Probe immobilization and target molecule capture

A wide range of biomolecular capture probes have been immobilized onto NPG, including enzymes, single-stranded DNA, aptamers, antibodies, and lectins (Figure 1c). Immobilization onto NPG has been achieved by simple physisorption within the porous structure,^{Reference Wu, Sun, Li, Liu, Du, Wang and Xu1} by physisorption/entrapment together with a polymer,^{Reference Salaj-Kosla, Pöller, Beyl, Scanlon, Beloshapkin, Shleev, Schuhmann and Magner2} cross-linking after physisorption,^{Reference Sanzó, Taurino, Antiochia, Gorton, Favero, Mazzei, De Micheli and Carrara3} direct thiol–Au interaction,^{Reference Yan, Wang, Zhao, Zhang, Xu and Ding4} and by conjugation to activated terminal ester groups of self-assembled monolayers (SAMs).^{Reference Xiao, Li, Wang, Zhang and Si5}

Enzymes immobilized on NPG by physisorption have been found to better retain their stability on NPG than when free in solution, as has been reported for laccase, a copper-containing oxidase enzyme.^{Reference Qiu, Xu, Huang, Ding, Qu and Gao6} While some redox enzymes have been adsorbed alone and reported to display direct electron transfer,^{Reference Qiu, Xu, Huang, Ding, Qu and Gao6} others have been co-adsorbed together with redox polymers.^{Reference Salaj-Kosla, Scanlon, Baumeister, Zahma, Ludwig, Conghaile, MacAodha, Leech and Magner7} Enzymes have been physisorbed onto NPG together with cross-linking polymers, or have been cross-linked after physisorption.^{Reference Siepenkoetter, Salaj-Kosla and Magner8} Enzymes such as laccase have also been covalently linked to SAMs formed from lipoic acid activated to produce reactive esters that form an amide linkage to a lysine residue on the enzyme.^{Reference Qiu, Xu, Huang, Ding, Qu and Gao9}

Thiolated single-stranded DNA oligonucleotides have been directly assembled onto NPG,^{Reference Daggumati, Appelt, Matharu, Marco and Seker10} as have thiolated aptamers.^{Reference Zhang, Chang, Hirata, Wu, Xue and Chen11} In the case of DNA and aptamer immobilization, subsequent exposure to a short chain alkanethiol such as mercaptohexanol has been applied to cover exposed sites, limit nonspecific adsorption, and promote better orientation of the DNA strand. Immobilization of antibodies has been achieved by physisorption followed by exposure to bovine serum albumin to block nonspecific adsorption.^{Reference Fan, Guo, Gao, Zhang, Fan, Ji, Du and Wei12} Antibodies have also been immobilized by conjugation to SAMs presenting activated ester groups for amide bond formation.^{Reference Li, Wang and Zhang13} Lectins (with specific sugars as targets) have been immobilized onto nanoporous gold modified by activated lipoic acid SAMs.^{Reference Alla, d’Andrea, Bhattarai, Cooper, Tan, Demchenko and Stine14} Reagents such as dithiobis(succinimidyl propionate) are available for direct formation of an activated SAM without need for the activation step.^{Reference Ding, Li, Hu and Lin15} The diversity of biomolecular probes that can be attached onto NPG offers the ability to capture a rich set of target biomolecules (e.g., nucleic acids, proteins, carbohydrates, metabolites, small molecules) (Figure 1c).

Once a solution containing the target molecule is introduced, the target molecule needs to reach the capture probe, which is a challenge in nanoporous metals due to the tortuous path the molecule needs to travel to reach the capture probes immobilized within deeper pores. This transport is generally driven by a diffusion-like process hindered by surface–molecule interactions. The transport process can be enhanced by convection (e.g., flow-through cells);^{Reference Alla, d’Andrea, Bhattarai, Cooper, Tan, Demchenko and Stine14} however, there are large backpressures involved. On the other hand, the nanoporous morphology offers additional attributes that assist with the capture process. For example, tunable morphology allows for introducing cracks in the film so target molecules can permeate the NPG both from top and sides. The transport phenomenon (i.e., diffusion- versus reaction-controlled processes) in NPG electrodes has been shown to enhance sensor selectivity (e.g., detection of dopamine in the presence of interfering ascorbic acid).^{Reference Qiu, Zhou, Ji, Zhang, Huang and Ding16} Another challenge is the presence of biofouling molecules, such as proteins that can nonspecifically adsorb on electrodes, blocking charge transfer between the electrode and reporter redox molecules. Several groups have reported that NPG films are highly biofouling-resilient and are able to sustain electrochemical function in protein solutions.^{Reference Daggumati, Appelt, Matharu, Marco and Seker10,Reference Patel, Radhakrishnan, Zhao, Uppalapati, Daniels, Ward and Collinson17} This has been attributed to NPG morphology at a certain pore size (tens of nanometers), which blocks the permeation of large proteins while still allowing the transport of small ions and reporter molecules, effectively serving as a biofouling-resilient electrode (Figure 1b and Figure 2c).

