Self-Assembly of Nanodiamonds and Plasmonic Nanoparticles for Nanoscopy.

Nanodiamonds have emerged as promising agents for sensing and imaging due to their exceptional photostability and sensitivity to the local nanoscale environment. Here, we introduce a hybrid system composed of a nanodiamond containing nitrogen-vacancy center that is paired to a gold nanoparticle via DNA hybridization. Using multiphoton optical studies, we demonstrate that the harmonic mode emission generated in gold nanoparticles induces a coupled fluorescence emission in nanodiamonds. We show that the flickering of harmonic emission in gold nanoparticles directly influences the nanodiamonds' emissions, resulting in stochastic blinking. By utilizing the stochastic emission fluctuations, we present a proof-of-principle experiment to demonstrate the potential application of the hybrid system for super-resolution microscopy. The introduced system may find applications in intracellular biosensing and bioimaging due to the DNA-based coupling mechanism and also the attractive characteristics of harmonic generation, such as low power, low background and tissue transparency.

Nonspecific Binding-Fundamental Concepts and Consequences for Biosensing Applications.

Nature achieves differentiation of specific and nonspecific binding in molecular interactions through precise control of biomolecules in space and time. Artificial systems such as biosensors that rely on distinguishing specific molecular binding events in a sea of nonspecific interactions have struggled to overcome this issue. Despite the numerous technological advancements in biosensor technologies, nonspecific binding has remained a critical bottleneck due to the lack of a fundamental understanding of the phenomenon. To date, the identity, cause, and influence of nonspecific binding remain topics of debate within the scientific community. In this review, we discuss the evolution of the concept of nonspecific binding over the past five decades based upon the thermodynamic, intermolecular, and structural perspectives to provide classification frameworks for biomolecular interactions. Further, we introduce various theoretical models that predict the expected behavior of biosensors in physiologically relevant environments to calculate the theoretical detection limit and to optimize sensor performance. We conclude by discussing existing practical approaches to tackle the nonspecific binding challenge in vitro for biosensing platforms and how we can both address and harness nonspecific interactions for in vivo systems.

An Approach for the Real-Time Quantification of Cytosolic Protein-Protein Interactions in Living Cells.

In recent years, cell-based assays have been frequently used in molecular interaction analysis. Cell-based assays complement traditional biochemical and biophysical methods, as they allow for molecular interaction analysis, mode of action studies, and even drug screening processes to be performed under physiologically relevant conditions. In most cellular assays, biomolecules are usually labeled to achieve specificity. In order to overcome some of the drawbacks associated with label-based assays, we have recently introduced "cell-based molography" as a biosensor for the analysis of specific molecular interactions involving native membrane receptors in living cells. Here, we expand this assay to cytosolic protein-protein interactions. First, we created a biomimetic membrane receptor by tethering one cytosolic interaction partner to the plasma membrane. The artificial construct is then coherently arranged into a two-dimensional pattern within the cytosol of living cells. Thanks to the molographic sensor, the specific interactions between the coherently arranged protein and its endogenous interaction partners become visible in real time without the use of a fluorescent label. This method turns out to be an important extension of cell-based molography because it expands the range of interactions that can be analyzed by molography to those in the cytosol of living cells.

Investigating Complex Samples with Molograms of Low-Affinity Binders.

In vitro diagnostics relies on the quantification of minute amounts of a specific biomolecule, called biomarker, from a biological sample. The majority of clinically relevant biomarkers for conditions beyond infectious diseases are detected by means of binding assays, where target biomarkers bind to a solid phase and are detected by biochemical or physical means. Nonspecifically bound biomolecules, the main source of variation in such assays, need to be washed away in a laborious process, restricting the development of widespread point-of-care diagnostics. Here, we show that a diffractometric assay provides a new, label-free possibility to investigate complex samples, such as blood plasma. A coherently arranged sub-micron pattern, that is, a peptide mologram, is created to demonstrate the insensitivity of this diffractometric assay to the unwanted masking effect of nonspecific interactions. In addition, using an array of low-affinity binders, we also demonstrate the feasibility of molecular profiling of blood plasma in real time and show that individual patients can be differentiated based on the binding kinetics of circulating proteins.

