Voltage- and Metal-assisted Chemical Etching of Micro and Nano Structures in Silicon: A Comprehensive Review.

Sculpting silicon at the micro and nano scales has been game-changing to mold bulk silicon properties and expand, in turn, applications of silicon beyond electronics, namely, in photonics, sensing, medicine, and mechanics, to cite a few. Voltage- and metal-assisted chemical etching (ECE and MaCE, respectively) of silicon in acidic electrolytes have emerged over other micro and nanostructuring technologies thanks to their unique etching features. ECE and MaCE have enabled the fabrication of novel structures and devices not achievable otherwise, complementing those feasible with the deep reactive ion etching (DRIE) technology, the gold standard in silicon machining. Here, a comprehensive review of ECE and MaCE for silicon micro and nano machining is provided. The chemistry and physics ruling the dissolution of silicon are dissected and similarities and differences between ECE and MaCE are discussed showing that they are the two sides of the same coin. The processes governing the anisotropic etching of designed silicon micro and nanostructures are analyzed, and the modulation of etching profile over depth is discussed. The preparation of micro- and nanostructures with tailored optical, mechanical, and thermo(electrical) properties is then addressed, and their applications in photonics, (bio)sensing, (nano)medicine, and micromechanical systems are surveyed. Eventually, ECE and MaCE are benchmarked against DRIE, and future perspectives are highlighted.

Optical-Quality Assessment of a Miniaturized Intraocular Telescope.

Age-related macular degeneration (AMD) causes severe vision impairments, including blindness. An option to improve vision in AMD patients is through intraocular lenses and optics. Among others, implantable miniaturized telescopes, which direct light to healthy lateral regions of the retina, can be highly effective in improving vision in AMD patients. Yet, the quality of the restored vision might be sensitive to the optical transmission and aberrations of the telescope. To shed light on these points, we studied the in vitro optical performance of an implantable miniaturized telescope, namely, the SING IMT™ (Samsara Vision Ltd., Far Hills, NJ, USA) designed to improve vision in patients affected by late-stage AMD. Specifically, we measured the optical transmission in the spectral range 350-750 nm of the implantable telescope with a fiber-optic spectrometer. Wavefront aberrations were studied by measuring the wavefront of a laser beam after passing through the telescope and expanding the measured wavefront into a Zernike polynomial basis. Wavefront concavity indicated that the SING IMT™ behaves as a diverging lens with a focal length of -111 mm. The device exhibited even optical transmission in the whole visible spectrum and effective curvature suitable for retinal images magnification with negligible geometrical aberrations. Optical spectrometry and in vitro wavefront analysis provide evidence supporting the feasibility of miniaturized telescopes as high-quality optical elements and a favorable option for AMD visual impairment treatments.

The revolution of PDMS microfluidics in cellular biology.

Microfluidics is revolutionizing the way research on cellular biology has been traditionally conducted. The ability to control the cell physicochemical environment by adjusting flow conditions, while performing cellular analysis at single-cell resolution and high-throughput, has made microfluidics the ideal choice to replace traditional in vitro models. However, such a revolution only truly started with the advent of polydimethylsiloxane (PDMS) as a microfluidic structural material and soft-lithography as a rapid manufacturing technology. Indeed, before the "PDMS age," microfluidic technologies were: costly, time-consuming and, more importantly, accessible only to specialized laboratories and users. The simplicity of molding PDMS in various shapes along with its inherent properties (transparency, biocompatibility, and gas permeability) has spread the applications of innovative microfluidic devices to diverse and important biological fields and clinical studies. This review highlights how PDMS-based microfluidic systems are innovating pre-clinical biological research on cells and organs. These devices were able to cultivate different cell lines, enhance the sensitivity and diagnostic effectiveness of numerous cell-based assays by maintaining consistent chemical gradients, utilizing and detecting the smallest number of analytes while being high-throughput. This review will also assist in identifying the pitfalls in current PDMS-based microfluidic systems to facilitate breakthroughs and advancements in healthcare research.

Nanopatterning with Photonic Nanojets: Review and Perspectives in Biomedical Research.

