5.4 Results and discussion
5.4.4 Surface modification using thiol chemistry
The study presented in this section looked to modify the chemical properties of the metallic nanopore layer. The objective was to observe the effects of modifying the layers influence on the cell. Here, interest was placed primarily upon reducing the nanopore membranes capacitive effects. For this, thiol self-assembled monolayers (SAMs) of 6-mercapto-hexan- 1-ol were formed across the surface by spontaneous adsorption of chemical molecules onto the Au nanopore; hence an ultra thin film was produced, as in Schwartz (2001). Thiols were used due to their strong gold-sulphur (Au-S) covalent bond formation. The method used to prepare these modified nanopores is described in Section 5.3.3.
Figure 5.15 shows the cyclic voltammograms of the 30 nm Au/Si3N4 nanopore mod-
ified with a SAM of 6-mercapto-hexan-1-ol (MCH) thin layer in 100 mM NaOH. Cyclic voltammograms were typically recorded by sweeping the potential between -500 mV to +500 mV at a scan rate of 50 mV s−1 at the Au nanopore surface before treatment and after chemical modification with thiol. The results demonstrate that the electrochemically active Au nanopore surface was fully passivated by the thiol layer which was covalently attached to it – their reductive desorption (release of thiolate is induced by the reduction
of surface Au(1) to Au(0) atoms) gives rise to distinctive peaks. A reductive desorption peak of Au-MCH SAMs is observed at -160 mV, while the bare Au electrode shows the typical features of reduction/oxidation of Au surface at peaks -200 mV; -440 mV and +220 mV, respectively. Once coated on the Au membrane, thiols are known to exhibit a blocking behaviour towards Faradaic processes at the electrode and this phenomenon can be observed in the case shown in Figure 5.15. Here, the CV trace for the thiolated mem- brane shows the suppression and disappearance of Au Faradaic reactions. Using the thiol modified CV trace, a charge of 3.78 x10−5 C is derived by integrating the area under the desorption peak. From this a surface coverage of Γ= 0.56 x10−10 mol cm−2 is calculated
using Equation 5.3.3 with a geometric area, A= 0.07 cm2.
−500 −400 −300 −200 −100 −0 100 200 300 400 500 −3 −2.5 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 Current/ µ A Potential/ mV bare Au/ Si 3N4 membrane 6−MCH thiolated membrane
Figure 5.15: Cyclic voltammetry measurements of the Au nanopore surface and the modified thiolated nanopore carried out before (blue curve, bare Au membrane) and after modification (green curve, Au-MCH modified surface) relative to Ag/AgCl in 100
mM NaOH. Scan rate= 50 mV s−1.
Once the modified Au membrane had been prepared it was assembled into the microflu- idic flow cell and filled with 1 M KCl salt solution. Two Ag/AgCl electrodes immersed in the top and bottom reservoir were used to apply a steady state current across the pore. I-V measurements were conducted as described before (by sweeping the potential between
±500 mV). The results of these measurements are plotted in Figure 5.16. In this case, the Au membrane was left floating.
−500 −400 −300 −200 −100 0 100 200 300 400 500 −60 −40 −20 0 20 40 60 Potential/ mV Current/ nA Before After
Figure 5.16: Figure: Typical I-V traces obtained through a 30 nm MCH-modified Au nanopore. Acquired via cyclic voltammetry in 1 M KCl; before (blue curve) and after
(green curve) chemical modification.
From these experiments, it is evident that the presence of the thiol layer gives a dis- tinctive curve with a rectification ratio of 1.10. In comparison to the bare Au trace (green curve), a 34% reduction in conductance is observed once the Au is coated with thiol. This decrease in conductance can be explained as a result of the charge transfer inhibition at the Au membrane-electrolyte interface, which occurs through a reduction in intimate contact with the charged surface. As mentioned, this inhibiting behaviour observed in the CV scans is characteristic of thiols, Love et al. (2005). Secondly, the chemically modified Au surface is expected to lead to weaker long range electrostatic interactions between elec- trolyte and charged surface (Barsoukov and Macdonald, 2005; Schwartz, 2001), thereby mobilising charges in the pore vicinity and potentially exercising a gating effect.
