Electrochemical Characterization of Nanogap Interdigitated Electrode Arrays for Lab-on-a-Chip Applications

In this work we present recent results on electrochemical characterization of the fabricated nanogap interdigitated electrode arrays (nIDAs) for Lab-on-a-Chip applications (LoC). The advantages of the presented nIDAs and their potential for application in bioelectrochemistry were studied using different electrochemical methods. Chronoamperometry is applied to achieve reversible redox processes which lead to an ampliﬁcation of the measured current compared to a single electrode conﬁguration and as a result, an increase in signal-to-noise ratio. For the electrochemical characterization of the created nIDAs ferrocenemethanol (FcMeOH) and p-aminophenol(pAP)were selected asredoxcouples.An ampliﬁcation factorabove160comparedtoa singleelectrode conﬁguration was obtained with FcMeOH for the nIDAs with 100 nm gap. An inverse correlation between gap size and ampliﬁcation was proven. In addition, the formation of a polymer ﬁlm during redox cycling could be veriﬁed to cause the lower ampliﬁcation factor observed in pAPmeasurements.Furthermore,differentelectrodecleaningproceduresweredemonstratedbycombinationofO 2 plasmaandcyclic voltammetry. The nIDAs presented here offer many advantages including low-cost fabrication, high ampliﬁcation and collection factors and thus are highly suitable for biosensor applications. © the rapid, and quantitative detection of

The development of screening tools for the rapid, sensitive and quantitative detection of the biologically relevant molecules is crucial in various fields of human and veterinary medicine (diagnostic of diseases), pharmacology (mechanism of drug action), food technology (detection and quantification of antibiotic residues in veterinary products) or agriculture (plant viruses). [1][2][3][4][5][6][7] In the last two decades, biosensors based on the electrochemical transduction methods have been extensively applied as a platform for the detection of such types of molecules. [6][7][8][9][10][11] Electrochemical biosensors provide an attractive way to analyze the content of a biological sample due to the direct conversion of a biological event to an electrical signal. 12,13 The operation principle of the electrochemical biosensors used for medical diagnostics is typically based on enzyme-linked bioassay systems. Here, the biomolecular interaction, which occurs in close proximity to the electrode surface of the sensor chip (transducer), leads to the enzyme-catalyzed conversion of an inactive mediator to a redox active substance. The formed redox active molecules are reduced or oxidized at an electrode polarized at the appropriate potential. The current measured during the redox process directly correlates with the concentration of the analyte. The detection and quantification limits of the biologically relevant species are mainly defined by the signalto-noise ratio (SNR) of the electrochemical measurement.
A significant signal amplification as well as an increase of the signal-to-noise ratio can be achieved due to the reversible redox process employing interdigitated electrode arrays (IDAs). 8 In that case, the multiple oxidation and reduction of the redox active substance (redox cycling) occur at closely spaced parallel finger electrodes. During redox cycling the species generated at one electrode (the generator) diffuses to the neighboring electrode (the collector) where it is regenerated back to the initial form present in the bulk solution. The enhanced cross-talk between electrodes leads to a reduced diffusion flux of species toward the bulk solution over time. Consequently, the steady-state regime is rapidly reached in the generator-collector mode. 14 The effectivity of that process can be characterized through collection efficiency representing the ratio of currents at the collector and generator electrodes. Bard et al. 15 and Aoki et al. 16 have shown that a short average diffusion length and a large number of alternating electrodes are necessary conditions for effective current amplification z E-mail: volha.matylitskaya@fhv.at in implementing reversible redox processes. A number of experimental and theoretical works have demonstrated the advantages of such electrode assemblies, which result from the specific mass transport of electroactive materials and diffusion regimes occurring at their interface. [14][15][16][17][18][19][20][21][22][23] In this manner, IDAs offer many distinctive features including fast response time, low capacitive current, high collection efficiency, and higher signal-to-noise ratio compared to the single electrode configuration. 23,24 Bard et al. 15 and later other research groups 16,[25][26][27][28][29][30][31] have shown that the gap between the electrodes determines the amplification factor during redox cycling, while the width of the IDAs only plays a minor role. Reducing the gap size leads to an intensified feedback between the electrodes and as result to a decrease of the equilibrium time and an increase in collection efficiency. 16,30 It is expected that the shrinking of gap width to nanometer range will lead to an exponential growth of the amplification factor. 9 For these reasons, the development of a simple and low-cost fabrication method of the nanogap IDAs (nIDAs) as well as the electrochemical study of these special devices has become essential. Here, we present the recent results on the electrochemical characterization of the fabricated nIDAs for Lab-on-a-Chip applications. The interdigitated electrode arrays with adjustable nanogaps between the finger electrodes were created using conventional and at the same time lowcost microfabrication techniques. The developed fabrication method is a combination of an initial structuring and two subsequent deposition steps. It is particular due to the complete elimination of the critical lift-off process. 31,32 Cyclic voltammetry (CV) and chronoamperometry (CA) were used for the electrochemical characterization of the fabricated nIDAs. Due to the fact that the operation principle of the final nIDA sensor is based on an enzyme-linked bioassay system, p-aminophenol (pAP) was selected as one of redox substances to be tested. The enzymatic production of pAP, a redox active molecule, from p-aminophenyl-beta-D-galactopyranoside (pAPG) related to the capture step has been shown to be a promising approach for biosensor application. [33][34][35][36] For the characterization of the nIDAs we used ferrocenemethanol (FcMeOH) as reference redox couple.

