the ph - sensing ion - sensitive field - effect transistor ( isfet ) , first proposed by bergveld in 1970 , has become the basis for many contemporary semiconductors and their respective biosensors.1 in an isfet , the ions within the sample media undergo multiple environment - influenced reactions .
the resulting molecules from these reactions accumulate upon the gate oxide layer ( gol ) , and the resulting change in charge affects the conductance in the isfet channel.2,3 consequently , the conductance between the source and the drain will differ , and this change is expressed as an electrical signal .
the gols of isfets are usually made up of dielectric materials , such as sno2,47 zno,8 sio2,9,10 al2o3,11 si3n4,12,13 ta2o5,14 tio2,15 and wo3,16 which readily react with the h and oh of the media .
dielectric materials that have a high reactivity with ions are used as either ph sensor or gas sensor.17 however , because other ions within the sample media also affect the surface charge on the gol , the electrical characteristics such as the threshold voltage and channel conductance will be influenced .
isfet - based biosensors , which detect bioanalytes , are affected by the undesirable ions in the media , and the resulting fluctuations or noise may cause the results to be deemed untrustworthy .
although the signal may stabilize as time progresses and the reaction becomes saturated ( reaches equilibrium state ) , the noise factor must be acknowledged when evaluating the final signal .
because the target bioanalyte is already in small amounts inside the target solution , any other factors that may affect the signal , such as the ions within the solution media , will affect the sensitivity of the biosensor .
when the biosensor applies highly reactive nanowires and thin films to enhance its sensitivity , the unwanted noise factor may also be increased and affect the result even more . in this study , we report a strategy to minimize the sensing voltage error in the isfet biosensor . to improve specific bioanalyte binding and to reduce undesirable ion reactions , an optimal presurface treatment on the gate oxide is demonstrated .
in addition , the sensing voltage drift error ( vdf ) of the isfet biosensor before presurface treatment and after presurface treatment is analyzed . finally , because multiple teams that study and utilize the isfet biosensor dilute the media to obtain a longer debye screening length , we analyzed the vdf for 1 phosphate buffered saline ( pbs ) and 0.01 pbs solutions
the gol of an isfet biosensor was fabricated by depositing an sno2 ( 99.9% ) thin film on the top of an indium tin oxide ( ito ) glass ( ito thickness : 300 nm ) .
the 80 nm sno2 film was deposited using a radio frequency ( rf ) magnetron sputtering system ( rf power : 50 w , base pressure : 210 torr , work pressure : 18 mtorr , ar gas flow : 5 sccm ) . the polydimethylsiloxane ( pdms ) block was manufactured from a sylgard 184 silicone elastomer kit ( dow corning , seoul , republic of korea ) , which was composed of a base and a curing agent .
the base and curing agent were mixed at a 10:1 ratio and baked at 60c for 4 h. the pdms block was then punctured with 6 mm holes to form a sample media reservoir . to attach the pdms to the surface of gol ,
the gol was sonicated in deionized water for 1 min and then sonicated in ethanol for 1 min .
the pdms block and gol were then placed inside an o2 plasma system ( cute - mpr ; femto science , gyeonggi - do , republic of korea ) and treated with o2 plasma ( dc power : 70 w , time : 1 min , o2 flow : 30 sccm , base pressure : 510 torr , working pressure : 510 torr ) .
the pdms block and gol were then attached together to form the sensing gate ( figure 1 ) .
the gol surface of the sensing gate was treated with plasma to form oh functional groups on the reservoir surface . then
, 5% 3-aminopropyltriethoxysilane ( aptes ) was prepared and quickly added to the surface of the gol for the formation of nh2 functional groups .
after the addition of the aptes , the gol was placed inside a petri dish and the petri dish was sealed with parafilm for 1 h. the chemical reaction should occur in a dark place .
after 1 h , the sensing gate was sonicated in ethanol for 1 min and dried with n2 gas .
the sensing gate was baked on a hot plate at 120c . to functionalize the gol surface of the sensing gate with cooh functional groups ,
5% 1 m succinic anhydride was made from succinic anhydride powder and dimethyl formamide ( dmf ) and added onto the surface of the gol .
