Letter pubs.acs.org/ac
Biofouling-Resilient Nanoporous Gold Electrodes for DNA Sensing Pallavi Daggumati, Zimple Matharu, Ling Wang, and Erkin Seker* Department of Electrical and Computer Engineering, University of California, Davis, California 95616, United States S Supporting Information *
ABSTRACT: Electrochemical nucleic acid sensors are promising tools for point-of-care diagnostic platforms with their facile integration with electronics and scalability. However, nucleic acid detection in complex biological fluids is challenging as biomolecules nonspecifically adsorb on the electrode surface and adversely affect the sensor performance by obscuring the transport of analytes and redox species to the electrode. We report that nanoporous gold (np-Au) electrodes, prepared by a microfabrication-compatible self-assembly process and functionalized with DNA probes, enabled detection of target DNA molecules (10−200 nM) in physiologically relevant complex media (bovine serum albumin and fetal bovine serum). In contrast, the sensor performance was compromised for planar gold electrodes in the same conditions. Hybridization efficiency decreased by 10% for np-Au with coarser pores revealing a pore-size dependence of sensor performance in biofouling conditions. This nanostructure-dependent functionality in complex media suggests that the pores with the optimal size and geometry act as sieves for blocking the biomolecules from inhibiting the surfaces within the porous volume while allowing the transport of nucleic acid analytes and redox molecules.
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surface and they suffer from low coating durability and repeatability.3 A recent approach to maintain the efficient transport of redox molecules onto the electrode surface has been to employ threedimensional electrode architectures with length scales relevant to biomolecule sizes.14,15 This approach, in some ways is reminiscent of the aforementioned membrane-based method, has the advantage of exploiting the intrinsic properties of the electrode and thereby it is conducive to microfabrication, highly repeatable, and scalable. The small interconnected pores (tens of nanometers) of nanoporous gold (np-Au) coatings have shown efficient transport of several redox molecules in the presence of biomolecules.14,15 Thin film np-Au electrodes can be easily produced by conventional microfabrication techniques for facile replacement of conventional gold electrodes. In addition, the porous morphology can be modulated by several different methods to tune the dynamic range of nucleic acid detection (500 pM−200 nM compared to 1 μM−10 μM for planar gold) by varying the molecular transport characteristics, as previously reported by our group.16 This work leverages enhanced detection sensitivity and inherent biosieving characteristics of np-Au electrodes for efficient detection of short DNA sequences in the presence of (i) an abundant protein in blood, bovine serum albumin (BSA), and (ii) fetal bovine serum (FBS), which emulates a rich library of biomolecules present in serum.
lectrochemical sensors offer high sensitivity and selectivity and are promising tools for building point-of-care diagnostic platforms.1,2 However, in many applications, these sensors are exposed to complex biological media along with the analyte of interest. The accumulation of biological components such as proteins, lipids, and polysaccharides on the sensor surface is referred to as biofouling.3 Biofouling is detrimental to the sensor performance as it impedes the transport of target analyte onto the electrode surface and hinders the coupling between redox moieties and the electrode. This has been a persistent challenge for electrochemical devices3 and has motivated the development of several methods to sustain sensor performance in biofouling conditions. These approaches include the use of semipermeable membranes and physical/ chemical modification of electrode surfaces with organic molecules. Several enzyme-based biosensors for glucose detection in blood4,5 employed membranes (e.g., polycarbonate,6 polypropelene,7 and Nafion8,9), which were enabled by the size exclusion principle where the analyte of interest can permeate the membrane while the confounding molecules are blocked. However, postprocessing steps necessary to reliably integrate membranes into microelectrodes remain as a challenge to the scalability of sensors. As for chemical modification of electrode surfaces, self-assembled monolayers of oligo- and polyethylene glycol (OEG and PEG) have been the most common approach to minimize biofouling.10−12 PEG layers prevent protein adsorption by minimizing the intermolecular forces between the biofouling molecules and the electrode surface.13 However, these nonconductive layers also impede redox molecules from reaching the electrode © XXXX American Chemical Society