Materials and Methods
Chip fabrication and design.-A 100 mm silicon substrate was spin coated with MEGAPOSIT SPR955-CM photoresist (purchased  from micro resist technology, Germany). The final thickness of the photosensitive layer was 650 nm (measured with DekTak 8, Bruker, USA). Afterwards, the photoresist was exposed in vacuum contact mode with a SUSS Mask Aligner MA6 utilizing MO Exposure Optics and an i-line filter. The required photomask with a minimum feature size of 1 μm was fabricated by Compugraphics Jena, Germany. A dose of 130 mJ cm −2 in combination with a 120 s post exposure bake on a hotplate (contact mode) provides an optimal pattern transfer. A subsequent development process in AZ 726MIF for 60 s leads to the final photoresist pattern. The pattern was then etched into silicon by a DRIE process (Bosch process) with gas chopping of SF 6 and C 4 F 8 in an ICP etcher Adixon AMS100 (Alcatel Vacuum technology, France). An etch time of 52 s results in an etch depth of 1 μm. The remaining photoresist was removed by O 2 plasma in the ICP etcher. The subsequent sputter deposition process was carried out on an LLS EVO system (Oerlikon Balzers, Liechtenstein). The deposited film forms the undercut and the nanogap between the elevated finger structures. An SiO 2 layer thickness of 750 nm results in nanogaps of about 100 nm in width. The second deposition step which forms the 100 nm thick platinum electrodes was done on a Univex 500 evaporator (Oerlikon Leybold Vacuum GmbH, Germany). To define the measurement area, the reference electrode and the contact pads, the wafer was spin coated with SU8-3005 with a final thickness of 5 μm. The soft bake was carried out on a hot plate in contact mode for 120 s at a temperature of 95 • C. A dose of 250 mJ cm −2 is utilized, followed by a post exposure bake for 120 s at 95 • C. The development takes place in mr-Dev 600 (purchased from micro resist technology, Germany) for 60 s and a subsequent isopropanol rinse. Individual sensor chips were prepared by a fs-laser dicing process (microSTRUCTvario, 3D-Micromac, Germany). The silver/silver chloride (Ag/AgCl) reference electrode with 90% of Ag and 10% of AgCl was manually dispensed (paste purchased from Gwent Electronic Materials Ltd., United Kingdom). Figure 1 illustrates the design of the fabricated nanogap IDA chip with its main elements. The size of a single chip is 3.7 mm × 20 mm.
Each created nIDA chip is a four-electrode system with two working electrodes (WE1 and WE2), a reference electrode (RE) and a counter electrode (CE). The counter and working electrodes are thin film platinum electrodes with a thickness of 100 nm. Both working electrodes represent an IDA pattern with 300 finger pairs. The distance between neighboring fingers is in the nanometer range.
Electrochemical characterization.-All chips were cleaned in O 2 plasma for 10 s before electrochemical measurements in order to remove photoresist residues after the lithography step. This step was performed on a deep reactive ion etching (DRIE) ICP system Adixen AMS100 (Alcatel Vacuum Technology, France).