The effects of using a thiol layer at the Au nanopore surface presented in this section can potentially help contribute towards an ohmic, symmetric and controlled potential drop across the membranes cylindrical aperture, where the overall reduction in surface capacitance can be modelled by a classical two plate capacitor, Bard and Faulkner (2000).
5.5
Summary
In this chapter, the metallic nanopores fabricated and discussed previously were charac- terised for their behaviour under various conditions. Tests were conducted for modifica- tions of four main variables; ionic solution concentration, bias potential, nanopore size and nanopore membrane properties. For the latter, the membrane properties were modified by applying thiols to the surface such that its capacitive effects were inhibited.
The first series of tests considered the effects of varying the ionic concentration for both metallic and Si4N4 nanopores. It was observed that an increase in ionic concentration lead
to an increase in pore conductance, as expected. Moreover, it was observed that the CV plots of both the metallic and non-metallic nanopores exhibited a linear profile. This suggested that the lack of any rectifying effects, further validating the observations that the applied metal surfaces were evenly distributed within the pore.
The next section considered the effects of varying salt concentration. It was expected that a higher salt concentration would increase the ionic current due to the increase in charge carriers present in the solution. Indeed such a result was observed. Theoretical values of the pore conductance were also calculated from Equation 5.1.2; however due to the neglect of the surface charge density, a large discrepancy between the model and experimental values was observed. Future work should look to consider methods of calcu- lating or estimating the surface charge density so that this term may also be included in future comparisons.
In parallel to these investigations, a model circuit was proposed so that the electro- chemical cells behaviour may be modelled by a theoretical circuit. The circuit consisted of two resistors in series (which represented the impedance effects of the ionic solution) and a resistor and capacitor in parallel between them (which represented the energy dissipating and storing effects of the nanopore). In order to calculate the resistive and capacitive values for these theoretical components, electrochemical impedance spectroscopy studies (EIS) were also applied to the cell. These studies consisted of applying an alternating current potential at various frequencies to gauge the cells response. It was found that the results of the model circuit, using the EIS-calculated component values, compared well with the experimental results of the cell.
In the final set of experiments, the surface properties of the nanopore membrane was modified by the addition of thiols. These served to produce an insulating layer on the surface of the membrane and so inhibit its capacitive effects. The results of this section
showed a clear influence of the thiols on the I-V trace suggesting and followed the expected behaviour. This test provided a preliminary investigation into the effects of surface mod- ification for metallic nanopores. Indeed future work should look to applying a potential to the nanopore membrane so that the ionic current can be controlled locally around the pore.
Detection of DNA using metallic
nanopores
This chapter investigates the translocation and detection of λ-DNA molecules through Pt deposited and Si3N4 nanopores. In Section 6.1 an overview is presented of the underlying
theory of DNA translocation through metallic nanopores. The primary focus of this section is to provide a brief outline of the fundamental theory associated with the drop in ionic current across nanopores that is induced by the translocation of DNA.
Section 6.2 then discusses the experimental set-up of the study presented in this chap- ter, a brief review of the various chemicals and DNA material employed has been provided as well as a description on the set-up of equipment used to conduct this study. Section 6.3 provides information regarding the data acquisition equipment employed and its set-up as well as the post-processing of the signal recorded during the experiments. Subsequently, Section 6.4 presents a discussion on the results achieved from the experiments. The find- ings of this study are then summarised in Section 6.5.