Nanostructured surfaces and devices offer astounding possibilities for biomedical research, including cellular and molecular biology, diagnostics, and therapeutics. However, the wide implementation of these systems is currently limited by the lack of cost-effective and easy-to-use nanopatterning tools. A promising solution is to use optical methods based on photonic nanojets, namely, needle-like beams featuring a nanometric width. In this review, we survey the physics, engineering strategies, and recent implementations of photonic nanojets for high-throughput generation of arbitrary nanopatterns, along with applications in optics, electronics, mechanics, and biosensing. An outlook of the potential impact of nanopatterning technologies based on photonic nanojets in several relevant biomedical areas is also provided.

Italian odonates in the Pandora's box: A comprehensive DNA barcoding inventory shows taxonomic warnings at the Holarctic scale.

The Odonata are considered among the most endangered freshwater faunal taxa. Their DNA-based monitoring relies on validated reference data sets that are often lacking or do not cover important biogeographical centres of diversification. This study presents the results of a DNA barcoding campaign on Odonata, based on the standard 658-bp 5' end region of the mitochondrial COI gene, involving the collection of 812 specimens (409 of which barcoded) from peninsular Italy and its main islands (328 localities), belonging to all the 88 species (31 Zygoptera and 57 Anisoptera) known from the country. Additional BOLD and GenBank data from Holarctic samples expanded the data set to 1,294 DNA barcodes. A multi-approach species delimitation analysis involving two distance (OT and ABGD) and four tree-based (PTP, MPTP, GMYC and bGMYC) methods was used to explore these data. Of the 88 investigated morphospecies, 75 (85%) unequivocally corresponded to distinct molecular operational units, whereas the remaining ones were classified as 'warnings' (i.e. showing a mismatch between morphospecies assignment and DNA-based species delimitation). These results are in contrast with other DNA barcoding studies on Odonata showing up to 95% of identification success. The species causing warnings were grouped into three categories depending on if they showed low, high or mixed genetic divergence patterns. The analysis of haplotype networks revealed unexpected intraspecific complexity at the Italian, Palearctic and Holarctic scale, possibly indicating the occurrence of cryptic species. Overall, this study provides new insights into the taxonomy of odonates and a valuable basis for future DNA and eDNA-based monitoring studies.

Impact of Fabrication and Bioassay Surface Roughness on the Performance of Label-Free Resonant Biosensors Based On One-Dimensional Photonic Crystal Microcavities.

Micro- and nanofabrication offer remarkable opportunities for the preparation of label-free biosensors exploiting optical resonances to improve sensitivity and reduce detection limit once specificity is imparted through surface biofunctionalization. Nonetheless, both surface roughness, peculiar of fabrication processes, and bioassay roughness, resulting from uneven molecular coverage of the sensing surfaces, produce light scattering and, in turn, deterioration of biosensing capabilities, especially in resonant cavities where light travels forth and back thousands to million times. Here, we present a quantitative theoretical analysis about the impact of fabrication and bioassay surface roughness on the performance of optical biosensors exploiting silicon-based, vertical one-dimensional (1D) photonic crystal resonant cavities, also taking noise sources into account. One-dimensional photonic crystal resonant cavities with different architectures and quality factors ranging from 102 to 106 are considered. The analysis points out that whereas sensitivity and linearity of the biosensors are not affected by the roughness level, either due to fabrication or bioassay, the limit of detection can be significantly degraded by both of them, depending on the quality factor of the cavity and noise level of the measurement system. The paper provides important insights into performance versus design, fabrication, and readout of biosensors based on resonant 1D photonic crystal cavities for real-setting operation.

Leaf-Inspired Authentically Complex Microvascular Networks for Deciphering Biological Transport Process.