Reporter molecules and transduction modalities

The majority of transduction events involving NPG have been carried out by either optical (e.g., plasmonic signals, fluorescence, color change) or electrochemical outputs (Figure 1a). One general detection strategy is variations of the enzyme-linked immunosorbent assay protocol,^{Reference Fan, Guo, Gao, Zhang, Fan, Ji, Du and Wei12} where a primary antibody immobilized onto NPG captures the target molecule, followed by a secondary antibody attaching to the target. The secondary antibody is tagged with a reporter molecule such as a fluorescent molecule, quantum dot, or enzyme (e.g., horseradish peroxidase) that leads to a color change in a solution. Similarly, the tag could be an electroactive molecule such as a redox reporter (e.g., methylene blue) where an electrical current is generated at a specific applied potential.^{Reference Daggumati, Appelt, Matharu, Marco and Seker10}

On the plasmonics front, NPG thin films have recently captured intense attention as plasmonic materials with propagating surface plasmon resonance (SPR),^{Reference Yu, Ahl, Caminade, Majoral, Knoll and Erlebacher18} localized surface plasmon resonance (LSPR),^{Reference Lang, Qian, Guan, Zi and Chen19} and use as a substrate for surface-enhanced Raman scattering.^{Reference Biener, Nyce, Hodge, Biener, Hamza and Maier20,Reference Qian, Yan, Fujita, Inoue and Chen21} For each plasmonic modality, the resonant frequency exhibits a strong pore-size dependence, which can be optimized by leveraging the highly tunable morphology of NPG. In order to add an additional length scale to improve coupling between light and plasmons, NPG nanoparticles/nanodisks were fabricated to yield large surface areas, tunable plasmonic resonances, and high-density hot spots.^{Reference Zhao, Zeng, Parvez Arnob, Sun, Qi, Motwani, Gheewala, Li, Paterson, Strych, Raja, Willson, Wolfe, Lee and Shih22,Reference Wi, Tominaka, Uosaki and Nagao23} These nanoporous disks have been integrated with microfluidic devices for chemical and biological sensing.^{Reference Zeng, Zhao, Li, Li, Lee and Shih24} A significant advantage of plasmonics-based techniques is that they allow for label-free detection, circumventing the need for reporter molecules. To that end, there are several different label-free detection options available, such as leveraging LSPR’s sensitivity to local refractive index changes near the metal surface (so-called LSPR spectroscopy) due to target molecule adsorption. Beyond refractive index sensing, electromagnetic field localization and enhancement near the nanostructures have been demonstrated to have profound impact on a variety of light–matter interactions, such as surface-enhanced Raman spectroscopy,^{Reference Biener, Nyce, Hodge, Biener, Hamza and Maier20,Reference Qian, Yan, Fujita, Inoue and Chen21} surface-enhanced infrared absorption,^{Reference Wang, Kundu and Halas25} surface-enhanced fluorescence,^{Reference Zhang, Song, Fujita, Zhang, Chen and Wang26} and surface-enhanced near-infrared absorption (Figure 2a).^{Reference Shih, Santos, Zhao, Zenasni and Arnob27} Each of the transduction modalities described here possess different strengths. For example, electrochemical transduction allows for easy integration with electronics for point-of-care applications, while plasmonics-based methods allow for label-free detection.

Bioseparation modalities

The tunable pore morphology and customizable surface functionalization of NPG has begun to be explored in applications that involve separations. NPG is well suited for flow of solutions through its internal pore structure. NPG monoliths of 0.25-mm thickness have been used for selectively regulating the transport of model analytes across them.^{Reference McCurry and Bailey28} The transport of methyl viologen (MV²⁺) or benzene sulfonate (BS^–) across NPG monoliths was monitored spectroscopically and found to be strongly dependent on the applied potential and on the ionic strength. The transport behavior was further regulated by modification of the NPG monoliths with alkanethiols with pH-sensitive terminal groups (–NH₂ or –COOH). The transport was sensitive to both pH and applied potential. NPG monoliths modified by alkanethiols with terminal mannose (a monosaccharide sugar of the aldose group) units were useful for capture and release of the lectin concanavalin A, a carbohydrate-binding protein originally extracted from the jack-bean, Canavalia ensiformis.^{Reference Tan, Fujikawa, Pornsuriyasak, Alla, Ganesh, Demchenko and Stine29}

