New Tools for Investigating Electromagnetic Hot Spots in Single‐Molecule Surface‐Enhanced Raman Scattering

Surface‐enhanced Raman scattering (SERS) is quickly growing as an analytical technique, because it offers both molecular specificity and excellent sensitivity. For select substrates, SERS can even be observed from single molecules, which is the ultimate limit of detection. This review describes recent developments in the field of single‐molecule SERS (SM‐SERS) with a focus on new tools for characterizing SM‐SERS‐active substrates and how they interact with single molecules on their surface. In particular, techniques that combine optical spectroscopy and microscopy with electron microscopy are described, including correlated optical and transmission electron microscopy, correlated super‐resolution imaging and scanning electron microscopy, and correlated optical microscopy and electron energy loss spectroscopy.     

Noble‐Metal‐Free Materials for Surface‐Enhanced Raman Spectroscopy Detection

Surface‐enhanced Raman spectroscopy (SERS) is an attractive tool for the sensing of molecules in the fields of chemical and biochemical analysis as it enables the sensitive detection of molecular fingerprint information even at the single‐molecule level. In addition to traditional coinage metals in SERS analysis, recent research on noble‐metal‐free materials has also yielded highly sensitive SERS activity. This Minireview presents the recent development of noble‐metal‐free materials as SERS substrates and their potential applications, especially semiconductors and emerging graphene‐based nanostructures. Rather than providing an exhaustive review of this field, possible contributions from semiconductor substrates, characteristics of graphene enhanced Raman scattering, as well as effect factors such as surface plasmon resonance, structure and defects of the nanostructures that are considered essential for SERS activity are emphasized. The intention is to illustrate, through these examples, that the promise of noble‐metal‐free materials for enhancing detection sensitivity can further fuel the development of SERS‐related applications.     

Direct Surface‐Enhanced Raman Scattering Analysis of DNA Duplexes

The exploration of the genetic information carried by DNA has become a major scientific challenge. Routine DNA analysis, such as PCR, still suffers from important intrinsic limitations. Surface‐enhanced Raman spectroscopy (SERS) has emerged as an outstanding opportunity for the development of DNA analysis, but its application to duplexes (dsDNA) has been largely hampered by reproducibility and/or sensitivity issues. A simple strategy is presented to perform ultrasensitive direct label‐free analysis of unmodified dsDNA with the means of SERS by using positively charged silver colloids. Electrostatic adhesion of DNA promotes nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at nanogram level. As potential applications, we report the quantitative recognition of hybridization events as well as the first examples of SERS recognition of single base mismatches and base methylations (5‐methylated cytosine and N6‐methylated Adenine) in duplexes.     

DNA-Origami-Based Assembly of Au@Ag Nanostar Dimer Nanoantennas for Label-Free Sensing of Pyocyanin

Early-stage detection of diseases caused by pathogens is a prerequisite for expedient patient care. Due to the limited signal-to-noise ratio, molecular diagnostics needs molecular signal amplification after recognition of the target molecule. In this present study, we demonstrate the design of plasmonically coupled bimetallic Ag coated Au nanostar dimers with controlled nanogap using rectangular DNA origami. We further report the utility of the designed nanostar dimer structures as efficient SERS substrate for the ultrasensitive and label-free detection of the pyocyanin molecule, which is a biomarker of the opportunistic pathogenic bacteria, Pseudomonas aeruginosa. The experimental results showed that the detection limit of pyocyanin with such nanoantenna based biosensor was 335 pM, which is much lower than the clinical range of detection. Thus, fast, sensitive and label-free detection of pyocyanin at ultralow concentration in an infected human body can pave a facile route for early stage warning for severe bacterial infections.     

Single‐Molecule Mechanochemical Sensing Using DNA Origami Nanostructures

While single‐molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single‐molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7‐tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet‐derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single‐molecule sensing with improved throughput.     

Size Effect of 3D Aggregates Assembled from Silver Nanoparticles on Surface‐Enhanced Raman Scattering

Size matters: Nanometer‐sized gaps in aggregates of silver nanoparticles are generated by covering the nanoparticle surface with a bilayer of cetyltrimethylammonium bromide. The nanometer‐ to micrometer‐sized wells are lithographically generated on polydimethylsiloxane surfaces. The wells filled with the modified nanoparticles (see picture) and the effect of the aggregate size on SERS enhancement are investigated.