The vascular transport of molecules, cells, and nanoconstructs is a fundamental biophysical process impacting tissue regeneration, delivery of nutrients and therapeutic agents, and the response of the immune system to external pathogens. This process is often studied in single-channel microfluidic devices lacking the complex tridimensional organization of vascular networks. Here, soft lithography is employed to replicate the vein system of a Hedera elix leaf on a polydimethilsiloxane (PDMS) template. The replica is then sealed and connected to an external pumping system to realize an authentically complex microvascular network. This satisfies energy minimization criteria by Murray's law and comprises a network of channels ranging in size from capillaries (∼50 µm) to large arterioles and venules (∼400 µm). Micro-PIV (micro-particle image velocimetry) analysis is employed to characterize flow conditions in terms of streamlines, fluid velocity, and flow rates. To demonstrate the ability to reproduce physiologically relevant transport processes, two different applications are demonstrated: vascular deposition of tumor cells and lysis of blood clots. To this end, conditions are identified to culture cells within the microvasculature and realize a confluent endothelial monolayer. Then, the vascular deposition of circulating breast (MDA-MB 231) cancer cells is documented throughout the network under physiologically relevant flow conditions. Firm cell adhesion mostly occurs in channels with low mean blood velocity. As a second application, blood clots are formed within the chip by mixing whole blood with a thrombin solution. After demonstrating the blood clot stability, tissue plasminogen activator (tPA) and tPA-carrying nanoconstructs (tPA-DPNs) are employed as thrombolytics. In agreement with previous data, clot dissolution is equally induced by tPA and tPA-DPNs. The proposed leaf-inspired chip can be efficiently used to study a variety of vascular transport processes in complex microvascular networks, where geometry and flow conditions can be modulated and monitored throughout the experimental campaign.

Fast Acoustic Light Sculpting for On-Demand Maskless Lithography.

Light interference is the primary enabler of a number of optical maskless techniques for the large-scale processing of materials at the nanoscale. However, methods controlling interference phenomena can be limited in speed, ease of implementation, or the selection of pattern designs. Here, an optofluidic system that employs acoustic standing waves in a liquid to produce complex interference patterns at sub-microsecond temporal resolution, faster than the pulse-to-pulse period of many commercial laser systems, is presented. By controlling the frequency of the acoustic waves and the motion of a translation stage, additive and subtractive direct-writing of tailored patterns over cm2 areas with sub-wavelength uniformity in periodicity and scalable spatial resolution, down to the nanometric range, are demonstrated. Such on-the-fly dynamic control of light enhances throughput and design flexibility of optical maskless lithography, helping to expand its application portfolio to areas as important as plasmonics, electronics, or metamaterials.

Near-Infrared Silicon Photonic Crystals with High-Order Photonic Bandgaps for High-Sensitivity Chemical Analysis of Water-Ethanol Mixtures.

Aqueous solutions of alcohols are used in several applications, from pharmaceutics and biology, to chemical, biofuel, and food industries. Nonetheless, development of a simple, inexpensive, and portable sensing device for the quantification of water in water-ethanol mixtures remains a significant challenge. Photonic crystals (PhCs) operating at very high-order photonic bandgaps (PBGs) offer remarkable opportunities for the realization of chemical sensors with high sensitivity and low detection limit. However, high-order PhC structures have been mostly confined to mere theoretical speculations so far, their effective realization requiring microfabrication tools enabling the control of periodic refractive index modulations at the submicrometric scale with extremely high accuracy and precision. Here, we report both experimental and theoretical results on high-sensitivity chemical analysis using vertical, silicon/air 1D-PhCs with spatial period of 10 and 20 µm (namely, over 10 times the operation wavelength) featuring ultra-high-order PBGs in the near-infrared region (namely, up to 50th at 1.1 µm). Fabrication of high-order 1D-PhCs was carried out by electrochemical micromachining (ECM) of silicon, which allowed both surface roughness and deviation from vertical of etched structures to be controlled below 5 nm and 0.1%, respectively. Optical characterization of ECM-fabricated 1D-PhCs, which was performed by acquiring reflectivity spectra over the wavelength range 1-1.7 µm, highlighted the presence of ultra-high-order PBGs with minor optical losses (i.e., <1 dB in reflectivity) separated by deep reflectivity notches with high Q-factors (i.e., >6000), in good agreement with theoretical calculations. Remarkably, the use of high-order 1D-PhCs as refractometric transducers for the quantitative detection of traces of water in water-ethanol mixtures, allowed high sensitivity (namely, either 1000 nm/RIU or ∼0.4 nm/% of water), good detection limit (namely, 5 × 10-3 RIU or ∼10% water), and excellent resolution (namely, either 6 × 10-4 RIU or 1.6% of water) to be reliably achieved on a detection volume of about 168 fL.