The electrochemical measurements were performed in a custommade Faraday cage using EmStat 3 4WE potentiostat (PalmSens BV, the Netherlands). The cyclic voltammetry with a scan rate of 0.1 V s −1 was performed to determine the oxidation and reduction potentials of the examined redox couples. All reported potentials are measured versus an Ag/AgCl reference electrode at room temperature. The cyclovoltammetric measurements in generator/collector mode were applied to obtain information about electron transfer during the reversible redox process. In this mode, one working electrode (generator) is scanned while the second working electrode (collector) is fixed at a constant potential (i.e., at the reduction potential of the employed redox couples). Furthermore, chronoamperometric measurements were carried out to determine signal amplification and collection efficiency by applying constant potentials to both generator and collector electrodes. On working electrode 1 (WE1) the oxidation potential and on working electrode 2 (WE2) the reduction potential were applied. Both cyclovoltammetric and chronoamperometric measurements were carried out in bulk solutions (the sensor was dipped into the solution).
The platinum nIDAs were characterized using 1 mM solutions of ferrocenemethanol and p-aminophenol. Both chemicals, FcMeOH and pAP, were purchased from Sigma-Aldrich, Germany. As a solvent 0.1 M phosphate buffered saline (PBS) solution containing 0.1 M sodium chloride (NaCl), 0.085 M sodium hydrogen phosphate (Na 2 HPO 4 ) and 0.015 M sodium dihydrogen phosphate (NaH 2 PO 4 ) was used. The substances used for preparation of the PBS solution were obtained from Lactan, Austria.

Parameters applied for the measurements with 1 mM FcMeOH.-
The scan range from 0.0 V to +0.3 V (scan rate 0.1 V s −1 ) was applied for the cyclovoltammetric measurements with FcMeOH.
For redox cycling experiments in generator/collector mode the generator (WE1) was swept from 0.0 V to +0.3 V (scan rate 0.1 V s −1 ) while the collector (WE2) was held at the reduction potential of 0.0 V.
For chronoamperometric measurements the oxidation potential of +0.3 V and the reduction potential of 0.0 V were applied to generator and collector electrodes respectively.
The two-step cleaning procedure (1 st step -cleaning in O 2 plasma for 10 s; 2 nd step -cleaning scan in 0.1 M PBS in potential range from −0.4 V to +0.4 V (CP I)) was developed and applied for the electrochemical characterization of Pt nIDAs with FcMeOH.
Parameters applied for the measurements with 1 mM pAP.-The cyclovoltammetric measurements were performed in the scan range from −0.3 V to +0.4 V (scan rate 0.1 V s −1 ). WE1 and WE2 were swept simultaneously.
For redox cyclic experiments in generator and collector mode WE1 was swept from −0.35 V to +0.5 V with the scan rate of 0.1 V s −1 while WE2 was fixed at −0.35 V.
The oxidation potential of +0.4 V (WE1) and reduction potential of −0.3 V were set as optimal for the chronoamperometric measurements with pAP.
The two-step cleaning procedure (1 st step -cleaning in O 2 plasma for 10 s; 2 nd step -cleaning scan in 0.1 M PBS in potential range from −0.7 V to +0.9 V (CP II)) was developed and applied for the electrochemical measurements with pAP.

Results and Discussion
Electrochemical characterization of platinum nIDAs in ferrocenemethanol.-The initial characterization of the fabricated platinum nanogap IDAs was performed using 1 mM solution of ferrocenemethanol in 0.1 M PBS. Here, various gap distances were tested. However, we are only referring to the 100 nm gap distance for FcMeOH.
Cyclovoltammetric measurements were carried out to determine the oxidation and reduction potentials of FcMeOH (see Fig. S1). Here, only WE1 was swept through the selected potential range while WE2 was switched off. On the basis of the obtained cyclic voltammograms, the oxidation potential of +0.3 V and the reduction potential of 0.0 V versus the Ag/AgCl reference electrode were set as optimal potentials for the subsequent electrochemical measurements of FcMeOH.
The redox cycling experiments in generator/collector mode were applied to obtain more information about the electron transfer between finger electrodes during reversible redox process (Fig. 2a, red dashed curve). The shape of the received diagram indicates that the redox process at the surface of the finger electrodes is limited by reaction kinetics. The increase in current at the generator electrode occurs over the entire potential range.