     

Photocontrolled DNA Origami Assembly by Using Two Photoswitches

Stimuli-responsive switching molecules have been widely investigated for the purpose of the mechanical control of biomolecules. Recently developed arylazopyrazole (AAP) shows photoisomerization activity, displaying a faster response to light-induced conformational changes and unique absorption spectral properties compared with those of conventionally used azobenzene. Herein, it is demonstrated that AAP can be used as a photoswitching molecule to control photoinduced assembly and disassembly of DNA origami nanostructures. An AAP-modified DNA origami has been designed and constructed. It is observed that the repeated assembly and disassembly of AAP-modified X-shaped DNA origami and hexagonal origami with complementary strands can be achieved by alternating UV and visible-light irradiation. Closed and linear assemblies of AAP-modified X-shaped origami were successfully formed by photoirradiation, and more than 1 μm linear assemblies were formed. Finally, it is shown that the two photoswitches, AAP and azobenzene, can be used in tandem to independently control different assembly configurations by using different irradiation wavelengths. AAP can extend the variety of available wavelengths of photoswitches and stably result in the assembly and disassembly of various DNA origami nanostructures.     

Raman Enhancement of Nanoparticle Dimers Self-Assembled Using DNA Origami Nanotriangles

Surface-enhanced Raman scattering is a powerful approach to detect molecules at very low concentrations, even up to the single-molecule level. One important aspect of the materials used in such a technique is how much the signal is intensified, quantified by the enhancement factor (EF). Herein we obtained the EFs for gold nanoparticle dimers of 60 and 80 nm diameter, respectively, self-assembled using DNA origami nanotriangles. Cy5 and TAMRA were used as surface-enhanced Raman scattering (SERS) probes, which enable the observation of individual nanoparticles and dimers. EF distributions are determined at four distinct wavelengths based on the measurements of around 1000 individual dimer structures. The obtained results show that the EFs for the dimeric assemblies follow a log-normal distribution and are in the range of 10⁶ at 633 nm and that the contribution of the molecular resonance effect to the EF is around 2, also showing that the plasmonic resonance is the main source of the observed signal. To support our studies, FDTD simulations of the nanoparticle’s electromagnetic field enhancement has been carried out, as well as calculations of the resonance Raman spectra of the dyes using DFT. We observe a very close agreement between the experimental EF distribution and the simulated values.     

Quantitative Assessment of Tip Effects in Single‐Molecule High‐Speed Atomic Force Microscopy Using DNA Origami Substrates

High‐speed atomic force microscopy (HS‐AFM) is widely employed in the investigation of dynamic biomolecular processes at a single‐molecule level. However, it remains an open and somewhat controversial question, how these processes are affected by the rapidly scanned AFM tip. While tip effects are commonly believed to be of minor importance in strongly binding systems, weaker interactions may significantly be disturbed. Herein, we quantitatively assess the role of tip effects in a strongly binding system using a DNA origami‐based single‐molecule assay. Despite its femtomolar dissociation constant, we find that HS‐AFM imaging can disrupt monodentate binding of streptavidin (SAv) to biotin (Bt) even under gentle scanning conditions. To a lesser extent, this is also observed for the much stronger bidentate SAv–Bt complex. The presented DNA origami‐based assay can be universally employed to quantify tip effects in strongly and weakly binding systems and to optimize the experimental settings for their reliable HS‐AFM imaging.     

Selectively Deposited Silver Coatings on Gold‐Capped Silicon Nanowires for Surface‐Enhanced Raman Spectroscopy

Gold caps on silicon nanowires are selectively coated with silver by autometallography (electroless deposition). Changing the conditions of silver deposition, a variety of different coating morphologies can be produced (see figure). The different silver coating morphologies are investigated in terms of their capabilities for surface enhanced Raman scattering (SERS) experiments.

     

Surface‐Assisted Large‐Scale Ordering of DNA Origami Tiles

The arrangement of DNA‐based nanostructures into extended higher order assemblies is an important step towards their utilization as functional molecular materials. We herein demonstrate that by electrostatically controlling the adhesion and mobility of DNA origami structures on mica surfaces by the simple addition of monovalent cations, large ordered 2D arrays of origami tiles can be generated. The lattices can be formed either by close‐packing of symmetric, non‐interacting DNA origami structures, or by utilizing blunt‐end stacking interactions between the origami units. The resulting crystalline lattices can be readily utilized as templates for the ordered arrangement of proteins.     

