6.1.1. Aptamer-Based Electrochemical Sensors
Aptamers are single strands of DNA that fold into a three-dimensional structure, which results in their binding with high specificity to a chosen target molecule. The optimal aptamer for a given target molecule is generated by the combinatorial evolutionary process, known as systematic evolution of ligands by exponential enrichment (SELEX) [87
]. Aptamers have advantages that include their stability, ease of re-use, broad range of possible targets, synthesis using well-established oligonucleotide technology, and relatively small size [88
]. Aptamers can be synthesized with a thiolated end for direct assembly onto np-Au or Au. In one study, a 5′-thiolated form of an aptamer binding to postoperative lung cancer tissue and related circulating tumor cells was assembled onto a gold electrode and then backfilled with mercaptoethanol. Clinical blood plasma samples from healthy and lung cancer patients were studied. The binding of the cancer-related proteins to the aptamers on the gold electrode resulted in a reduction of the peak current due to the ferrocyanide/ferricyanide redox couple, as detected by square-wave voltammetry [89
]. The signal was enhanced by the use of hydrophobic beads binding to the proteins bound to the aptamers. The samples from the cancer patients inhibited the peak current significant more than samples from healthy patients. In another study, aptamers against cardiac troponin I (cTnI), which is a biomarker for acute myocardial infarction, were developed using SELEX and then immobilized on Au in their 5′-thiolated form. The electrode surface was then filled in with mercaptohexanol to limit nonspecific binding. The binding of cTnI to the aptamers reduced the peak current from the oxidation of ferrocene on ferrocene-modified silica nanoparticles, as detected by square-wave voltammetry.
Aptamers have been immobilized on np-Au in a small number of studies thus far. An aptamer for the protein thrombin was immobilized onto np-Au prepared by applying a square-wave pulse to a gold electrode [90
]. The thiolated aptamer was immobilized on np-Au so that it could bind to one binding site on thrombin. Subsequently, after binding of thrombin, Au nanoparticles decorated with aptamer and non-binding ssDNA were allowed to bind to another binding site on the thrombin. Detection was achieved by chronocoulometry oxidation of [Ru(NH3
electrostatically bound to anionic phosphodiester bonds of the oligonucleotide backbone with the amount bound proportional to the amount of bound ssDNA which was proportional to the amount of bound thrombin. The aptasensor had a linear range from 0.01 to 22 nM and could detect thrombin to as low as 30 fM with good selectivity. In another study, an aptamer for adenosine triphosphate (ATP) was split into two fragments prior to immobilization [91
]. The first thiolated fragment was immobilized on np-Au that had been prepared by anodization, followed by reduction by ascorbic acid. After binding of ATP, the second fragment of the aptamer was bound and then conjugated to the redox marker 3, 4-diaminobenzoic acid, which was detected by differential pulse voltammetry. The response, although nonlinear, covered a wide range of ATP concentration from 0.1 to 3000 µM. An aptamer against bisphenol A was immobilized on np-Au electrode that was prepared by affixing dealloyed gold leaf onto a glassy carbon electrode [92
]. Filling in the surface with mercaptopropionic acid was used to limit nonspecific adsorption. The steps of the surface modification were characterized using electrochemical impedance spectroscopy. In the case of this aptasensor, the bound bisphenol A was electroactive and was detected by differential pulse voltammetry. Selectivity against molecules of similar structure, such as bisphenol B, was demonstrated.
In addition to the synthesis of aptamers, it is also possible to create ssDNA with catalytic activity, referred to as a DNAzyme [93
]. A Pb2+
dependent DNAzyme was immobilized onto np-Au that was prepared from dealloyed foils affixed onto glassy carbon. A capture probe ssDNA was immobilized onto np-Au, followed by binding of Au nanoparticles modified by a partially complementary ssDNA. Addition of Pb2+
activated the capture probe ssDNA to give DNAzyme activity whereby cleavage of the complementary strand was achieved. Detection was achieved by chronocoulometry after exposure of the electrode to [Ru(NH3
. An increase in Pb2+
resulted in enhanced DNAzyme activity, cleavage of the complementary strands, and hence, binding of less [Ru(NH3
and a lower total charge detected by chronocoulometry.
