ar carrier gas was injected into a bubble-type canister containing TiCl 4 to inject a Ti precursor into a stabilized plasma flame. The resulting TiO 2 NPs were injected into the chamber and deposited on an Pt IDE fabricated substrate.

To prepare the active layer, the surface of the TiO 2 NPs layer was functionalized with 0.02 M APTES by self-assembly inside a fume hood [34] [35] [36] [37] [38] . Next, 25 µL of APTES was dropped onto the surface of the TiO 2 -NPs-based transducer and stayed for 3 h in a drying cabinet followed by washing the unreacted APTES with deionized water (DI, > 18 MΩ). Any other chemicals for activation were not used because the picoammeter could measure the very small current changes during the immobilization of probe DNA and the hybridization of target DNA molecules.

A carboxyl-functionalized synthetic 30-base oligonucleotide probe and 30-base target oligonucleotides were used for E. coli O157:H7 detection. The base sequences of the synthetic oligonucleotides probe and targets were as follows: probe oligonucleotide: (5 - (COOH) AAC GCC GAT ACC ATT ACT TAT ACC GCG ACG-3 ), complementary  oligonucleotide: (5 -CGT CGC GGT ATA AGT AAT GGT ATC GGC GTT-3 ), noncomple-mentary oligonucleotide: (5 -GCA GCG CCA TAT TCA TTA CCA TAG CCG CAA-3 ), and  the single base mismatch oligonucleotide: (5 -CGT CGC GGT ATA ACT AAT GGT ATC  GGC GTT-3 ) . The oligonucleotide stock solutions (10 µM) were prepared using deionized ultrapure water (>18 MΩ). The probe oligonucleotide was immobilized on the surface of the TiO 2 -NPs-deposited Pt IDE by dropping 2.0 µL of the probe oligonucleotide solution and spreading according to the previously reported method [34] [35] [36] [37] [38] . Remaining unbound probe DNA was removed by rinsing with deionized water.

For this, 2.0 µL of the complementary DNA solution was dropped on the TiO 2 -NPsdeposited Pt IDE on which the probe DNA was immobilized and spread well to perform a hybridization reaction between the probe and the complementary DNA, followed by rinsing the surface with deionized water and performing the electrical measurements using a Keithley 6487 picoammeter. Other 30-base oligonucleotides were tested with the same method.

Conventional electrochemical methods suffer from noise because of the use of various chemicals for obtaining the detection results. To overcome this problem, a device which detects the specific DNA without using other chemicals was developed. The Pt interdigitated electrode (IDE) was patterned on a glass substrate with negative photoresist by the lift-off lithography method instead of the conventional photolithography method. The Pt film was deposited at a pressure of~10 −6 Torr using a DC magnetron sputtering system, and the photoresist was stripped. Figure 1a ,b shows a photograph and schematic diagram of the interdigitated electrode sensor array with dimensions 5 × 12 mm 2 . The width of the interdigitated Pt finger electrodes was fixed to 315 µm and a gap of 15 µm was used between two fingers in the active area. The size of each contact pad was 2 × 5 mm 2 .

A carboxyl-functionalized synthetic 30-base oligonucleotide probe and 30-base target oligonucleotides were used for E. coli O157:H7 detection. The base sequences of the synthetic oligonucleotides probe and targets were as follows: probe oligonucleotide: (5′-(COOH) AAC GCC GAT ACC ATT ACT TAT ACC GCG ACG-3′), complementary oligonucleotide: (5′-CGT CGC GGT ATA AGT AAT GGT ATC GGC GTT-3′), noncomplementary oligonucleotide: (5′-GCA GCG CCA TAT TCA TTA CCA TAG CCG CAA-3′), and the single base mismatch oligonucleotide: (5′-CGT CGC GGT ATA ACT AAT GGT ATC GGC GTT-3′). The oligonucleotide stock solutions (10 μM) were prepared using deionized ultrapure water (>18 MΩ). The probe oligonucleotide was immobilized on the surface of the TiO2-NPs-deposited Pt IDE by dropping 2.0 μL of the probe oligonucleotide solution and spreading according to the previously reported method [34] [35] [36] [37] [38] . Remaining unbound probe DNA was removed by rinsing with deionized water.