the petri dish was sealed with parafilm and placed inside an incubator at 37c for overnight .
the presurface - treated sensing gate was washed with dmf and deionized water for 20 s. this gol was then treated with 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide ( edc ) hcl and sulfo - n - hydroxysulfosuccinimide ( nhs ) chemistry.18 gol after the addition of edc and sulfo - nhs was identified as a surface - treated gol ( st - gol ) .
antibodies were then added to the wells . here , we used prostate - specific membrane antigen ( psma ) antibodies
it had an igg2b isotype structure and was specific to the c - terminal of a psma antigen .
the psma antibody was conjugated onto the sensing gate surface at a concentration of 100 nm . to prevent unwanted reactions with unreacted chemical cross - linkers left on the surface
, 1 m ethanolamine ( ph 8.5 ) was added . after adding the ethanolamine to the st - gol ,
the st - gol was washed with 1 pbs . in order to protect the st - gol from nonspecific binding ,
10% bovine serum albumin ( bsa ) was added and left to undergo the chemical reaction for 1 h. finally , the st - gol was washed with 1 pbs ( figure 2 ) . a commercial ag / agcl reference electrode ( qrins , mf-2097 ) , a commercial cmos transistor ( on semiconductor , mc14007ub , n - type mosfet ) , and a high precision semiconductor parameter analyzer ( 4200-scs ; keysight technologies , santa rosa , ca
the experiment was conducted under different conditions . in the initial condition , the vdr of the i 
v curves was measured at the 0 , 1 , 3 , and 5 min marks after inserting a 0.01 pbs solution into pdms reservoirs on all the sensing gates . in the second condition ,
the 0.01 pbs was replaced with 1 pbs and the solution was added into the pdms reservoirs on all the sensing gates .
the measurement was taken at the same time points as the initial condition ( 0 , 1 , 3 , and 5 min marks ) .
the chemical compositions of the pbs solution are as follows : 1 ) potassium phosphate monobasic ( kh2po4 ) 1.0589 mm , 2 ) sodium chloride ( nacl ) 155.172 mm , and 3 ) sodium phosphate dibasic ( na2hpo47h2o ) 2.9665 mm .
in addition , to measure the vdf difference between the different surface treatments of the gol , 1 pbs was added to bare gol , st - gol without antibodies , and st - gol with antibodies .
the vdr was measured at the 0 , 1 , 3 , 5 , and 10 min marks ( figure 1 ) .
the gol of an isfet biosensor was fabricated by depositing an sno2 ( 99.9% ) thin film on the top of an indium tin oxide ( ito ) glass ( ito thickness : 300 nm ) .
the 80 nm sno2 film was deposited using a radio frequency ( rf ) magnetron sputtering system ( rf power : 50 w , base pressure : 210 torr , work pressure : 18 mtorr , ar gas flow : 5 sccm ) . the polydimethylsiloxane ( pdms ) block was manufactured from a sylgard 184 silicone elastomer kit ( dow corning , seoul , republic of korea ) , which was composed of a base and a curing agent .
the base and curing agent were mixed at a 10:1 ratio and baked at 60c for 4 h. the pdms block was then punctured with 6 mm holes to form a sample media reservoir . to attach the pdms to the surface of gol ,
the gol was sonicated in deionized water for 1 min and then sonicated in ethanol for 1 min .
the pdms block and gol were then placed inside an o2 plasma system ( cute - mpr ; femto science , gyeonggi - do , republic of korea ) and treated with o2 plasma ( dc power : 70 w , time : 1 min , o2 flow : 30 sccm , base pressure : 510 torr , working pressure : 510 torr ) .
the pdms block and gol were then attached together to form the sensing gate ( figure 1 ) .
the gol surface of the sensing gate was treated with plasma to form oh functional groups on the reservoir surface . then , 5% 3-aminopropyltriethoxysilane ( aptes ) was prepared and quickly added to the surface of the gol for the formation of nh2 functional groups .
after the addition of the aptes , the gol was placed inside a petri dish and the petri dish was sealed with parafilm for 1 h. the chemical reaction should occur in a dark place .