The surface coverages of SAMs within the monoliths of bound lectin were determined using thermogravimetric analysis. NPG monoliths modified by lipoic acid and linked to the concanavalin A were suitable for selective capture and release of the mannose-rich glycoprotein ovalbumin.^{Reference Alla, d’Andrea, Bhattarai, Cooper, Tan, Demchenko and Stine14} In each case, release of protein was achieved by flow of free mannose ligand through the monolith. Solid-phase microextraction experiments using a nanoporous gold fiber combined with analysis by gas chromatography have been reported.^{Reference Hafez and Wenclawiak30} In this study, the nanoporous fiber more efficiently extracted dodecanethiol from ethanol than the standard polymer fiber. Additionally, the nanoporous gold fiber was used to extract 10 sulfur-bearing compounds from the headspace above an onion sample with the compounds identified by gas chromatography/mass spectrometry. NPG has been modified by thiolated single-stranded DNA and used to capture a 26-mer target DNA from tobacco mosaic virus.^{Reference Daggumati, Appelt, Matharu, Marco and Seker10} Electrochemical desorption was used to release the double-stranded DNA from within NPG for collection and analysis. The strategy was able to efficiently capture the specific target DNA in the presence of competing nonspecific DNA and serum proteins of various sizes.

Electrode impedance

The first challenge of obtaining high-density electrode arrays with low electrical impedance has been addressed by employing traditional photolithography to fabricate NPG electrodes on glass and silicon substrates.^{Reference Chapman, Wang, Chen, Garrison, Lein and Seker32,Reference Kim, Kim, Kim, Han, Chung and Jung33} In electrophysiological recordings, the sensing is due to capacitive coupling between the ionic changes (created by neurons) and the electric double layer at the electrode–electrolyte interface. While reduction of electrical impedance due to electrodes with high effective surface area is commonly observed, this reduction was sustained even in biofouling solutions, likely by the aforementioned biofouling resilience (Figure 2c).^{Reference Daggumati, Appelt, Matharu, Marco and Seker10,Reference Patel, Radhakrishnan, Zhao, Uppalapati, Daniels, Ward and Collinson17}

Topographical cues

As discussed earlier, another factor that influences the recording SNR is the proximity of neurons to the electrode. One approach to enhance this has been by introducing micro- and nanoscale features onto electrode surfaces. Cells have the ability to sense the underlying substrate and regulate their adhesion in part as a function of the underlying surface topography (Figure 3b). Studies on the influence of NPG topographical cues on cellular spreading have revealed an interesting phenomenon, where the NPG surface reduced astrocyte (a star-shaped glial cell of the central nervous system) spreading while it did not affect neuronal coverage (Figure 4a).^{Reference Chapman, Wang, Chen, Garrison, Lein and Seker32} Similarly, the mobility of another important neural cell, microglia, which promotes spreading of astrocytes, was limited on NPG surfaces.^{Reference Tan, Terrill, Paranjape, Stine and Nichols34} These are important findings that point to NPG’s potential as a neural interface coating for enhanced electrode reliability and SNR for recording and electrical stimulation (Figure 4b).^{Reference Chapman, Wang, Chen, Garrison, Lein and Seker32,Reference Kim, Kim, Kim, Han, Chung and Jung33}

Soluble factor delivery

Another approach to manage tissue response to electrodes is releasing soluble factors, such as drug molecules in situ from the device coatings. This approach, in some ways, is the opposite physical problem of that discussed for biosensing, where here the reaction is the surface–molecule association/dissociation, and transport is the outflux of the molecule through the porous network. In this case, drug molecules are loaded into the porous matrix and retained largely by weak intermolecular interaction between the surface and the molecules. As expected, the large effective surface area of NPG drastically increases the loading capacity.^{Reference Polat and Seker35} In addition, this surface–molecule interaction largely dictates the molecular release kinetics, extending the release half-lives to almost half of one day from submicron thin films.

The surface–molecule interactions can be modulated by various external stimuli opening the door to on-demand release of drugs. For example, it has been shown that by leveraging the high photothermal-light-harvesting efficiency of NPG nanodisks, molecular release from NPG was triggered by exposure to a near-infrared laser (Figure 4c).^{Reference Santos, Zhao, Zeng and Shih36} Analogously, by varying the surface charge of NPG via an applied electric potential between NPG and a reference electrode, charged drug molecules were successfully released.^{Reference Gittard, Pierson, Ha, Wu, Narayan and Robinson37} The molecular release was also gated by the presence of halides that have a higher affinity for the gold surface than physisorbed dye molecules.^{Reference Polat and Seker35} The biological relevance of the drug delivery capability of NPG was demonstrated by loading NPG-coated glass coverslips with a drug molecule inhibiting cell division. When astrocytes were cultured on the coverslips, the release of the inhibitory drug molecules suspended the spreading of astrocytes compared to control samples.^{Reference Seker, Berdichevsky, Staley and Yarmush38} Along with surface–molecule interaction-mediated molecular release, it is also possible to modulate the hydrophilicity of NPG by applying electrical potentials and enabling electrically controlled wicking and dispensing of extremely small liquid volumes (Figure 4d).^{Reference Xue, Markmann, Duan, Weissmüller and Huber39}