For this, 2.0 μL of the complementary DNA solution was dropped on the TiO2-NPsdeposited Pt IDE on which the probe DNA was immobilized and spread well to perform a hybridization reaction between the probe and the complementary DNA, followed by rinsing the surface with deionized water and performing the electrical measurements using a Keithley 6487 picoammeter. Other 30-base oligonucleotides were tested with the same method.

Conventional electrochemical methods suffer from noise because of the use of various chemicals for obtaining the detection results. To overcome this problem, a device which detects the specific DNA without using other chemicals was developed. The Pt interdigitated electrode (IDE) was patterned on a glass substrate with negative photoresist by the lift-off lithography method instead of the conventional photolithography method. The Pt film was deposited at a pressure of ~10 −6 Torr using a DC magnetron sputtering system, and the photoresist was stripped. Figure 1a ,b shows a photograph and schematic diagram of the interdigitated electrode sensor array with dimensions 5 × 12 mm 2 . The width of the interdigitated Pt finger electrodes was fixed to 315 μm and a gap of 15 μm was used between two fingers in the active area. The size of each contact pad was 2 × 5 mm 2 . After patterning Pt IDE on the glass substrate, the TiO2-DNA biosensor was fabricated in four steps as shown in Figure 1b : the thermal plasma deposition of pure TiO2 NPs, After patterning Pt IDE on the glass substrate, the TiO 2 -DNA biosensor was fabricated in four steps as shown in Figure 1b : the thermal plasma deposition of pure TiO 2 NPs, the silanization of TiO 2 NPs with APTES, the immobilization of probe DNA, and the hybridization of target DNA. Single-stranded DNA (ss-DNA) was immobilized on the surface of TiO 2 NPs through coordinated cross-linkages [39] .

Pure crystalline TiO 2 NPs were deposited directly on the Pt IDE patterned glass substrate using self-developed thermal plasma deposition equipment [33] . The injected Ti precursor (TiCl 4 ) was quickly mixed with the O 2 radical and the swirling gas to produce TiO 2 NPs. The produced NPs were injected into the chamber and deposited on the Pt IDE fabricated substrate.

A TEM image of the deposited TiO 2 NPs by the thermal plasma process is shown in Figure 2a . The average size of the prepared TiO 2 NPs was measured as around 20 nm. The purity of TiO 2 NPs synthesized by the thermal plasma process was confirmed by the result of TEM-EDX observations as shown in Figure 2b .

bridization of target DNA. Single-stranded DNA (ss-DNA) was immobilized on the surface of TiO2 NPs through coordinated cross-linkages [39] .

Pure crystalline TiO2 NPs were deposited directly on the Pt IDE patterned glass substrate using self-developed thermal plasma deposition equipment [33] . The injected Ti precursor (TiCl4) was quickly mixed with the O2 radical and the swirling gas to produce TiO2 NPs. The produced NPs were injected into the chamber and deposited on the Pt IDE fabricated substrate.

A TEM image of the deposited TiO2 NPs by the thermal plasma process is shown in Figure 2a . The average size of the prepared TiO2 NPs was measured as around 20 nm. The purity of TiO2 NPs synthesized by the thermal plasma process was confirmed by the result of TEM-EDX observations as shown in Figure 2b . The crystal structure of the TiO2 NPs deposited on the Pt IDE through the microwave thermal plasma process was analyzed by X-ray diffraction (XRD) (Figure 3 ). The XRD spectrum exhibited typical patterns of crystalline TiO2 NPs with high peak intensities, indicating that the deposited TiO2 NPs had a high degree of crystallinity. The peak values of the TiO2 XRD pattern agree well with the standard values of anatase and rutile phases (JCPDS data cards No. 21-1272 and 21-1276, respectively). The quantification of phase proportions was calculated by the method of Spurr and Myers [40] , which utilizes the ratio of the rutile peak at 27.355° 2θ to the anatase peak at 25.176° 2θ as shown in Equation (1):