6.1.2. DNA Hybridization-Based Electrochemical Sensors
The hybridization of ssDNA immobilized on np-Au with a complementary strand has also been used as the basis for the development of electrochemical biosensors. Many of these biosensors have used for detection of complementary ssDNA strands, and the hybridization events are detected by the electrochemical signal from electroactive bound or covalently attached redox probes. For example, a thiolated ssDNA capture probe can be assembled onto a gold electrode such that an outer partial length of the capture probe will hybridize with a portion of the target DNA strand [94
]. The reporter DNA strands, which are bound to Au nanoparticles, will then bind to the remainder of the target probe length. Signal amplification was achieved by the numerous reporter strands on the Au nanoparticles. Exposure of this sandwich-like assembly to [Ru(NH3
resulted in the binding of [Ru(NH3
by electrostatic interaction with the negatively charged phosphates of the oligonucleotides. Chronocoloumetry was used to determine a total charge that was associated with bound [Ru(NH3
, which was proportional to the amount of bound DNA. The method was able to detect target DNA down to femtomolar levels. DNA that was associated with the breast cancer gene BRCA1 was used as a target. Signals due to DNA with single oligonucleotide mismatches were less than 20% those for the exact complementary sequences. In the preparation of these modified electrodes, the ssDNA capture probe assembled on the Au surface is subject to additional assembly of a molecule, such as mercaptohexanol, which makes the DNA strands stand up better and inhibits nonspecific interactions. The electrostatic binding of [Ru(NH3
to ssDNA on Au, as assessed by chronocoulometry, has been used to determine the surface density of ssDNA in mixed monolayers with mercaptohexanol on Au [96
]. The method was also used to determine surface coverage of dsDNA, and hence the hybridization efficiency as a function of surface coverage of ssDNA. Hybridization was found to be efficient up to a threshold probe surface density value.
A np-Au electrode prepared using square-wave oxidation reduction cycling (SWORC) was used to create a hybridization-based sensor for DNA of the gene for the surviving protein that is associated with osteosarcoma [97
]. The capture probe DNA was immobilized and then oriented and backfilled with mercaptohexanol. The hybridization of the target DNA was monitored by chronocoulometric detection of the additional bound [Ru(NH3
to the hybridized structure. Detection of the target DNA down to 5.6 fM was possible, and was selective against single base mismatches. In another study, a np-Au electrode was modified with the DNA capture probe [98
]. Au nanoparticles were prepared being modified with both reporter DNA complementary to the target strand and also DNA not complementary to the target strand whose purpose was to reduce cross-reaction between the target and reporter. Detection of DNA binding was done by chronocoulometric detection of [Ru(NH3
electrostatically bound, and a detection limit of 28 aM was achieved with selectivity against single base pair mismatches. The scheme for this analysis is reproduced in Figure 6
In another approach, ferrocene modified DNA probes were used to develop a sensor based on hybridization on a np-Au electrode [99
]. Ferrocene carboxylic acid was conjugated to the amino-labeled DNA probe using EDC/NHS coupling chemistry. In this study, differential pulse voltammetry was used to measure the current after exposure of the immobilized capture probe to target DNA that was complementary, singly mismatched, or non-complementary, and then to the ferrocene labeled reporter DNA strand. The sensor, as designed, had good selectivity and could detect as little as 25 pmol of target DNA. A sensor for the genomic DNA of E. coli
was developed on np-Au electrodes using methylene blue as a redox probe, as it binds with higher affinity to ssDNA than to dsDNA [100
]. Using differential pulse voltammetry (DPV), successively lower peak currents from methylene blue were found upon exposure of the capture probe modified DNA with genomic DNA from greater amounts of colony forming units (CFU) per µL of E. coli
. The decrease in DPV peak current due to decreased methylene blue binding to hybridized DNA was also used to create a sensor on np-Au for the PML/RAR fusion gene associated with acute promyelocytic leukemia [101
]. In this case, the np-Au electrode was created using SWORC. A detection limit of 6.7 pM target DNA was achieved.
