Electrochemical Characterization for Determination of PNA Surface Density and Rate Electron Transfer Constant Based on Methylene Blue Accumulation Charge

Here we report the PNA surface density and ratio of EDC/NHS was used as coupling agent in electrochemical application based on methylene blue (MB) accumulation charge. In this study, chronouclometry technique has been used to calculate PNA probe density to obtain optimum PNA surface density for electrochemical sensor application. Furthermore, kinetic behavior of PNA electrochemical biosensor also have been investigated to determine the rate electron transfer of MB before and after hybridization. The obtained data revealed that the PNA bound to modified electrode at optimum probe concentration activated at 5/5 mM of EDC/NHS to give highest electrochemical signal. Meanwhile the MB rate electron transfer was obtained in this study was about 2.9 s−1 before and 2.5 s-1 after hybridization by using laviron approach. These result demonstrated that MB has intercalated into DNA through long range charge transfer.


Introduction
Probe density is one of important factor in order to improve detection signal in electrochemical biosensor [1]. Instead of DNA, PNA has difference properties due to its neutral backbone and proper interbase spacing, PNA binds to its complementary nucleic acid sequence with higher affinity and specificity compared to traditional oligonucleotides [2]. The use cross-linking and conjugation of method to immobilization of NH 2 -containing biomolecules onto carboxyl-containing substrates via covalent amide bond by using EDC and NHS most widely used to control over the immobilization of the probe oligonucleotides. However, the amount of EDC and NHS been used to bound amine biomolecule in previous study seem different between modified electrode.
In previous report, wide range of EDC/NHS concentration from M to the mM range from one study to another has been studied.
Voicu, et al. [3] used 0.1M NHS and 0.4 M EDC in order to activate silicon surface. In another study, equal amounts of EDC and NHS (100mM) were used for activation of carboxylic acid terminated self-assembled monolayers [4]. Therefore, in this study, we shared result obtained of amount of 1-ethyl-(dimethylaminopropyl) carbodiimide hydrcholoride (EDC) and N-hydroxy succinimide (NHS) used as controlling agent to determine PNA probe density on SPCE modified graphene quantum dots nanomaterial once the carboxyl groups to produce NHSS-ester [5] (Figure 1). Additionally, the rate of methylene blue (MB) also been investigated. Q nFAT = where Q is the charge which was obtained from choronouclometry measurement, n is the number of electrons in the reaction, and A is the surface area of fabricated electrode (0.12cm 2 ).
While based on scan rate study, calculation on laviron approach [7] to determine the electron transfer rate has been determined by cyclic voltammetry. The measurement was carried out by Autolab PGSTAT30 (Netherlands) which is con-trolled using computer. showed decreasing of DNA probe density has led to increasing electrochemical signal due to lack of bulk steric effect [9].

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In addition, PNA is also well known with the natural charge contradict with DNA properties typically with carry negative charge, therefore electrostatic repulsion would not be an issue at high density probe. Therefore, higher probe density ought to yield higher MB accumulation charge and enhanced sensor signal. This can be seen in chronouclometry response studies which had shown larger accumulation charge was obtained after hybridization events on modified electrode as shown in Figure 2d. Moreover, PNA-DNA surface density approximately about 74.8% more than PNA surface density on modified electrode. Furthermore, kinetic analysis of the ET reactions in the system MB-PNA-modified electrodes was performed by processing the CV data within the Laviron formalism in Figure 3. The plotted on the cathodic and anodic peak currents of MB increased gradually with the increase of scan rate which were linearly proportional to scan rate in the range from 50 to 350 mV s-1. Beside two linear regression equations based on anodic log peak current (ipa) and log scan rate were plotted before and after hybridization stage. The result has shown that slope exhibit more than 0.5 as Ipa (µA)= 1.026x-1.2105 (R 2 =0.996) and Ipa (µA)= 0.7884x + 0.004 (R 2 =0.997) (Figures 3a & 3b) suggest that the electrochemical the electron transfer process of MB in fabricated electrode before and after hybridization was a typical surfaceconfined electrochemical behavior or absorption control process [11]. . Thus the value of αn was calculated to be 1.11 and 0.80, respectively. It well known that the transfer electron number in MB to LB involves two-electron transfer [11] therefore the rate coefficient (α) were determine is 0.55 and 0.34 respectively before and after hybridization. The value α is assumed to be 0.5 before hybridization indicated that symmetry behavior of transition state between reactant and product. However, the value of 0.34 suggesting that asymmetry transition state between MB to LB occurred due to external force influence activated complex at the transition state after hybridization with DNA target [12].