after 1 h , the sensing gate was sonicated in ethanol for 1 min and dried with n2 gas . the sensing gate was baked on a hot plate at 120c . to functionalize the gol surface of the sensing gate with cooh functional groups , 5% 1 m succinic anhydride was made from succinic anhydride powder and dimethyl formamide ( dmf ) and added onto the surface of the gol .
the petri dish was sealed with parafilm and placed inside an incubator at 37c for overnight .
the presurface - treated sensing gate was washed with dmf and deionized water for 20 s. this gol was then treated with 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide ( edc ) hcl and sulfo - n - hydroxysulfosuccinimide ( nhs ) chemistry.18 gol after the addition of edc and sulfo - nhs was identified as a surface - treated gol ( st - gol ) .
antibodies were then added to the wells . here , we used prostate - specific membrane antigen ( psma ) antibodies .
it had an igg2b isotype structure and was specific to the c - terminal of a psma antigen .
the psma antibody was conjugated onto the sensing gate surface at a concentration of 100 nm . to prevent unwanted reactions with unreacted chemical cross - linkers left on the surface
, 1 m ethanolamine ( ph 8.5 ) was added . after adding the ethanolamine to the st - gol ,
the st - gol was washed with 1 pbs . in order to protect the st - gol from nonspecific binding ,
10% bovine serum albumin ( bsa ) was added and left to undergo the chemical reaction for 1 h. finally , the st - gol was washed with 1 pbs ( figure 2 ) .
a commercial ag / agcl reference electrode ( qrins , mf-2097 ) , a commercial cmos transistor ( on semiconductor , mc14007ub , n - type mosfet ) , and a high precision semiconductor parameter analyzer ( 4200-scs ; keysight technologies , santa rosa , ca , usa ) were utilized for the measurement of the vdf .
the experiment was conducted under different conditions . in the initial condition , the vdr of the i 
v curves was measured at the 0 , 1 , 3 , and 5 min marks after inserting a 0.01 pbs solution into pdms reservoirs on all the sensing gates . in the second condition ,
the 0.01 pbs was replaced with 1 pbs and the solution was added into the pdms reservoirs on all the sensing gates .
the measurement was taken at the same time points as the initial condition ( 0 , 1 , 3 , and 5 min marks ) .
the chemical compositions of the pbs solution are as follows : 1 ) potassium phosphate monobasic ( kh2po4 ) 1.0589 mm , 2 ) sodium chloride ( nacl ) 155.172 mm , and 3 ) sodium phosphate dibasic ( na2hpo47h2o ) 2.9665 mm .
in addition , to measure the vdf difference between the different surface treatments of the gol , 1 pbs was added to bare gol , st - gol without antibodies , and st - gol with antibodies .
the vdr was measured at the 0 , 1 , 3 , 5 , and 10 min marks ( figure 1 ) .
we investigated the vdf of a bare gol , an st - gol without antibodies , and an st - gol with antibodies in 0.01 pbs ( figure 3a  c ) . after adding 0.01 pbs into all respective pdms reservoirs of the gol and waiting for 1 min , the voltage values were measured .
subsequent analyses were conducted in 2 min intervals and repeated two times to identify the vdf . in the case of the bare gol , a large vdf of 21.5 mv for 5 min ( 4.3 mv / min ) was observed .
this error value reaches 36.3% of an fet s nernst limit ( 59.3 mv / ph).19,20 this result implies that in a situation where sensing is conducted in a low antigen concentration range , which is lower than the ionic concentration change range following a change in 1 ph , it is hard to obtain a trustworthy sensing signal .
an additional phenomenon that was observed was the consistent shift of the vdf as time passes .
this is because the nonpreconditioned gol has an unstable surface and , thus , reacts with numerous ions of the pbs ( oh , h , and o2 ) , for a long time .
this phenomenon contributes to sensors being unable to produce reliable results when exposed to extended periods of testing . in the cases of the st - gol without antibodies and the st - gol with antibodies ,
the st - gol without antibodies showed a vdf of 11.37 mv/5 min ( 2.3 mv / min ) , and the st - gol with antibodies showed a vdf 7.13 mv/5 min ( 1.4 mv / min ) .