For further characterization of the Pt nIDAs and to get an efficiency estimation of the reversible redox process chronoamperometric measurements were carried out. Generally, two parameters are significant for the characterization of chronoamperometric measurements: the amplification factor (ampl(t)) and the collection efficiency (coll(t)). The amplification factor is defined as the ratio between the current at the generator electrode during redox cycling and the current at the working electrode in single mode. 14 The current values in steady state were used for the calculations (Equation 1). The redox cycling amperometry of the IDA with 100 nm electrode gap leads to a signal amplification of about 130-fold compared to the single mode amperometry (Fig. 2b, red dashed curve). The collection efficiency reflects the capture yield of an electroactive substance produced at the generator (WE1) and gathered at the collector (WE2) during active redox cycling (Equation 2). 14 The collection efficiency of 99.6% is determined for the tested Pt nIDA in 1 mM FcMeOH. The obtained value is in agreement with previous results received for gold nIDAs. 31 ampl(t) = I 180 s / I 90 s [1] coll(t) = I WE2 at 180 s / I WE1 at 180 s [2] Despite the high amplification factor achieved in the chronoamperometric measurements of 1 mM FcMeOH, the results of the cyclovoltammetric measurements in generator/collector mode point out a reduced electron transfer during redox cycling at the Pt nIDA (Fig. 2a, red dashed curve). Since the redox reaction occurs in close proximity to the electrode surface, clearness of the electrode surface plays a crucial role in the electron exchange between electroactive substance and electrode. Therefore, all fabricated chips were cleaned in O 2 plasma before the electrochemical measurements. However, it is also known that treatment of platinum in O 2 plasma leads to the formation of an ultra-thin platinum oxide film on the metal surface. [37][38][39] In order to remove the oxide film from the surface of the Pt nIDAs, electrochemical cleaning procedure using cyclic voltammetry in 0.1 M PBS solution in a potential range from −0.4 V to +0.4 V was applied (Fig. 3). A reduction peak at −0.36 V can be observed at the first scan of a Pt nIDA (Fig. 3,   blue dashed curve). We attribute this peak to a reduction process of platinum oxide formed during the cleaning step in oxygen plasma. The absence of such a reduction peak in the second scan (Fig. 3, black solid curve) and the following scans supports our assumption.
After the electrochemical cleaning procedure, the CV measurement in generator/collector mode as well as the CA measurement was repeated by applying the same parameters as before. The cyclic voltammogram obtained in redox cycling mode (Fig. 2a, black solid curve) shows relatively stable current at potentials above +0.2 V. This indicates that the redox process is only limited by diffusion of electroactive species to the electrodes (electron transfer is not kinetically hindered). This can be also confirmed by the significant increase of the amplification factor (Fig. 2b, black solid curve). Here, a signal amplification of 161-fold is achieved. This is the highest amplification factor measured by redox cycling with interdigitated electrode arrays so far.
Electrochemical characterization of platinum nIDAs in p-aminophenol.-As already mentioned, the operation principle of the final nIDA biosensors is based on the enzyme-linked bioassay protocol. p-Aminophenol is frequently used as an electroactive substance for this kind of analysis. [33][34][35][36] Herein, the fabricated Pt nIDAs were measured with 1 mM pAP in 0.1 M PBS solution. According to the previous results obtained with FcMeOH (indication of thin platinum oxide layer formation during O 2 plasma cleaning) the influence of the electrochemical cleaning scan in PBS (CP I) on the amplification factor achievable with pAP was also investigated.
In contrast to FcMeOH, no effect of this cleaning step on the signal amplification at chronoamperometric measurement with pAP was observed. On the other hand, the cyclovoltammetric measurement in generator/collector mode as well as the chronoamperometric measurement (Fig. 4, red dashed curves) proves a kinetically limited redox reaction of pAP. The permanently increasing current in the CV measurement in generator/collector mode (Fig. 4a, red dashed curve) and the low (34-fold) amplification factor (Fig. 4b, red dashed curve) indicate a reduced electron transfer for the redox cycling of pAP. This leads us to the conclusion that the two-step cleaning procedure CP I developed for the electrochemical characterization of Pt nIDAs in FcMeOH is not sufficient for electrochemical measurements of Pt nIDAs in pAP.