Environmentally Benign Production of Stretchable and Robust Superhydrophobic Silicone Monoliths.

Superhydrophobic materials hold an enormous potential in sectors as important as aerospace, food industries, or biomedicine. Despite this great promise, the lack of environmentally friendly production methods and limited robustness remain the two most pertinent barriers to the scalability, large-area production, and widespread use of superhydrophobic materials. In this work, highly robust superhydrophobic silicone monoliths are produced through a scalable and environmentally friendly emulsion technique. It is first found that stable and surfactantless water-in-polydimethylsiloxane (PDMS) emulsions can be formed through mechanical mixing. Increasing the internal phase fraction of the precursor emulsion is found to increase porosity and microtexture of the final monoliths, rendering them superhydrophobic. Silica nanoparticles can also be dispersed in the aqueous internal phase to create micro/nanotextured monoliths, giving further improvements in superhydrophobicity. Due to the elastomeric nature of PDMS, superhydrophobicity can be maintained even while the material is mechanically strained or compressed. In addition, because of their self-similarity, the monoliths show outstanding robustness to knife-scratch, tape-peel, and finger-wipe tests, as well as rigorous sandpaper abrasion. Superhydrophobicity was also unchanged when exposed to adverse environmental conditions including corrosive solutions, UV light, extreme temperatures, and high-energy droplet impact. Finally, important properties for eventual adoption in real-world applications including self-cleaning, stain-repellence, and blood-repellence are demonstrated.

Ordered Silicon Pillar Arrays Prepared by Electrochemical Micromachining: Substrates for High-Efficiency Cell Transfection.

Ordered arrays of silicon nano- to microscale pillars are used to enable biomolecular trafficking into primary human cells, consistently demonstrating high transfection efficiency can be achieved with broader and taller pillars than reported to date. Cell morphology on the pillar arrays is often strikingly elongated. Investigation of the cellular interaction with the pillar reveals that cells are suspended on pillar tips and do not interact with the substrate between the pillars. Although cells remain suspended on pillar tips, acute local deformation of the cell membrane was noted, allowing pillar tips to penetrate the cell interior, while retaining cell viability.

3D Silicon Microstructures: A New Tool for Evaluating Biological Aggressiveness of Tumor Cells.

In this work, silicon micromachined structures (SMS), consisting of arrays of 3- µ m-thick silicon walls separated by 50- µm-deep, 5- µ m-wide gaps, were applied to investigate the behavior of eight tumor cell lines, with different origins and biological aggressiveness, in a three-dimensional (3D) microenvironment. Several cell culture experiments were performed on 3D-SMS and cells grown on silicon were stained for fluorescence microscopy analyses. Most of the tumor cell lines recognized in the literature as highly aggressive (OVCAR-5, A375, MDA-MB-231, and RPMI-7951) exhibited a great ability to enter and colonize the narrow deep gaps of the SMS, whereas less aggressive cell lines (OVCAR-3, Capan-1, MCF7, and NCI-H2126) demonstrated less penetration capability and tended to remain on top of the SMS. Quantitative image analyses of several fluorescence microscopy fields of silicon samples were performed for automatic cell recognition and count, in order to quantify the fraction of cells inside the gaps, with respect to the total number of cells in the examined field. Our results show that higher fractions of cells in the gaps are obtained with more aggressive cell lines, thus supporting in a quantitative way the observation that the behavior of tumor cells on the 3D-SMS depends on their aggressiveness level.

On the performance of label-free biosensors based on vertical one-dimensional photonic crystal resonant cavities.