where WR, IR, WA, and IA are the weight fractions and peak intensities of rutile and anatase, respectively. The calculated weight fraction of anatase phase (90.4%) was much higher than that of rutile phase (9.6%). This result implies that the formation of an anatase structure as a kinetic product is preferred over the formation of a rutile structure, which is thermodynamically more stable than an anatase structure due to the deposition at high temperature with short reaction time. The average size of crystalline structures can also be calculated employing the Scherer equation [40, 41] as shown in Equation (2):

where λ, B, and 2θ are the wavelength of X-ray radiation, the full-width at the half-maximum of the diffraction peak, and the diffraction angle, respectively. The calculated crystallite size of deposited TiO2 was 22.2 nm, and this result agrees well with the TEM result (vide supra). The crystal structure of the TiO 2 NPs deposited on the Pt IDE through the microwave thermal plasma process was analyzed by X-ray diffraction (XRD) (Figure 3 ). The XRD spectrum exhibited typical patterns of crystalline TiO 2 NPs with high peak intensities, indicating that the deposited TiO 2 NPs had a high degree of crystallinity. The peak values of the TiO 2 XRD pattern agree well with the standard values of anatase and rutile phases (JCPDS data cards No. 21-1272 and 21-1276, respectively). The quantification of phase proportions was calculated by the method of Spurr and Myers [40] , which utilizes the ratio of the rutile peak at 27.355 • 2θ to the anatase peak at 25.176 • 2θ as shown in Equation (1):

where W R , I R , W A , and I A are the weight fractions and peak intensities of rutile and anatase, respectively. The calculated weight fraction of anatase phase (90.4%) was much higher than that of rutile phase (9.6%). This result implies that the formation of an anatase structure as a kinetic product is preferred over the formation of a rutile structure, which is thermodynamically more stable than an anatase structure due to the deposition at high temperature with short reaction time. The average size of crystalline structures can also be calculated employing the Scherer equation [40, 41] as shown in Equation (2):

where λ, B, and 2θ are the wavelength of X-ray radiation, the full-width at the halfmaximum of the diffraction peak, and the diffraction angle, respectively. The calculated crystallite size of deposited TiO 2 was 22.2 nm, and this result agrees well with the TEM result (vide supra). Biosensors 2021, 11, x 6 of 12 Self-assembly of the monolayer to introduce the oligomeric DNA on the contact layer is very important for the fabrication of a DNA sensor (Scheme 1). The surface of the TiO2 NPs is terminated by a hydroxyl group (-OH) under atmospheric conditions, allowing the molecule to attach through a condensation reaction. For this reason, the transducer was silanized by dropping 25 μL of 0.02 M APTES solution. The aim of silanization using APTES is to modify the TiO2 layer by the hydrolyzation of -OH group on the surface of TiO2 NPs and form siloxane bonds (Si-O-Si) to immobilize the probe DNA at the surface of TiO2 NPs. The silane layer formed over TiO2 can be thin and uniform because APTES is fragile and can be easily washed away by the buffer solution [34] [35] [36] [37] [38] 42 ].

