The first possible application of the DAP we want to evaluate is to detect the DNA concentration by using the electrochemical sensor. As discussed in Chapter 1, a rise in electric signal will indicate the occurrence of hybridization event between aptamers.
However, as the signal is tiny, some modifications must be applied for better sensitivity and selectivity of the sensor. Conductive polymers are well known for its good electrical conductivity, ease of preparation and chemical stability. Their ability to bind with metals as well as biology substances makes them excellent candidate for immobilization of biomolecules and rapid electron transfer for the fabrication of biosensors [12]. In this thesis, poly-pyrrole nanowire was used for surface modification of working electrode.
The nanowire form will increase the contacting area of aptamers with the sensor, thus enhance the signal to noise ratio and overall performance of the sensor.
The sensor was firstly surface-modified by electrochemical synthesis of Poly-pyrrole nanowire followed by the immobilization of the DNA robep . Finally, the target DNA will be dropped onto the probe-DNA-ready sensor where the hybridization occurred leading to a change in current of the interface.
Electrochemical synthesis of Poly-pyrrole nanowire
Monomer Pyrrole solution was prepared by mixing phosphate buffer solution (pH 7.4), Gelatin Solution 0.08% weight, and Monomer Pyrrole 1M in order.
Poly-pyrrole nanowire will be grown on working electrode by lectroce hemical deposition method. The electrode will be immersed in pyrrole solution, a voltage at fixed value will be applied to working electrode to start the deposition process. The fixed voltage will be 0.75 V compared to Ag/AgCl reference electrode and the applying time will be 200 s. The SEM image of the working electrode with poly-pyrrole nanowire was shown in Figure 3.9:
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Figure 3.9 - SEM Image of working electrode with PPy-NWs
PPy-NWs synthesized were uniformly distributed on the surface of the electrode. The size of PPy-NWs was from 60 nm to 105 nm.
DNA Probe Immobilization
Both DNA Probe and DNA Target are chemicals from Integrated DNA Technologies (IDT). The aptamer order was shown in Table 3.1:
Probe Sequence Thiol-C6- -AGACCTCCAGTCTCCATGGTACGTC- 5’ 3’
Target Sequence 5’-GACGTACCATGGAGACTGGAGGTCT- 3’
Table 3.1- Aptamer Sequence
The DNA probe is normally preserved in fridge at 40C. For immobilization process, DNA probe was taken out of the fridge to defrost, then it was dropped onto the surface of working electrode which was now covered by poly-pyrrole nanowire. The electrodes would be stored in a dry place at room temperature ( ) for one day. After that, they were taken out and rinsed off to clean the unlinked probe.
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Detection of the DNA target using DAP and DNA sensor
In this thesis, we will use cyclic voltammetry technique to determine the hybridization process of DNA. For each steps (Pyrrole nanowire synthesis DNA probe – immobilization – DNA target detection), a CV scan from both instruments (EC301 and DAP) will be performed and the change in amplitude as well as the voltage where current peak appears could be observed.
Figure 3. - CV Voltammogram after each steps at Target C = 1010 -6 mol/l by EC301
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Figure 3. - CV Voltammogram after each steps at C = 1011 -6 mol/l by the DAP A similarity between the two voltammogram summary could be seen, which is a reduction in current amplitude after each steps, as indicated on Figure 3.10 and 3.11.
There are two approaches we can stand on to explain the reduction of peak current after each steps.
In term of electrochemical approach, as the theories on double layer capacitance were presented in the previous section, when adding a layer of chemical component to the electrode surface, a charge barrier was created. PPy-NWs are semiconducting polymer [3], which of course will inhibit the charge transfer process between the bulk solution and the metal surface of the electrode. Similarly, when DNA probe binds to the PPy- NWs, the capacitance and resistance at the electrode surface also increase, resulting in a peak current decrement. The same process happens when complementary DNA target
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binds to DNA probe, the density of chemical materials increases, resulting in the inhibition of electrons transfer. Thus, after each immobilization steps, the peak current decreases.
Meanwhile, in the physic point of view, as PPy-NWs are p-type semiconductor [3] and their dominant charge carriers are holes, when they are first grown on the surface of working electrode, the transfer process of electrical charge carriers will be inhibited.
Dominant charge carriers of DNA aptamers are electrons, so that when DNA probe binds to PPy-NWs, the concentrations of basic electrical charge carriers (holes) will decrease.
As the DNA target binds to DNA probe, more and more electrons were pumped into the interface between PPy-NWs and bulk solution, thus strongly decrease the hole concentration. As a result, conduction ability of the materials will decrease, leading to the reduction of peak current after each immobilization step.
A series of tests on different concentrations was conducted, which are , , , and . For this application, the value as calculated in equation 3.1 will be employed to view on the change after each steps:
) (3.1)
Figure 3.12 shows the dependence of target concentration to the peak current for both device:
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Figure 3. - 12 Relation between ∆Ip to concentrations of DNA target when measured with EC301 and PSoC circuit
We can see a similar tendency between the two regression lines. The reason for the decrease of peak current for each concentrations was presented. As the concentration decreases from to , the number of bindings between DNA probe and DNA target also decreases. Naturally, this affects the amplitude of current reduction. The less DNA bindings, the less inhibition they caused to the charge transfer process, thus the difference between the probe peak current and target peak current decreased.
It can be seen that the detection limit of the DAP is lower than that of EC301, which indicates the noise level of EC301 is lower, for detection limit could be seen as three times of noise magnitude. Also, the steeper slope of EC301 to the DAP show that the sensitivity of EC301 is higher than that of the DAP.
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