Acid-Modulated Peptide Synthesis for Application on Oxide Biosensor Interfaces.

In this paper we report an acid-modulated strategy for novel peptide microarray production on biosensor interfaces. We initially selected a controlled pore glass (CPG) as a support for solid-phase peptide synthesis (SPPS) to implement a chemistry that can be performed at the interface of multiple field effect transistor (FET) sensors, eventually to generate label-free peptide microarrays for protein screening. Our chemistry uses a temporary protection of the N-terminal amino function of each amino acid building block with a tert-butyloxycarbonyl (Boc) group that can be removed after each SPPS cycle, in combination with semi-permanent protection of the side chains of trifunctional amino acid residues. Such a protection scheme with a well-proven record of application in conventional, batchwise SPPS has been fine-tuned for optimal performance on CPG and, from there, translated to SPR chips that allow layer-by-layer monitoring of amino acid coupling. Our results validate this acid-modulated synthesis as a feasible approach for producing peptides in high yields and purity on flat glass surfaces, such as those in bio-FETs.

Planar Junctionless Field-Effect Transistor for Detecting Biomolecular Interactions.

Label-free field-effect transistor-based immunosensors are promising candidates for proteomics and peptidomics-based diagnostics and therapeutics due to their high multiplexing capability, fast response time, and ability to increase the sensor sensitivity due to the short length of peptides. In this work, planar junctionless field-effect transistor sensors (FETs) were fabricated and characterized for pH sensing. The device with SiO2 gate oxide has shown voltage sensitivity of 41.8 ± 1.4, 39.9 ± 1.4, 39.0 ± 1.1, and 37.6 ± 1.0 mV/pH for constant drain currents of 5, 10, 20, and 50 nA, respectively, with a drain to source voltage of 0.05 V. The drift analysis shows a stability over time of -18 nA/h (pH 7.75), -3.5 nA/h (pH 6.84), -0.5 nA/h (pH 4.91), 0.5 nA/h (pH 3.43), corresponding to a pH drift of -0.45, -0.09, -0.01, and 0.01 per h. Theoretical modeling and simulation resulted in a mean value of the surface states of 3.8 × 1015/cm2 with a standard deviation of 3.6 × 1015/cm2. We have experimentally verified the number of surface sites due to APTES, peptide, and protein immobilization, which is in line with the theoretical calculations for FETs to be used for detecting peptide-protein interactions for future applications.

Engineering Electromagnetic Hot-Spots in Nanoparticle Cluster Arrays on Reflective Substrates for Highly Sensitive Detection of (Bio)molecular Analytes.

Intense electromagnetic (EM) hot-spots arising at the junctions or gaps in plasmonic nanoparticle assemblies can drive ultrahigh sensitivity in molecular detection by surface-enhanced spectroscopies. Harnessing this potential however requires access to the confined physical space at the EM hot-spots, which is a challenge for larger analytes such as biomolecules. Here, we demonstrate self-assembly derived gold nanoparticle cluster arrays (NCAs) on gold substrates exhibiting controlled interparticle (<1 nm wide) and intercluster (<10 nm wide) hot-spots as highly promising in this direction. Sensitivity of the NCAs toward detection of small (<1 nm) or large (protein-receptor interactions) analytes in surface-enhanced Raman and metal-enhanced fluorescence assays is found to be strongly impacted by the size of the cluster and the presence of reflective substrates. Experiments supported by numerical simulations attribute the higher sensitivity to higher EM field enhancements at the hot-spots, as well as greater analyte leverage over EM hot-spots. The best-performing arrays could push the sensitivity down to picomolar detection limits for sub-nanometric organic analytes as well as large protein analytes. The investigation paves the way for rational design of plasmonic biosensors and highlights the unique capabilities of a molecular self-assembly approach toward catering to this objective.

Analyte Co-localization at Electromagnetic Gap Hot-Spots for Highly Sensitive (Bio)molecular Detection by Plasmon Enhanced Spectroscopies.