An immobilization process of the DNA probe was carried out after silanization of the TiO2 NPs as shown in Scheme 1. For detection of the target DNA containing a complementary nucleotide sequence to the probe DNA, single-stranded probe DNA (ss-DNA) modified with a carboxyl group (-COOH) was immobilized on the surface of the silanized TiO2 NPs through the formation of an amide bond between APTES and the probe DNA. Figure 5 shows the X-ray photoelectron spectroscopy (XPS) analysis results of the electrode surface. The Ti and O are from deposited TiO2 NPs; the O, N, Si, C, and P are from APTES and probe DNA. Self-assembly of the monolayer to introduce the oligomeric DNA on the contact layer is very important for the fabrication of a DNA sensor (Scheme 1). The surface of the TiO2 NPs is terminated by a hydroxyl group (-OH) under atmospheric conditions, allowing the molecule to attach through a condensation reaction. For this reason, the transducer was silanized by dropping 25 μL of 0.02 M APTES solution. The aim of silanization using APTES is to modify the TiO2 layer by the hydrolyzation of -OH group on the surface of TiO2 NPs and form siloxane bonds (Si-O-Si) to immobilize the probe DNA at the surface of TiO2 NPs. The silane layer formed over TiO2 can be thin and uniform because APTES is fragile and can be easily washed away by the buffer solution [34] [35] [36] [37] [38] 42 ].

An immobilization process of the DNA probe was carried out after silanization of the TiO2 NPs as shown in Scheme 1. For detection of the target DNA containing a complementary nucleotide sequence to the probe DNA, single-stranded probe DNA (ss-DNA) modified with a carboxyl group (-COOH) was immobilized on the surface of the silanized TiO2 NPs through the formation of an amide bond between APTES and the probe DNA. Figure 5 shows the X-ray photoelectron spectroscopy (XPS) analysis results of the electrode surface. The Ti and O are from deposited TiO2 NPs; the O, N, Si, C, and P are from APTES and probe DNA. Self-assembly of the monolayer to introduce the oligomeric DNA on the contact layer is very important for the fabrication of a DNA sensor (Scheme 1). The surface of the TiO 2 NPs is terminated by a hydroxyl group (-OH) under atmospheric conditions, allowing the molecule to attach through a condensation reaction. For this reason, the transducer was silanized by dropping 25 µL of 0.02 M APTES solution. The aim of silanization using APTES is to modify the TiO 2 layer by the hydrolyzation of -OH group on the surface of TiO 2 NPs and form siloxane bonds (Si-O-Si) to immobilize the probe DNA at the surface of TiO 2 NPs. The silane layer formed over TiO 2 can be thin and uniform because APTES is fragile and can be easily washed away by the buffer solution [34] [35] [36] [37] [38] 42 ].