these values are only 19.2% of the nernst limit and 12% of the nernst limit , respectively .
the reasons for the decreased vdf can be thought to be due to the following .
first , the random chemical reactions between the chemical links on the surface of gol and ions of the pbs were restricted by the presurface treatment .
second , the number of residual chemical links that can react and bind to the ions within the pbs is limited and this causes saturation to occur very quickly .
figure 4a and b shows the vdf values of adding undiluted 1 pbs solution to an st - gol without antibodies and to an st - gol with antibodies .
the experiment was conducted under the same conditions as the experiment of figure 3 . as can be seen from the results ,
the vdf results are much smaller than the vdf of its 0.01 pbs counterpart : the st - gol without antibodies has a vdf of 0.37 mv / min and the st - gol with antibodies has a vdf of 0.33 mv / min .
the significant reduction of the vdf values is because of the debye screening length in the 1 pbs .
the 1 pbs has the much smaller debye screening length of 0.7 nm than 0.01 pbs s debye screening length of ~7.3 nm.21,22 consequentially , the gol can only detect much smaller ions in 1 pbs , and as a result , the chemical reaction is rapidly saturated ( or stabilized ) .
figure 5 shows the results for all gol vdf values that were measured at the 0 , 1 , 3 , 5 , 10 , 15 , and 20 min marks in 1 pbs . for the bare gol ,
the chemical links of the sno2 surface reacted continuously with the ions in the pbs and the vdf increased as time passed .
. however , gol that has undergone presurface treatment showed complete saturation by the ions in 1 pbs by the 1 min mark . consequently , a presurface - treated gol utilizing 1 pbs can decrease the noise from unwanted ions and produce a reliable biosensing signal .
the results clearly indicate that in scenarios where the isfet is used to sense biomarkers , the undiluted 1 pbs could produce trustworthy biosensing signals . however , the 1 pbs will lead to a much shorter debye screening length and subsequent biomarker detection limit .
this can lead to problems if a lengthy or bulky bioconjugate , such as whole antibodies , is utilized . to solve the problem from the short debye screening length , small capture linkers , such as antibody fragments , aptamers , and peptides ,
this study used sno2 as a gate oxide sensing layer in an isfet biosensor to capture biomarkers and analyze their biosignals .
the results demonstrate that proper surface treatment of a gate oxide sensing layer in an isfet can significantly reduce the sensing signal error . because the amounts of biomarkers in samples are usually minuscule , a biosensor must be sensitive enough to detect these biomarkers at low concentrations . to produce a longer debye screening length for the isfet biosensor ,
the sample media , which were the pbs solutions in this study , are diluted .
however , this could lead to a situation where the media itself can cause both a large vdf and a large margin of error , thus making the resulting biosensing signal unreliable . to decrease such error from the media , a more concentrated medium , such as 1 pbs ,
the 1 pbs medium quickly reacts and saturates the surface of sensing gate and subsequently stabilizes the electrical potential .
an unfortunate side result from adding the concentrated medium is the shortening of the debye screening length . to overcome
this obstacle , aptamers , peptides , and antibody fragments and immobilization materials that are shorter than a whole antibody need to be utilized to maintain a reliable sensor sensitivity outcome .
 an ion - sensitive field - effect transistor ( isfet ) biosensor is thought to be the center of the next era of health diagnosis . 
 however , questions are raised about its functions and reliability in liquid samples . 
 consequently , real - life clinical applications are few in number . in this study 
 , we report a strategy to minimize the sensing signal drift error during bioanalyte detection in an isfet biosensor . 
 a nanoscale sno2 thin film is used as a gate oxide layer ( gol ) , and the surface of the gol is chemically modified for improving bioanalyte - specific binding and for reducing undesirable ion reactions in sample solutions . 
 the isfet biosensor with surface - modified gol shows significantly reduced sensing signal error compared with an isfet with bare gol in both diluted and undiluted phosphate buffered saline solutions . 
Introduction
Experimental
Sensing gate fabrication
Presurface treatment of GOL and antibody fixation
Measurement
Results and discussion
Conclusion