INHIBIT‐Inspired Two‐Output DNA Logic Gates Based on Surface‐Enhanced Raman Scattering

Herein, we presented a novel logic gate based on an INHIBITION gate that performs parallel readouts. Logic gates performing INHIBITION and YES/OR were constructed using surface‐enhanced Raman scattering as optical outputs for the first time. The strategy allowed for simultaneous reading of outputs in one tube. The applicability of this strategy has been successfully exemplified in the construction of half‐adder using the two‐output logic gates as reporting gates. This reporting strategy provides additional design flexibility for dynamic DNA devices.     

Site‐Directed,On‐Surface Assembly of DNA Nanostructures

Two‐dimensional DNA lattices have been assembled from DNA double‐crossover (DX) motifs on DNA‐encoded surfaces in a site‐specific manner. The lattices contained two types of single‐stranded protruding arms pointing into opposite directions of the plane. One type of these protruding arms served to anchor the DNA lattice on the solid support through specific hybridization with surface‐bound, complementary capture oligomers. The other type of arms allowed for further attachment of DNA‐tethered probe molecules on the opposite side of the lattices exposed to the solution. Site‐specific lattice assembly and attachment of fluorophore‐labeled oligonucleotides and DNA–protein conjugates was demonstrated using DNA microarrays on flat, transparent mica substrates. Owing to their programmable orientation and addressability over a broad dynamic range from the nanometer to the millimeter length scale, such supramolecular architecture might be used for presenting biomolecules on surfaces, for instance, in biosensor applications.     

Reversible Reconfiguration of DNA Origami Nanochambers Monitored by Single‐Molecule FRET

Today, DNA nanotechnology is one of the methods of choice to achieve spatiotemporal control of matter at the nanoscale. By combining the peculiar spatial addressability of DNA origami structures with the switchable mechanical movement of small DNA motifs, we constructed reconfigurable DNA nanochambers as dynamic compartmentalization systems. The reversible extension and contraction of the inner cavity of the structures was used to control the distance‐dependent energy transfer between two preloaded fluorophores. Interestingly, single‐molecule FRET studies revealed that the kinetics of the process are strongly affected by the choice of the switchable motifs and/or actuator sequences, thus offering a valid method for fine‐tuning the dynamic properties of large DNA nanostructures. We envisage that the proposed DNA nanochambers may function as model structures for artificial biomimetic compartments and transport systems.     

Direct Measurement of Single‐Molecule DNA Hybridization Dynamics with Single‐Base Resolution

Herein, we report label‐free detection of single‐molecule DNA hybridization dynamics with single‐base resolution. By using an electronic circuit based on point‐decorated silicon nanowires as electrical probes, we directly record the folding/unfolding process of individual hairpin DNAs with sufficiently high signal‐to‐noise ratio and bandwidth. These measurements reveal two‐level current oscillations with strong temperature dependence, enabling us to determine the thermodynamic and kinetic properties of hairpin DNA hybridization. More importantly, successive, stepwise increases and decreases in device conductance at low temperature on a microsecond timescale are successfully observed, indicating a base‐by‐base unfolding/folding process. The process demonstrates a kinetic zipper model for DNA hybridization/dehybridization at the single base‐pair level. This measurement capability promises a label‐free single‐molecule approach to probe biomolecular interactions with fast dynamics.     

Assembly and Purification of Enzyme‐Functionalized DNA Origami Structures

The positioning of enzymes on DNA nanostructures for the study of spatial effects in interacting biomolecular assemblies requires chemically mild immobilization procedures as well as efficient means for separating unbound proteins from the assembled constructs. We herein report the exploitation of free‐flow electrophoresis (FFE) for the purification of DNA origami structures decorated with biotechnologically relevant recombinant enzymes: the S‐selective NADP⁺/NADPH‐dependent oxidoreductase Gre2 from S. Cerevisiae and the reductase domain of the monooxygenase P450 BM3 from B. megaterium. The enzymes were fused with orthogonal tags to facilitate site‐selective immobilization. FFE purification yielded enzyme–origami constructs whose specific activity was quantitatively analyzed. All origami‐tethered enzymes were significantly more active than the free enzymes, thereby suggesting a protective influence of the large, highly charged DNA nanostructure on the stability of the proteins.