Received: August 3, 2015 Accepted: August 14, 2015
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DOI: 10.1021/acs.analchem.5b02969 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
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RESULTS AND DISCUSSION Np-Au electrodes were prepared as described previously.17 Briefly, a stack of metal layers (160 nm chrome, 80 nm gold, and 600 nm silver−gold alloy) were sequentially deposited onto acid-cleaned glass substrates. The samples were then dealloyed in nitric acid to produce the np-Au films. In order to obtain samples with larger pores (annealed np-Au), a group of dealloyed chips were thermally treated for 3 min at 225 °C. The morphology of the np-Au electrodes was characterized via scanning electron microscopy (Figure 1). The median pore
hampers the electron transfer between the redox molecule and the electrode surface.3 A concentration of 2 mg/mL was chosen for BSA, as it is equivalent to the amount of albumin present in 10% serum. The redox signal depleted by ∼30% for pl-Au electrodes within 1 h, whereas the peak current remained stable for np-Au electrodes (Figure S3) consistent with results reported by others.14,15 This may be due to BSA accumulation on the pl-Au electrode surface, resulting in a diminished coupling between the electrode and redox marker and hence a decrease in the peak current. On the contrary, the nanostructured surface of np-Au may act as a sieve allowing the transport of [Fe(CN)6]3‑/4‑ marker while blocking the macromolecule, BSA. The median pore radius of unannealed np-Au is comparable to the size of BSA while it is much larger than the redox molecule. It is probable that BSA molecules adsorb onto the top pitted surface but do not penetrate into the deeper pores. This in turn allows for the redox marker to access the nonbiofouled deeper surfaces, where efficient electrode-redox marker coupling is sustained. We next expanded the redox study to evaluate DNA detection performance of the DNA probe-functionalized npAu and pl-Au electrodes in the presence of BSA and FBS. Electrochemical reactions involving potassium ferrocyanide are diffusion-limited due to fast electron transfer rate constant of these molecules.21 In such cases, the redox molecules do not have enough time to permeate through the porous structure and thus enhanced surface area no longer is a benefit. On the other hand, methylene blue (MB) is a good redox marker for DNA sensing owing to its reaction-limited nature and the ability to discriminate dsDNA from ssDNA, as previously demonstrated.22 The np-Au and pl-Au electrodes were immobilized with ssDNA probes (26 bp) and their response to MB was interrogated via square wave voltammetry (SWV). The electrodes were then challenged with different target DNA concentrations in their respective dynamic ranges of detection identified in our previous study16 (Scheme S1). The molecular transport characteristics influenced by np-Au morphology dictated the dynamic range of different np-Au electrodes. The np-Au electrodes exhibited a dynamic range of detection between 10 nM and 100 nM. Annealed np-Au electrodes resulted in a further improvement in target hybridization efficiency due to better transport enabling a 10-fold shift in dynamic range. A decrease in SWV amplitude (compared to the baseline current for probe-modified electrode) upon target
Figure 1. Scanning electron micrographs of top-views of (a) unannealed and (b) annealed np-Au electrodes. Insets display the corresponding cross-sectional views.
radius of unannealed np-Au was 14 nm and 30 nm in the case of annealed np-Au. Pore size distributions are shown in Figure S1. The median pore radius for unannealed np-Au is comparable to the BSA length (14 nm)18 while it is smaller than other complex components (proteins) present in serum. The total surface area of the three different electrodes was determined via electrochemical analysis,16,19 where unannealed and annealed np-Au samples exhibited 10 and 2.4 times larger surface area, respectively, in comparison to the planar gold (plAu) counterpart (Figure S2). In order to assess electrochemical coupling of redox markers with the different electrodes in the presence of biofouling agents, we employed potassium ferro/ferri cyanide (5 mM) ([Fe(CN)6]3‑/4‑), which is a commonly used small molecule redox marker (hydrodynamic radius