Ultra-Stable Molecular Sensors by Sub-Micron Referencing and Why They Should Be Interrogated by Optical Diffraction-Part I. The Concept of a Spatial Affinity Lock-in Amplifier.

Label-free optical biosensors, such as surface plasmon resonance, are sensitive and well-established for the characterization of molecular interactions. Yet, these sensors require stabilization and constant conditions even with the use of reference channels. In this paper, we use tools from signal processing to show why these sensors are so cross-sensitive and how to overcome their drawbacks. In particular, we conceptualize the spatial affinity lock-in as a universal design principle for sensitive molecular sensors even in the complete absence of stabilization. The spatial affinity lock-in is analogous to the well-established time-domain lock-in. Instead of a time-domain signal, it modulates the binding signal at a high spatial frequency to separate it from the low spatial frequency environmental noise in Fourier space. In addition, direct sampling of the locked-in sensor's response in Fourier space enabled by diffraction has advantages over sampling in real space as done by surface plasmon resonance sensors using the distributed reference principle. This paper and part II hint at the potential of spatially locked-in diffractometric biosensors to surpass state-of-the-art temperature-stabilized refractometric biosensors. Even simple, miniaturized and non-stabilized sensors might achieve the performance of bulky lab instruments. This may enable new applications in label-free analysis of molecular binding and point-of-care diagnostics.

Ultra Stable Molecular Sensors by Submicron Referencing and Why They Should Be Interrogated by Optical Diffraction-Part II. Experimental Demonstration.

Label-free optical biosensors are an invaluable tool for molecular interaction analysis. Over the past 30 years, refractometric biosensors and, in particular, surface plasmon resonance have matured to the de facto standard of this field despite a significant cross reactivity to environmental and experimental noise sources. In this paper, we demonstrate that sensors that apply the spatial affinity lock-in principle (part I) and perform readout by diffraction overcome the drawbacks of established refractometric biosensors. We show this with a direct comparison of the cover refractive index jump sensitivity as well as the surface mass resolution of an unstabilized diffractometric biosensor with a state-of-the-art Biacore 8k. A combined refractometric diffractometric biosensor demonstrates that a refractometric sensor requires a much higher measurement precision than the diffractometric to achieve the same resolution. In a conceptual and quantitative discussion, we elucidate the physical reasons behind and define the figure of merit of diffractometric biosensors. Because low-precision unstabilized diffractometric devices achieve the same resolution as bulky stabilized refractometric sensors, we believe that label-free optical sensors might soon move beyond the drug discovery lab as miniaturized, mass-produced environmental/medical sensors. In fact, combined with the right surface chemistry and recognition element, they might even bring the senses of smell/taste to our smart devices.

Quantification of Molecular Interactions in Living Cells in Real Time using a Membrane Protein Nanopattern.

Molecular processes within cells have traditionally been studied with biochemical methods due to their high degree of specificity and ease of use. In recent years, cell-based assays have gained more and more popularity since they facilitate the extraction of mode of action, phenotypic, and toxicity information. However, to provide specificity, cellular assays rely heavily on biomolecular labels and tags while label-free cell-based assays only offer holistic information about a bulk property of the investigated cells. Here, we introduce a cell-based assay for protein-protein interaction analysis. We achieve specificity by spatially ordering a membrane protein of interest into a coherent pattern of fully functional membrane proteins on the surface of an optical sensor. Thereby, molecular interactions with the coherently ordered membrane proteins become visible in real time, while nonspecific interactions and holistic changes within the living cell remain invisible. Due to its unbiased nature, this new cell-based detection method presents itself as an invaluable tool for cell signaling research and drug discovery.

Single-Nanoparticle Thermometry with a Nanopipette.