In this work three Fabry-Perot (FP) resonant cavities based on vertical silicon/air one-dimensional photonic crystals (1DPhCs) featuring different architectures and fluidic functionalities are designed, and the role of key design parameters on their ideal biosensing performance, i.e. surface sensitivity, limit of detection, range of linearity, is investigated. Numerical calculations of the transmission spectra of the 1DPhC FP resonant cavities using the Transfer Matrix Method (TMM), versus thickness of a biolayer simulating biomolecules (e.g. proteins) adsorbed on the 1DPhC FP cavity surfaces, show that biosensors with surface sensitivity up to 300 pm/nm, limit of detection down to 0.07 nm, and high linearity over the range 0-50 nm of biolayer thickness can be achieved.

Label-free optical detection of cells grown in 3D silicon microstructures.

We demonstrate high aspect-ratio photonic crystals that could serve as three-dimensional (3D) microincubators for cell culture and also provide label-free optical detection of the cells. The investigated microstructures, fabricated by electrochemical micromachining of standard silicon wafers, consist of periodic arrays of silicon walls separated by narrow deeply etched air-gaps (50 µm high and 5 µm wide) and feature the typical spectral properties of photonic crystals in the wavelength range 1.0-1.7 µm: their spectral reflectivity is characterized by wavelength regions where reflectivity is high (photonic bandgaps), separated by narrow wavelength regions where reflectivity is very low. In this work, we show that the presence of cells, grown inside the gaps, strongly affects light propagation across the photonic crystal and, therefore, its spectral reflectivity. Exploiting a label-free optical detection method, based on a fiberoptic setup, we are able to probe the extension of cells adherent to the vertical silicon walls with a non-invasive direct testing. In particular, the intensity ratio at two wavelengths is the experimental parameter that can be well correlated to the cell spreading on the silicon wall inside the gaps.

A new cell-selective three-dimensional microincubator based on silicon photonic crystals.

In this work, we show that vertical, high aspect-ratio (HAR) photonic crystals (PhCs), consisting of periodic arrays of 5 µm wide gaps with depth of 50 µm separated by 3 µm thick silicon walls, fabricated by electrochemical micromachining, can be used as three-dimensional microincubators, allowing cell lines to be selectively grown into the gaps. Silicon micromachined dice incorporating regions with different surface profiles, namely flat silicon and deeply etched PhC, were used as microincubators for culturing adherent cell lines with different morphology and adhesion properties. We extensively investigated and compared the proliferative behavior on HAR PhCs of eight human cell models, with different origins, such as the epithelial (SW613-B3; HeLa; SW480; HCT116; HT29) and the mesenchymal (MRC-5V1; CF; HT1080). We also verified the contribution of cell sedimentation into the silicon gaps. Fluorescence microscopy analysis highlights that only cell lines that exhibit, in the tested culture condition, the behavior typical of the mesenchymal phenotype are able to penetrate into the gaps of the PhC, extending their body deeply in the narrow gaps between adjacent silicon walls, and to grow adherent to the vertical surfaces of silicon. Results reported in this work, confirmed in various experiments, strongly support our statement that such three-dimensional microstructures have selection capabilities with regard to the cell lines that can actively populate the narrow gaps. Cells with a mesenchymal phenotype could be exploited in the next future as bioreceptors, in combination with HAR PhC optical transducers, e.g., for label-free optical detection of cellular activities involving changes in cell adhesion and/or morphology (e.g., apoptosis) in a three-dimensional microenvironment.

Fibrillogenesis of human ß2 -microglobulin in three-dimensional silicon microstructures.

The authors describe the interaction of biological nanostructures formed by ß(2) -microglobulin amyloid fibrils with three-dimensional silicon microstructures consisting in periodic arrays of vertical silicon walls (≈3 µm-thick) separated by 50 µm-deep air gaps (≈5 µm-wide). These structures are of great interest from a biological point of view since they well mimic the interstitial environment typical of amyloid deposition in vivo. Moreover, they behave as hybrid photonic crystals, potentially applicable as optical transducers for label-free detection of the kinetics of amyloid fibrils formation. Fluorescence and atomic force microscopy (AFM) show that a uniform distribution of amyloid fibrils is achieved when fibrillogenesis occurs directly on silicon. The high resolution AFM images also demonstrate that amyloid fibrils grown on silicon are characterized by the same fine structure typically ensured by fibrillogenesis in solution.