Electromagnetic hot-spots at ultranarrow plasmonic nanogaps carry immense potential to drive detection limits down to few molecules in sensors based on surface-enhanced Raman or fluorescence spectroscopies. However, leveraging the EM hot-spots requires access to the gaps, which in turn depends on the size of the analyte in relation to gap distances. Herein, we leverage a well-calibrated process based on self-assembly of block copolymer colloids on a full-wafer level to produce high-density plasmonic nanopillar arrays exhibiting a large number (>1010 cm-2) of uniform interpillar EM hot-spots. The approach allows convenient handles to systematically vary the interpillar gap distances down to a sub-10 nm regime. The results show compelling trends of the impact of analyte dimensions in relation to the gap distances toward their leverage over interpillar hot-spots and the resulting sensitivity in SERS-based molecular assays. Comparing the detection of labeled proteins in surface-enhanced Raman and metal-enhanced fluorescence configurations further reveal the relative advantage of fluorescence over Raman detection while encountering the spatial limitations imposed by the gaps. Quantitative assays with limits of detection down to picomolar concentrations are realized for both small organic molecules and proteins. The well-defined geometries delivered by a nanofabrication approach are critical to arriving at realistic geometric models to establish meaningful correlation between the structure, optical properties, and sensitivity of nanopillar arrays in plasmonic assays. The findings emphasize the need for the rational design of EM hot-spots that takes into account the analyte dimensions to drive ultrahigh sensitivity in plasmon-enhanced spectroscopies.

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

Controlled Nanoscale Topographies for Osteogenic Differentiation of Mesenchymal Stem Cells.

Nanotopography with length scales of the order of extracellular matrix elements offers the possibility of regulating cell behavior. Investigation of the impact of nanotopography on cell response has been limited by the inability to precisely control geometries, especially at high spatial resolutions and across practically large areas. In this paper, we demonstrate well-controlled and periodic nanopillar arrays of silicon and investigate their impact on osteogenic differentiation of human mesenchymal stem cells (hMSCs). Silicon nanopillar arrays with critical dimensions in the range of 40-200 nm, exhibiting standard deviations below 15% across full wafers, were realized using the self-assembly of block copolymer colloids. Immunofluorescence and quantitative polymerase chain reaction measurements reveal clear dependence of osteogenic differentiation of hMSCs on the diameter and periodicity of the arrays. Further, the differentiation of hMSCs was found to be dependent on the age of the donor. While osteoblastic differentiation was found to be promoted by the pillars with larger diameters and heights independent of donor age, they were found to be different for different spacings. Pillar arrays with smaller pitch promoted differentiation from a young donor, while a larger spacing promoted those of an old donor. These findings can contribute for the development of personalized treatments of bone diseases, namely, novel implant nanostructuring depending on patient age.

Nanostructured sensors for biomedical applications--a current perspective.

Nanostructured sensors have unique capabilities that can be tailored to advantage in advancing the diagnosis, monitoring and cure of several diseases and health conditions. This report aims at providing a current perspective on, (a) the emerging clinical needs that defines the challenges to be addressed by nanostructured sensors, with specific emphasis on early stage diagnosis, drug-diagnostic combinations, and predictive models to design therapy, (b) the emerging industry trends in in vitro diagnostics, mobile health care, high-throughput molecular and cell-based diagnostic platforms, and (c) recent instances of nanostructured biosensors, including promising sensing concepts that can be enhanced using nanostructures that carry high promise towards catering to the emerging clinical needs, as well as the market/industry trends.

Hierarchically built gold nanoparticle supercluster arrays as charge storage centers for enhancing the performance of flash memory devices.

Flash memory devices with high-performance levels exhibiting high charge storage capacity, good charge retention, and high write/erase speeds with lower operating voltages are widely in demand. In this direction, we demonstrate hierarchical self-assembly of gold nanoparticles based on block copolymer templates as a promising route to engineer nanoparticle assemblies with high nanoparticle densities for application in nanocrystal flash memories. The hierarchical self-assembly process allows systematic multiplication of nanoparticle densities with minimal increase in footprint, thereby increasing the charge storage density without an increase in operating voltage. The protocol involves creation of a parent template composed of gold nanoclusters that guides the self-assembly of diblock copolymer reverse micelles which in turn directs electrostatic assembly of gold nanoparticles resulting in a three-level hierarchical system. Capacitance-voltage (C-V) measurements of the hierarchical nanopatterns with a metal-insulator-semiconductor capacitor configuration reveal promising enhancement in memory window as compared to nonhierarchical nanoparticle controls. Capacitance-time (C-t) measurements show that over half the stored charges were retained when extrapolated to 10 years. The fabrication route can be readily extended to programmed density multiplication of features made of other potential charge storage materials such as platinum, palladium, or hybrid metal/metal oxides for next generation, solution-processable flash memory devices.