Thermal measurements at the nanoscale are key for designing technologies in many areas, including drug delivery systems, photothermal therapies, and nanoscale motion devices. Herein, we present a nanothermometry technique that operates in electrolyte solutions and, therefore, is applicable for many in vitro measurements, capable of measuring and mapping temperature with nanoscale spatial resolution and sensitive to detect temperature changes down to 30 mK with 43 µs temporal resolution. The methodology is based on local measurements of ionic conductivity confined at the tip of a pulled glass capillary, a nanopipettete, with opening diameters as small as 6 nm. When scanned above a specimen, the measured ion flux is converted into temperature using an extensive theoretical support given by numerical and analytical modeling. This allows quantitative thermal measurements with a variety of capillary dimensions and is applicable to a range of substrates. We demonstrate the capabilities of this nanothermometry technique by simultaneous mapping of temperature and topography on sub-micrometer-sized aggregates of thermoplasmonic nanoparticles heated by a laser and observe the formation of micro- and nanobubbles upon plasmonic heating. Furthermore, we perform quantitative thermometry on a single-nanoparticle level, demonstrating that the temperature at an individual nanoheater of 25 nm in diameter can reach an increase of about 3 K.

Image reversal reactive immersion lithography improves the detection limit of focal molography: erratum.

Some minor issues were discovered after publication of Opt Lett. 43, 5801 (2018) and are corrected here. They do not change the main message of the paper.

Image reversal reactive immersion lithography improves the detection limit of focal molography.

Focal molography is a label-free optical biosensing method that relies on a coherent pattern of binding sites for biomolecular interaction analysis. Reactive immersion lithography (RIL) is central to the patterning of molographic chips but has potential for improvements. Here, we show that applying the idea of image reversal to RIL enables the fabrication of coherent binding patterns of increased quality (i.e., higher analyte efficiency). Thereby the detection limit of focal molography in biological assays can be improved.

Focal molography is a new method for the in situ analysis of molecular interactions in biological samples.

Focal molography is a next-generation biosensor that visualizes specific biomolecular interactions in real time. It transduces affinity modulation on the sensor surface into refractive index modulation caused by target molecules that are bound to a precisely assembled nanopattern of molecular recognition sites, termed the 'mologram'. The mologram is designed so that laser light is scattered at specifically bound molecules, generating a strong signal in the focus of the mologram via constructive interference, while scattering at nonspecifically bound molecules does not contribute to the effect. We present the realization of molograms on a chip by submicrometre near-field reactive immersion lithography on a light-sensitive monolithic graft copolymer layer. We demonstrate the selective and sensitive detection of biomolecules, which bind to the recognition sites of the mologram in various complex biological samples. This allows the label-free analysis of non-covalent interactions in complex biological samples, without a need for extensive sample preparation, and enables novel time- and cost-saving ways of performing and developing immunoassays for diagnostic tests.

Fully Tunable Silicon Nanowire Arrays Fabricated by Soft Nanoparticle Templating.

We demonstrate a fabrication breakthrough to produce large-area arrays of vertically aligned silicon nanowires (VA-SiNWs) with full tunability of the geometry of the single nanowires and of the whole array, paving the way toward advanced programmable designs of nanowire platforms. At the core of our fabrication route, termed "Soft Nanoparticle Templating", is the conversion of gradually compressed self-assembled monolayers of soft nanoparticles (microgels) at a water-oil interface into customized lithographical masks to create VA-SiNW arrays by means of metal-assisted chemical etching (MACE). This combination of bottom-up and top-down techniques affords excellent control of nanowire etching site locations, enabling independent control of nanowire spacing, diameter and height in a single fabrication route. We demonstrate the fabrication of centimeter-scale two-dimensional gradient photonic crystals exhibiting continuously varying structural colors across the entire visible spectrum on a single silicon substrate, and the formation of tunable optical cavities supported by the VA-SiNWs, as unambiguously demonstrated through numerical simulations. Finally, Soft Nanoparticle Templating is combined with optical lithography to create hierarchical and programmable VA-SiNW patterns.

Capacitive soft strain sensors via multicore-shell fiber printing.

A new method for fabricating textile integrable capacitive soft strain sensors is reported, based on multicore-shell fiber printing. The fiber sensors consist of four concentric, alternating layers of conductor and dielectric, respectively. These wearable sensors provide accurate and hysteresis-free strain measurements under both static and dynamic conditions.