To overcome this issue, an extended electrochemical cleaning procedure with a wide potential range between −0.7 V and +0.9 V was applied to the Pt nIDAs after the O 2 plasma treatment (Fig. 5). Two reduction peaks at −0.31 V and at −0.4 V were observed at the curve representing the first scan in PBS (Fig. 5, blue dashed curve). In contrast, the cyclic voltammogram obtained for a smaller cleaning potential range (−0.4 V to +0.4 V) shows only one reduction peak at −0.36 V (Fig. 3, blue dashed curve). This leads us to the conclusion that a complete reduction of the oxide formed in the O 2 plasma treatment occurs at the 1 st scan in PBS in the potential range of −0.7 V to +0.9 V.
In order to validate this hypothesis, the previously used chip for p-aminophenol measurements (Fig. 4, red dashed curves) was electrochemically cleaned again in 0.1 M PBS applying the extended potential range (from −0.7 V to +0.9 V). After this cleaning step the CV measurement in generator/collector mode as well as the CA measurement was repeated. An almost two-fold increase of the measured current was observed in both generator/collector experiments (Fig. 4, black solid curves). This leads to an increase of the amplification factor to the same level (Fig. 4b). Furthermore, the improved cleaning procedure results in an enhancement of the collection efficiency from 98.5% to 99.8%. The CV in generator/collector mode (Fig. 4a, black solid curve) indicates that the previously observed reduced electron transfer increases and therefore, results in a higher current density.
On the basis of the obtained results all chips were cleaned in O 2 plasma followed by a subsequent electrochemical cleaning in the wide potential range (CP II). Based on the experimental data the oxidation potential of +0.4 V and the reduction potential of −0.3 V versus the Ag/AgCl reference electrode were set as the optimal conditions for the electrochemical measurements of pAP.
We observed that p-aminophenol shows some phenomena during redox cycling compared to other tested redox couples such as ferrocenemethanol, 1,1´-ferrocenedimethanol and hexaammineruthenium(III) chloride. Figure 6 illustrates two cyclic voltammograms of a Pt nIDA measured under identical conditions: WE1 and WE2 were swept simultaneously from −0.3 V to +0.4 V. In the first case (Fig. 6a) the cyclic voltammetry was carried out directly after O 2 plasma and electrochemical cleaning of the Pt nIDA. Here, the same current flows through both electrodes, the curves of WE1 (black solid line) and WE2 (red dashed line) overlap with each other (Fig. 6a). In the second case (Fig. 6b) the cyclovoltammetric measurement was performed after the chip cleaning procedure described above and several subsequent electrochemical measurements. The current at WE1 (Fig. 6b, black solid line) is significantly lower than the current at WE2 (Fig. 6b, red dashed line). This signal decrease of WE1 could occur due to the previously performed chronoamperometric measurements, where the WE1 was set as anode at a constant potential.
The chronoamperometric measurements in pAP solution were performed in reverse order compared to the measurements with ferrocenemethanol. The measurement starts with the redox cycling of pAP (two electrode mode), after that single mode amperometry is done. In redox cycling mode WE1 is fixed at the oxidation potential of +0.4 V and WE2 is kept at the reduction potential of −0.3 V. In single mode only WE1 is switched on. Equation 3 was used for the calculation of the signal amplification in pAP. ampl(t) = I 90 s / I 180 s [3] It was observed that the measuring sequence significantly influences the amplification factor of the Pt nIDAs for pAP. Figure 7a represents two chronoamperometric measurements. The first measurement (Fig. 7a, black solid line) was performed directly after the two-step cleaning procedure developed for the electrochemical characterization of the Pt nIDAs with pAP. The red dashed line (Fig. 7a) shows the results obtained for the cleaned chip after two cyclovoltammetric measurements with pAP, in normal and in generator/collector modes. In the second case the amplification factor is significantly lower than the measurement after the cleaning procedure (78 instead of 99). Furthermore, a decrease in both current and signal amplification after each performed CA measurement has been observed. Figure 7b represents three of six CA measurements of such experiment series. Here, the amplification factor decreases from 99 at the 1 st measurement down to an 83 at the 6 th measurement.