An immobilization process of the DNA probe was carried out after silanization of the TiO 2 NPs as shown in Scheme 1. For detection of the target DNA containing a complementary nucleotide sequence to the probe DNA, single-stranded probe DNA (ss-DNA) modified with a carboxyl group (-COOH) was immobilized on the surface of the silanized TiO 2 NPs through the formation of an amide bond between APTES and the probe DNA. Figure 5 shows the X-ray photoelectron spectroscopy ( To monitor the performance of the developed device, instead of using the conventional electrochemical methods which have poor signal-to-noise ratios because of the use of various chemicals, the very small electric current flowing through the TiO2 NPs was measured with a picoammeter in a voltage range of 0 to 1 V ( Figure 6 ). Since this method only uses deionized water for the dilution of DNA, it can greatly reduce the effects of noise generated by the various chemical substances used in conventional methods. To monitor the performance of the developed device, instead of using the conventional electrochemical methods which have poor signal-to-noise ratios because of the use of various chemicals, the very small electric current flowing through the TiO2 NPs was measured with a picoammeter in a voltage range of 0 to 1 V ( Figure 6 ). Since this method only uses deionized water for the dilution of DNA, it can greatly reduce the effects of noise generated by the various chemical substances used in conventional methods. To monitor the performance of the developed device, instead of using the conventional electrochemical methods which have poor signal-to-noise ratios because of the use of various chemicals, the very small electric current flowing through the TiO 2 NPs was measured with a picoammeter in a voltage range of 0 to 1 V ( Figure 6 ). Since this method only uses deionized water for the dilution of DNA, it can greatly reduce the effects of noise generated by the various chemical substances used in conventional methods. Figure 6 shows current-voltage curves of TiO2-NPs-based biosensors. In order to build a DNA sensor that does not damage the sensitive DNA molecules, the current flowing through the sensor must be very low, and quantitative detection should be possible with a high signal-to-noise ratio. At 0.5 V, as shown in Figure 6b , a very low current of Figure 6 shows current-voltage curves of TiO 2 -NPs-based biosensors. In order to build a DNA sensor that does not damage the sensitive DNA molecules, the current flowing through the sensor must be very low, and quantitative detection should be possible with a high signal-to-noise ratio. At 0.5 V, as shown in Figure 6b , a very low current of 0.18 nA flowed through the sensor with only TiO 2 NPs deposited, and only a slightly increased current of 0.36 nA flowed even after the silanization by APTES. When ss-DNA was immobilized on the sensor surface, however, the current value increased to 14.0 nA as the surface charge density was increased by DNA, which has negative charges on its backbone [43] . Figure 6a and Table 1 exhibit the current-voltage curves and average currents of the ss-DNA-immobilized sensors according to the different deposition times of TiO 2 NPs on the surface of the Pt IDEs. As the deposition time of the TiO 2 NPs increased, the number of TiO 2 NPs that could immobilize the DNA molecules increased. As a result, the active surface area of the Pt IDEs increased, and accordingly, more probe DNA could be attached, and the current increased. The selectivity of the sensor was further investigated by hybridizing three different types of DNA, each at a concentration of 10 µM individually (Figure 6b ). When the ss-DNA with a complementary nucleotide sequence and the immobilized probe DNA was completely hybridized to form double-stranded DNA (ds-DNA), the current signal was greatly reduced to 4.6 nA because the negative charges became difficult to transfer to the sensor surface due to the ion blockage by the excessive negative charges of the ds-DNA. Therefore, the output current was inversely proportional to the concentration of the complementary DNA [44] . When non-complementary DNA was exposed to the probe DNA, the change in current was not observed because the hybridization had not occurred. This result shows the high selectivity of the DNA biosensor built for detection by the hybridization of DNA. Meanwhile, when the single-base-mismatched DNA was exposed to the probe DNA, the measured current was 8.6 nA. Therefore, it was confirmed that the fabricated DNA sensor using TiO 2 NPs deposited on Pt IDE can be used as a very effective indicator which is able to distinguish abnormally mismatched oligonucleotides.

To evaluate the detection range, E. coli O157:H7 DNA solutions with varied concentrations were prepared and 2.0 µL of DNA solution was taken at each concentration and dropped onto the IDE sensor. Figure 7a shows the measured currents of the fabricated TiO 2 biosensors at different E. coli O157:H7 DNA concentrations. The results show that there was a high correlation between the concentration of E. coli O157:H7 DNA and the output current. The output current of the probe DNA-immobilized IDE sensor decreased as the concentration of E. coli O157:H7 DNA increased. The peak current exhibited a linear relationship in the wide concentration range of 1 pM to 10 µM with the current values of 18.4 nA to 6.9 nA. The R 2 value of the calibration curve was 0.997, indicating that the reactivity according to the target DNA concentration was excellent (Figure 7b ). The sensitivity and the limit of detection (LOD) of the IDE sensor were determined as 286 nA logM −1 cm −2 and 0.43 pM (S/N = 15.5), respectively. The sensitivity was calculated using Equation (3), and LOD was obtained by applying 6 sigmas to the standard deviation value: Sensitivity = ∆nA/(∆logM × sample sensing area (cm 2 ))

where ∆nA is the measured current value and ∆logM is the concentration of target DNA.

