Nanoelectrode Array Based Electrochemical Biosensors

The embedded CNT array minimizes background from the sidewalls while the well-defined graphitic chemistry at the exposed open ends allows the selective functionalization of -COOH groups with primary amine-terminated oligonucleotide probes through amide bonds. The wide electropotential window of carbon makes it possible to measure directly the oxidation signal of guanine bases immobilized at the electrode surface. The probe [Cy3]5'-CTIIAT TTCICAIITCCT-3'[AmC7-Q] and the target [Cy5]5'-AGGACCTGCGAA ATCCAGGGGGGGGGGG-3' are used, which are related to the wild-type (Arg1443stop) BRCA1 gene (107). A 10-bp polyG is attached to the target sequence as the signal moiety and the guanine bases in the probe are replaced with inosine to ensure that guanine signal is only from target DNA. Hybridization is carried out at 40°C for about 1 h in approx 100 nM target solution in

Fig. 6. Schematic of MWCNT nanoelectrode array combined with Ru(bpy)32+-mediated guanine oxidation for ultrasensitive DNA detection.

X3 saline sodium citrate (SSC) buffer solution. Rigorous washing using X3 SSC, X2 SSC with 0.1% sodium dodecyl sulfate, and X1 SSC, respectively, at 40°C for 15 min after each probe functionalization and target hybridization process are applied to get rid of the nonspecifically bound DNA molecules, which is critical for obtaining reliable electrochemical data.

Such a solid-state nanoelectrode array has great advantages in stability and processing reliability over other electrochemical DNA sensors based on mixed self-assembled monolayers of small organic molecules. The density of a nanoelectrode can be controlled precisely using lithographic techniques, which, in turn, define the number of probe molecules. The detection limit can be optimized by lowering the nanoelectrode density. However, the electrochemical signal is defined by the number of electrons that can be transferred between the electrode and analytes, which is observable only if it is over the level of the background. In particular, guanine oxidation occurs at rather high potential (approx 1.05 V vs saturated calomel electrode [SCE]) at which a high background is produced by carbon oxidation and water electrolysis. This problem can be solved by introducing Ru(bpy)32+ mediators to amplify the signal based on an electrocatalytic mechanism (108). Combining the CNT nanoelectrode array with the Ru(bpy)32+-mediated guanine oxidation method (as schematically shown in Fig. 6), Li et al. (35) demonstrated that the hybridization of less than a few attomoles of oligonucleotide targets can be easily detected with a 20 x 20 ^m2 electrode, with orders-of-magnitude improvement in sensitivity compared to previous electrochemically based DNA detections.

Fig. 7. (A) Three consecutive ACV measurements of low-density MWCNT array electrode functionalized with oligonucleotide probes with sequence [Cy3]5'-CTIIAT TTCICAIITCCT-3'[AmC7-Q] and hybridized with oligonucleotide targets with sequence [Cy5]5'-AGGACCTGCGAAATCCAGGGGGGGGGGG-3'. The thick, thin, and dotted lines correspond to the first, second, and third scan, respectively. Measurements were carried out in 5 mM Ru(bpy)32+ in 0.20 M NaOAC supporting electrolyte (at pH 4.8) with an AC sinusoidal wave of 10 Hz and 25-mV amplitude on top of a staircase DC ramp. (B) Difference between first and second scans (solid line), and between second and third scans (dotted line), respectively. The positive peak corresponds to the increase in Ru(bpy)32+ oxidation signal owing to the guanine bases on the surface. The negative peak serves as a control representing the behavior of a bare electrode.

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Fig. 7. (A) Three consecutive ACV measurements of low-density MWCNT array electrode functionalized with oligonucleotide probes with sequence [Cy3]5'-CTIIAT TTCICAIITCCT-3'[AmC7-Q] and hybridized with oligonucleotide targets with sequence [Cy5]5'-AGGACCTGCGAAATCCAGGGGGGGGGGG-3'. The thick, thin, and dotted lines correspond to the first, second, and third scan, respectively. Measurements were carried out in 5 mM Ru(bpy)32+ in 0.20 M NaOAC supporting electrolyte (at pH 4.8) with an AC sinusoidal wave of 10 Hz and 25-mV amplitude on top of a staircase DC ramp. (B) Difference between first and second scans (solid line), and between second and third scans (dotted line), respectively. The positive peak corresponds to the increase in Ru(bpy)32+ oxidation signal owing to the guanine bases on the surface. The negative peak serves as a control representing the behavior of a bare electrode.

Figure 7A shows three consecutive alternating current voltammetry (ACV) scans in 5 mM Ru(bpy)32+ in 0.20 M NaOAc buffer solutions after hybridizing the polyG-tagged BRCA1 targets on an MWCNT array electrode (with average tube-tube spacing of approx 1.5 ^m). The AC current is measured by applying a sinusoidal wave of 10-Hz frequency and 25-mV amplitude on a staircase potential ramp. Well-defined peaks are observed around 1.04 V, with the first scan clearly higher than the almost superimposed subsequent scans. The background is almost a flat line at zero. As shown in Fig. 7B, subtracting the second scan from the first gives a well-defined positive peak (continuous line), whereas subtracting the third scan from the second gives a small negative peak (dashed line) serving as an unambiguous negative control. The high quality of the data indicates that there is still plenty of room to lower the detection limit of target DNAs. In practical diagnosis, the target DNA consists of hun dreds of guanine bases as active signal moieties giving a much higher signal. Thus, the detection limit could be reduced well below 1 attomol. Successful electrochemical detection of PCR products using this platform was recently demonstrated. Other advantages of electrochemical detection, such as the ability to apply extra stringency control using an electrical field, could possibly be realized with this system.

The nanoelectrode array platform can also be extended to other electrode materials such as Au and Pt NWs. Different functionalization schemes and signal moieties have to be employed for different applications to take advantage of the spatial and temporal resolution of nanoelectrodes. This platform is also applicable for enzyme-based biosensors or for electrochemically based pathogen detection by immobilizing proteins such as enzymes and antibodies at the electrodes.

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