Hierarchically built hetero-superstructure arrays with structurally controlled material compositions.

Hierarchical assemblies are repeatedly encountered in nature, and when replicated in synthetic patterns and materials, can enhance their functionality or impart multifunctionality. In order to assemble a hierarchical superstructure that consists of components made up of multiple nanostructures, control over placement and stoichiometry is desirable. Macroscopic arrays that present up to three levels of hierarchy are demonstrated here and are achieved using the self-assembly of soft, collapsible block copolymer nanospheres for the first two levels, followed by directed self-assembly of metal nanospheres for the third. The fabrication approach combines advantages of soft sphere self-assembly to yield non-close-packed and variable array pitch values, with the inherent chemical functionality presented by the polymer-based soft spheres; these assemblies can then be transformed into a range of different materials, including metal or semiconductor nanostructures, or further tailored with an additional level of complexity. Structural investigation shows the superstructure formation to be governed by generic design rules that can be extended across different material combinations.

In situ synthesis of high density sub-50 nm ZnO nanopatterned arrays using diblock copolymer templates.

Fabrication of high density (~155 Gbit in(-2)) ZnO nanopatterns through in situ decomposition of Zn precursors inside diblock copolymer templates and their application as charge storage centers in nonvolatile memory devices is described. The fabrication is performed in a highly controlled fashion with the resulting ZnO nanopatterned arrays exhibiting diameters of 38 nm and heights of 14 nm offering sub-50 nm feature resolutions. The ZnO nanopatterns are naturally n-type due to the presence of zinc interstitials and oxygen vacancies that act as defect levels in trapping charge carriers. Test capacitors (metal-oxide-semiconductor, MOS) constructed using nanopatterns formed on p-Si exhibited a large flatband voltage shift of about ~2.2 V for a low operating voltage of 10 V. A high charge trap density of 3.47 × 10(18) cm(-3) combined with a good retention capacity is observed with low tunneling oxide (thermally grown) thickness of 3 nm. This demonstrates the significant promise of the ZnO nanopatterned arrays to act as charge storage centers for potential application in nonvolatile flash memory devices. The charge trapping characteristics, the capacitance-voltage measurements, and the potential of ZnO nanopatterns as charge storage centers in fabricating nonvolatile memory devices are discussed.

Enhancing charge-storage capacity of non-volatile memory devices using template-directed assembly of gold nanoparticles.

We demonstrate the controlled fabrication of aggregates of gold nanoparticles as a means of enhancing the charge-storage capacity of metal-insulator-semiconductor (MIS) devices by up to 300% at a low biasing voltage of ±4 V. Aggregates of citrate stabilized gold nanoparticles were obtained by directed electrostatic self-assembly onto an underlying nanopattern of positively charged centers. The underlying nanopatterns consist of amine functionalized gold nanoparticle arrays formed using amphiphilic diblock copolymer reverse micelles as templates. The hierarchical self-organization leads to a twelve-fold increase in the number density of the gold nanoparticles and therefore significantly increases the charge storage centers for the MIS device. The MIS structure showed counterclockwise C-V hysteresis curves indicating a good memory effect. A memory window of 1 V was obtained at a low biasing voltage of ±4 V. Furthermore, C-t measurements conducted after applying a charging bias of 4 V showed that the charge was retained beyond 20,000 s. The proposed strategy can be readily adapted for fabricating next generation solution processible non-volatile memory devices.

Nanoparticle cluster arrays for high-performance SERS through directed self-assembly on flat substrates and on optical fibers.