cm −2 and 0.43 pM (S/N = 15.5), respectively. The sensitivity was calculated using Equation (3) , and LOD was obtained by applying 6 sigmas to the standard deviation value: Sensitivity = ΔnA/(ΔlogM × sample sensing area (cm 2 ))

where ΔnA is the measured current value and ΔlogM is the concentration of target DNA. Figure 8 shows the results of observing the amount of current obtained periodically for 8 months. The fabricated IDE sensor showed almost consistent responses for 6 months, and the relative standard deviation (RSD) was calculated as 3.9%. After that, a decrease in current amount of about 7.5% was observed for 2 months. This is a similar result to the previous report in which current responses decreased due to the stacking interaction of base pairs present in the probe DNA immobilized on the sensor surface [43] . Figure 8 shows the results of observing the amount of current obtained periodically for 8 months. The fabricated IDE sensor showed almost consistent responses for 6 months, and the relative standard deviation (RSD) was calculated as 3.9%. After that, a decrease in current amount of about 7.5% was observed for 2 months. This is a similar result to the previous report in which current responses decreased due to the stacking interaction of base pairs present in the probe DNA immobilized on the sensor surface [43] . 

A high-purity TiO2-NPs-based microchip biosensor array plasma-deposited on Pt IDE for the detection of E. coli O157:H7 DNA hybridization was successfully fabricated. The biosensor was fabricated by synthesizing TiO2 nanoparticles in thermal plasma, depositing them directly onto a Pt IDE, and then silanizing them to combine single-stranded DNA with complementary nucleotide sequences with target DNA. The fabricated biosensor was able to detect target DNA with high sensitivity and selectivity using a picoammeter. This picoammeter biosensor is distinguished from conventional electrochemical methods by its simple yet low-noise biosensing method that does not require any chemicals other than DI water. In addition, for the production of TiO2 NPs, it was possible to obtain high sensitivity compared to a conventional biosensor by using direct deposition using a plasma process instead of the conventional sol-gel method. For this reason, we judge that the purity of TiO2 nanoparticles deposited by the plasma process is higher than that of those produced by the sol-gel method, and the surface area is wider. The results of this study are expected to be utilized in the development of a field diagnostic biosensor that can diagnose high-risk infectious diseases such as foot-and-mouth disease and African swine fever without complicated pre-treatment and cloning procedures in the field. 

A high-purity TiO 2 -NPs-based microchip biosensor array plasma-deposited on Pt IDE for the detection of E. coli O157:H7 DNA hybridization was successfully fabricated. The biosensor was fabricated by synthesizing TiO 2 nanoparticles in thermal plasma, depositing them directly onto a Pt IDE, and then silanizing them to combine single-stranded DNA with complementary nucleotide sequences with target DNA. The fabricated biosensor was able to detect target DNA with high sensitivity and selectivity using a picoammeter. This picoammeter biosensor is distinguished from conventional electrochemical methods by its simple yet low-noise biosensing method that does not require any chemicals other than DI water. In addition, for the production of TiO 2 NPs, it was possible to obtain high sensitivity compared to a conventional biosensor by using direct deposition using a plasma process instead of the conventional sol-gel method. For this reason, we judge that the purity of TiO 2 nanoparticles deposited by the plasma process is higher than that of those produced by the sol-gel method, and the surface area is wider. The results of this study are expected to be utilized in the development of a field diagnostic biosensor that can diagnose high-risk infectious diseases such as foot-and-mouth disease and African swine fever without complicated pre-treatment and cloning procedures in the field. Author Contributions: Conceptualization, J.Y. and S.P.; methodology, J.Y. and S.K.P.; writingoriginal draft preparation, J.Y. and H.J.; writing-review and editing, S.K.P. and J.S.L.; supervision, S.P. and J.S.L.; funding acquisition, S.P. and J.S.L. All authors have read and agreed to the published version of the manuscript. 

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Institutional Review Board Statement: Not applicable.

Data Availability Statement: Not applicable.

The authors declare no conflict of interest.