To explain these phenomena occurring during redox cycling of pAP, the measured nIDAs were inspected by SEM (Fig. 8). On the surface of some chips residual fibers were found (Fig. 8b). The width of these fibers (see Fig. S2a) is comparable to the width of the finger electrodes (Fig. 8a) (about 1.5 μm). Energy dispersive X-ray analysis (EDX) was used to estimate the chemical composition of these fibers (see Fig. S2b) and was compared to the composition of the fiber-free surface (see Fig. S2c). The presence of carbon, nitrogen and an increased content of oxygen compared to the fiber-free surface was detected. The ratio of elements present in the fiber correlates with the ratio of elements in p-aminophenol. Therefore, we conclude that the formation of a polymer film takes place during CV measurements of pAP. Our observations are in good agreement with previously published works of other research groups. [40][41][42][43][44][45][46][47] The chemical and electrochemical pathways of multistage electrooxidation of pAP via p-quinoneimine and p-benzoquinone to amine-substituted quinones films are well described in the literature. 42,[47][48][49][50][51] The polymer film formed during redox cycling influences the electron transfer between pAP and electrodes. Therefore, the maximum signal amplification with pAP was achieved in those measurements carried out directly after the chip cleaning procedure. The formation of the polymer film explains also the lower amplification factor which has been obtained with pAP for the 100 nm gap IDA compared to ferrocenemethanol. The amplification factor of 118 was measured with pAP (see Fig. S3) while a 161 amplification for the same chip was achieved with FcMeOH ( Fig. 2b, black solid line). Because of the formation of a polymer film during redox cycling of pAP the reverse mode order for the CA measurements with pAP was applied in contrast to the measurements with FcMeOH (comparison of Fig. 2b and Fig. 7). If the chronoamperometric measurement starts with the single mode amperometry, the polymer film formed at the anode hinders the electron transfer during following redox cycling of pAP (two electrode mode).
The current gain during active redox cycling of pAP (two electrode mode) was studied over a concentration range of 100 nM to 2 mM. The calibration plot (see Fig. S4) shows nearly constant signal amplification in the range of 1 μM to 2 mM. For the concentration of 100 nM a decrease of the amplification was observed.

Correlation between the gap size and obtained amplification
factor.-For the selected chips the correlation between gap size and amplification factor was estimated. Table I represents our results obtained from the chronoamperometric measurements of Pt nIDAs in 1 mM solution of FcMeOH. The mean of the amplification factor and standard deviation were calculated for three successive chronoam-  Table II were achieved by the chronoamperometric measurements done directly after CV cleaning procedure. The mean and standard deviation values weren't calculated for pAP since decrease of current with each performed measurement was observed. This behavior can be attributed to the formation of polymer film during redox cycling of pAP. In both cases, FcMeOH and pAP, the amplification factor increases when the gap size between the electrodes shrinks.

Conclusions
In this work we present our recent results on the electrochemical characterization of the fabricated Pt nanogap IDAs. We demonstrated the potential of high signal amplification of our fabricated nIDAs by chronoamperometric measurements of the nowadays used redox couples. Two electroactive substances, 1 mM solutions of ferrocenemethanol and p-aminophenol in 0.1 M PBS, were used for this purpose. It was shown that the cleanness of the chip surface influences the signal amplification achievable with FcMeOH as well as with pAP. Therefore, suitable cleaning procedures were developed and optimized for both tested substances. In both cases the cleaning process is a combination of an initial cleaning in O 2 plasma for 10 s and a subsequent electrochemical cleaning in 0.1 M PBS solution. The optimal scan range for the cleaning of Pt nIDAs measured with FcMeOH is from −0.4 V to +0.4 V. The chips characterized with pAP were electrochemically cleaned in a wider potential range (−0.7 V to +0.9 V).
It was also confirmed that an inverse correlation between gap size and achievable amplification factor exists. The highest amplification was measured for a Pt IDA with 100 nm gap size between electrodes. A signal amplification of 161-fold was determined for this chip with FcMeOH. To the best of our knowledge, this is the highest amplification achieved by CA measurements in bulk solutions using interdigitated electrodes (the sensor was dipped into the solution) reported so far. For the same nIDA a significantly lower amplification factor was observed for p-aminophenol compared to FcMeOH. This lower value could be explained by the formation of a polymer film during redox cycling of pAP which results in a reduced electron transfer. The results of the electrochemical measurements as well as the results of the SEM and EDX prove the presence of such a polymer film. It has been verified that the cleanness of the IDA surface and measuring sequence strongly influence the signal amplification for pAP.
To conclude, the results presented in this work prove not only high signal amplification but also high collection efficiency of more than 99% for both tested redox couples. Therefore, the fabricated nIDA is a promising candidate for biosensing applications.