Stripping voltammetry has also been used to create a hybridization-based sensor on np-Au [102
]. In this study, Au nanoparticles were labeled with both reporter DNA strands, and with DNA strands that were linked to lead sulfide (PbS) nanoparticles. After target hybridization to the immobilized capture probe DNA and binding of the reporter probe modified nanoparticles, the assemblies were dissolved using nitric acid. The lead ion content was detected by differential pulse anodic stripping voltammetry. A detection limit of 0.26 fM was achieved with good selectivity.
The interaction of Hg2+
with stem-loop DNA probes immobilized on np-Au was used to create an electrochemical hybridization based sensor for Hg2+
can bind between two thymine bases and promote the formation of stable thymine–Hg2+
–thymine base pairs and the opening of the stem-loop capture probe. The binding of a complementary strand, labeled with ferrocene, resulted in a peak current in DPV that increased with Hg2+
concentration. A detection limit of 0.0036 nM was achieved with excellent selectivity against other dissolved metal ions.
The Seker lab has explored the effects of np-Au morphology and pore size on DNA hybridization sensing [7
]. The decreased binding of methylene blue upon hybridization was detected by square-wave voltammetry [7
]. It was found that there were different optimal square-wave frequencies for maximizing the current response on np-Au as prepared and thermally annealed. The hybridization could be detected to as low as 500 pM on annealed np-Au, which was a 10× lower detection limit than on np-Au, as prepared. Np-Au, with pore size that as comparable to that of proteins such as bovine serum albumin or those in fetal bovine serum used as a simulant of human serum, was found to resist biofouling when used as an electrochemical sensor for DNA hybridization [11
]. The np-Au with pore size 14 nm was shown to show a minimal effect of concentrations of bovine serum albumin of 2 mg·mL−1
on the response of methylene blue redox probe to DNA hybridization (26 bp), as assessed by square-wave voltammetry. The capture of target DNA from fetal bovine serum by np-Au modified with capture probe DNA was followed by the release of the hybridized DNA by reductive desorption, carried out by cyclic voltammetry scans between 0 and −1.5 V (vs. Ag/AgCl) [16
]. Creation of a library of np-Au morphologies on a chip was used to optimize the performance of the DNA hybridization sensors [7
]. Table 2
summarizes the analytical response characteristics, and the type of recognition, either aptamer or hybridization, for the DNA based sensors that are discussed in these two sections.
Besides large active surface area, np-Au with the optimal size and geometry was found to act as sieves, allowing for only targeted analyte and redox probe inside np-Au, while blocking the transportation of the complex media. This process highly reduces the biofouling and nonspecific interactions with the receptors [28
]. Seker’s group used SWV to detect target DNA molecules using capture probe immobilized on np-Au surface [28
]. They were able to detect 10 nM to 200 nM DNA molecules in physically relevant complex media. Unlike for chronocoulometry (CC) and DPV, the tagging/labeling step is unnecessary in SWV based on np-Au. The group has shown that the electrochemical current response during probe and target DNA hybridization can be amplified 10-fold using np-Au when compared to planar Au electrodes [11
]. In a recent work by this group, multiple np-Au electrode arrays having different morphology were microfabricated on a single chip to study DNA hybridization [7
]. Using SWV, it was concluded that np-Au, with a range of average pore radii of 25–30 nm, facilitates the permeation of target DNA, while inhibiting the nonspecific adsorption of the background proteins. The comparison among some of the electrochemical techniques in terms of their sensitivity for the detection of varieties of analytes is shown (Table 1
). However, there are other electrochemical techniques, such as EIS and CV, which frequently are used as complementary tools along with CC, DPV, and SWV for electrochemical aptasensing [91