We demonstrate template-guided self-assembly of gold nanoparticles into ordered arrays of uniform clusters suitable for high-performance SERS on both flat (silicon or glass) chips and an optical fiber faucet. Cluster formation is driven by electrostatic self-assembly of anionic citrate-stabilized gold nanoparticles (~11.6 nm diameter) onto two-dimensionally ordered polyelectrolyte templates realized by self-assembly of polystyrene-block-poly(2-vinylpyridine). A systematic variation is demonstrated for the number of particles (N ≈ 5, 8, 13, or 18) per cluster as well as intercluster separations (S(c) ≈ 37-10 nm). Minimum interparticle separations of <5 nm, intercluster separations of ~10 nm, and nanoparticle densities on surfaces as high as ~7 × 10(11)/in.(2) are demonstrated. Geometric modeling is used to support experimental data toward estimation of interparticle and intercluster separations in cluster arrays. Optical modeling and simulations using the finite difference time domain method are used to establish the influence of cluster size, shape, and intercluster separations on the optical properties of the cluster arrays in relation to their SERS performance. Excellent SERS performance, as evidenced by a high enhancement factor, >10(8) on flat chips and >10(7) for remote sensing, using SERS-enabled optical fibers is demonstrated. The best performing cluster arrays in both cases are achievable without the use of any expensive equipment or clean room processing. The demonstrated approach paves the way to significantly low-cost and high-throughput production of sensor chips or 3D-configured surfaces for remote sensing applications.

Inherently reproducible fabrication of plasmonic nanoparticle arrays for SERS by combining nanoimprint and copolymer lithography.

We present an inherently reproducible route to realizing high-performance SERS substrates by exploiting a high-throughput top-down/bottom-up fabrication scheme. The fabrication route employs self-assembly of amphiphilic copolymers to create high-resolution molds for nanoimprint lithography (NIL) spanning entire 100 mm Si wafers. The nanoporous polymer templates obtained upon NIL are subjected to galvanic displacement reactions to create gold nanorod arrays. Nanorods are subsequently converted to nanodiscs by thermal annealing. The nanodiscs were found to perform as robust SERS substrates as compared with the nanorods. The SERS performance of these substrates and its generality for catering to diverse molecules is demonstrated through the excellent Raman peak resolution and intensity for three different molecules, exhibiting different interaction modes on surface. Numerical simulations using FDTD shows plasmonic coupling between the particles and also brings out the influence due to size distribution. The approach combines distinct advantages of high-precision and repeatability offered by NIL with low-cost fabrication of high-resolution NIL molds by copolymer self-assembly.

Optical sensors based on whispering gallery modes in fluorescent microbeads: response to specific interactions.

Whispering gallery modes (WGMs) in surface-fixated fluorescent polystyrene microbeads are studied in view of their capability of sensing the formation of biochemical adsorption layers on their outer surface with the well-established biotin-streptavidin specific binding as the model system. Three different methods for analysis of the observed shifts in the WGM wavelength positions are applied and used to quantify the adsorbed mass densities, which are then compared with the results of a comparative surface plasmon resonance (SPR) study.

Confinement-Induced Enhancement of Antigen-Antibody Interactions within Binary Nanopatterns to Achieve Higher Efficiency of On-Chip Immunosensors.

By combining molecular self-assembly, nanosphere lithography and reactive ion etching, large-scale nanopatterns of antibodies are fabricated for direct application in state-of-the-art on-chip immunosensors. Using in-situ surface plasmon resonance, the patterns are studied in view of their antigen binding capacity, which shows an increase of up to 120% solely in the case of antibody confinement into the nanopatches by means of a nonfouling embedding matrix.

Block copolymer micelles as switchable templates for nanofabrication.

Block copolymer inverse micelles from polystyrene-block-poly-2-vinylpyridine (PS-b-P2VP) deposited as monolayer films onto surfaces show responsive behavior and are reversibly switchable between two states of different topography and surface chemistry. The as-coated films are in the form of arrays of nanoscale bumps, which can be transformed into arrays of nanoscale holes by switching through exposure to methanol. The use of these micellar films to act as switchable etch masks for the structuring of the underlying material to form either pillars or holes depending on the switching state is demonstrated.