6.1
Background theory
Changes in the ionic current
As explained previously in Chapter 1 detection of DNA using a nanopore consists of an experimental set-up whereby a single nanopore provides the only passage between two compartments containing an aqueous electrolyte. By inducing a current across the nanopore, ions are transferred from one compartment to the other. Hence, observations of the current across the nanopore provide the ionic current from one compartment to the
other, as shown in Figure 6.1. By introducing DNA the ionic current passing through the nanopore will decrease as the molecule translocates through the pore. The translocation of each single molecule is observed as a single translocation ‘event’.
Figure 6.1: General set-up for translocation experiment. A charged polymer is electrophoretically driven through a nanometer-sized aperture, located between two reservoirs kept at a potential difference. Top: transmembrane potential causes a constant
flow of ions through a single pore. Bottom: analytes that pass the pore cause temporary current blockades which are further detected and characterised.
Translocation of a charged molecule such as λ-DNA through a pore can affect the ionic current by (i) changing the effective ionic concentration in the nanopore due to charges carried by the molecule, and by (ii) physically restricting the nanopore cross sectional area (Chang et al., 2004, and Smeets et al., 2006). Since DNA is negatively charged, additional conducting counterions are introduced into the pore when the molecule enters a nanopore, this results in an increase in the ionic current I, (Smeets et al., 2006) given by:
I = µ b ∆n e V /L2pore, (6.1.1) where ∆n is the number of charges introduced uniformly into a nanopore of length Lporewith a voltage bias V applied across it, e is the charge of an electron, µ is the ionic
mobility, and b is the fraction of counterions that are mobile. This mechanism dominates at low ionic concentrations where the total number of ions in the bulk solution is small. At higher salt concentrations, physical blockage of the nanopore by the DNA molecule dominates, resulting in a current decrease during translocation, Figure 6.1.
To a first order approximation, the reduction in current induced by the physical block- age of the nanopore by the molecule is equivalent to the total ionic current that can be carried by the displaced electrolyte:
Iblock = K A Vbias/Lpore (6.1.2)
where K is the solution conductivity, Vbias the applied voltage, Lporethe effective pore
length, and A the hydrodynamic cross section of the translocating molecule.
Since it is assumed that the reduction in current is linearly proportional to the hy- drodynamic cross section of the translocating material, (Li et al., 2003; Muthukumar, 2007) the translocation events exhibiting only one blockade level can be interpreted as the translocation of a λ-DNA molecule through the pore in a linear, single-file manner. Events exhibiting two or more quantised blockade levels can be interpreted as λ-DNA that translocate through the pore with portions of the molecule folded in on itself, or where two or even three parallel lengths of the λ-DNA translocate through the pore simultaneously, Li et al. (2003).
Frequency of DNA translocations
Each translocation event whereby DNA molecules are transported from a solution on one side of the nanopore to the other side can be divided into three steps: (i) transport of DNA molecules from the solution to the opening of the nanopore, (ii) entrance of the molecule into the nanopore, and (iii) translocation of the molecule across the nanopore to the solution on the other side. The transport of DNA molecules to the nanopore can occur by diffusion, electrokinetic transport, or by convection. Therefore the frequency of arrival of the molecules at the entrance of the nanopore is linearly proportional to the DNA concentration and the electric field at the entrance, (Chen et al., 2004b) and can be given by:
farrival∝ c /timesV (6.1.3)
where farrival is the frequency of DNA arrival at the pore entrance, c is the DNA
concentration, and V is the applied voltage bias. Entrance of the DNA molecule into the nanopore requires crossing an entropic free energy barrier since the DNA conformation is constrained inside the nanopore when the pore size is smaller than the DNA. Thus the
timescale for translocation depends exponentially on the voltage bias and the free energy barrier, (Henrickson et al., 2000):
1/τ ∝ exp(zeV − ∆G∗/kT ) (6.1.4) where z is the number of charges that interact with the electric field, V is the potential difference in the entrance region of the pore, and ∆G∗ is the free energy barrier. Thus, the frequency of DNA translocating across the nanopore depends on the applied voltage bias and the mobility of DNA molecules through the nanopore, Chen et al. (2004b), given by 1/τtrans ∝ V .