Multiplex Quantification of Exosomes via Multiple Types of Nanobeads Labeling Combined with Laser Scanning Detection.

In recent years, exosomes have attracted attention in many aspects from basic research to clinical application, including therapeutic reagents or biomarkers for liquid biopsy. The increasing understanding of exosome's heterogeneous properties is expected to lead to more advanced exosome research, and there is therefore a need for a multiplex system that can easily classify and analyze exosomes in complex biological samples according to their properties. In this study, we developed a simple and sensitive multiplexed exosome quantification system based on ExoCounter, an exosome quantification system utilizing optical disk technology, by introducing nanobeads made of different materials as exosome labeling substances. The refractive indices suitable for nanobead materials were analyzed by computer simulation of optical diffraction generated by nanobeads. The results showed that polymer (FG), Au, and Ag nanobeads exhibited superior discrimination capability in terms of the amplitude and polarity of detection pulses generated by each nanobead. The specificity and detection sensitivity of three types of nanobeads were confirmed by detecting HER2-positive exosomes with anti-HER2 antibody-conjugated nanobeads. Furthermore, CD147-positive, HER2-positive, and CD81-positive exosomes in 12.5 µL of serum were simultaneously quantified with high discrimination performance using the anti-CD147 antibody, anti-HER2 antibody, or anti-CD81 antibody conjugated for FG beads, Au nanobeads, or Ag nanobeads, respectively. A limit of detection was also evaluated as low as 210 exosomes/µL. This system is a promising tool for advanced exosome research because it enables multiplexed detection of heterogeneous exosomes in serum with high specificity, accuracy, and sensitivity without purification.

Estimation of the Depletion Layer Thickness in Silicon Nanowire-Based Biosensors from Attomolar-Level Biomolecular Detection.

Silicon nanowire (SiNW) biosensors have attracted a lot of attention due to their superior sensitivity. Recently, the dependence of biomolecule detection sensitivity on the nanowire (NW) width, number, and doping density has been partially investigated. However, the primary reason for achieving ultrahigh sensitivity has not been elucidated thus far. In this study, we designed and fabricated SiNW biosensors with different widths (10.8-155 nm) by integrating a complementary metal-oxide-semiconductor process and electron beam lithography. We aimed to investigate the detection limit of SiNW biosensors and reveal the critical effect of the 10-nm-scaled SiNW width on the detection sensitivity. The sensing performance was evaluated by detecting antiovalbumin immunoglobulin G (IgG) with various concentrations (from 6 aM to 600 nM). The initial thickness of the depletion region of the SiNW and the changes in the depletion region due to biomolecule binding were calculated. The basis of this calculation are the resistance change ratios as functions of IgG concentrations using SiNWs with different widths. The calculation results reveal that the proportion of the depletion region over the entire SiNW channel is the essential reason for high-sensitivity detection. Therefore, this study is crucial for an indepth understanding on how to maximize the sensitivity of SiNW biosensors.

Design and Fabrication of Silicon Nanowire-Based Biosensors with Integration of Critical Factors: Toward Ultrasensitive Specific Detection of Biomolecules.

As critical factors affecting the sensing performance of silicon nanowire (SiNW) biosensors, the structure, functional interface, and detection target were analyzed and designed to improve sensing performance. For an improved understanding of the dependence of sensor structure on sensitivity, a simple theoretical analysis was proposed to predict the sensitivity of biosensors with different SiNW types, widths, and doping concentrations. Based on the theoretical analysis, a biosensor integrating optimized critical factors was designed and fabricated. Optimizations focusing on the following aspects are considered: (1) employing n-type SiNW and controlling the impurity doping concentration of SiNW at approximately 2 × 1016-6 × 1016 atoms/cm3 to obtain a suitable charge density, (2) minimizing the SiNW width to 16.0 nm to increase the surface area-to-volume ratio, (3) using a native oxide layer on SiNW as a gate insulator to transport the captured charge molecules closer to the SiNW surface, (4) modifying the SiNW surface by 2-aminoethylphosphonic acid coupling to form a high-density self-assembled monolayer for enhancing the stability bound molecules, and (5) functionalizing the SiNW with ovalbumin molecules for specifically capturing the target immunoglobulin G (IgG) molecules. The sensing performance was evaluated by detecting IgG with concentrations ranging from 6 aM to 600 nM and control experiments. The SiNW biosensor revealed ultrahigh sensitivity and specific detection of target IgG with a measured limit of detection of 6 aM. The integration of the critical SiNW biosensor factors provides a significant possibility of a rapid and ultrasensitive diagnosis of diseases at their early stages.

Nanometer resolution stress measurement of the Si gate using illumination-collection-type scanning near-field Raman spectroscopy with a completely metal-inside-coated pyramidal probe.

We have proposed an illumination-collection-type scanning near-field Raman spectroscopy (SNRS) with a completely gold metal-inside-coated (MIC) pyramidal probe without an optical aperture in order to detect the Raman spectra of fine Si devices for local stress measurements. The gold MIC pyramidal probe has been studied to act as a plasmon resonance near-field optical probe with high power using a finite differential time domain (FDTD) simulation and the prototyped SNRS. In the simulation, the propagated optical power can be made available for SNRS. In the experiments, it is clear that the prototyped SNRS enhanced the Si Raman peak signal by plasmon resonance and could measure the Si Raman peak shift by line scanning the Si gate region and the Si active layer. Furthermore, compressive and tensile stresses localized around the Si gate were demonstrated by the Si Raman peak shift with a resolution of about 10 nm. It is clarified that the proposed SNRS has the possibility of detecting the Raman spectra of a local area.

Observation of Si pattern sidewall using inclination atomic force microscope for evaluation of line edge roughness.

Inclination atomic force microscope (AFM) imaging has been studied on the possibility to observe a pattern sidewall in contact mode or digital probing (step-in) mode for a line edge roughness (LER) or line width roughness (LWR). Analysis of the AFM tip bending and slipping indicates that it is serious problem to measure and control very fine patterns within an error of less than 1 nm in contact of the tip on the steep slop of the pattern, and it is very important directly to observe the sidewall at inclination angle. In experiments using pyramidal tip and steep Si pattern with about 90 degrees slop, it has demonstrated that the inclination angle is 35-40 degrees for faithful observation of the sidewall. We have observed the etched strip lines on the sidewall with a width of about 100 nm and a depth of about 6.4 nm. We have demonstrated that the inclination AFM is very useful for evaluation of the LER or LWR.

Formation of dot arrays with a pitch of 20 nm × 20 nm for patterned media using 30 keV EB drawing on thin calixarene resist.

We studied the possibility of achieving very fine-pitch dot arrays with a pitch of 20 nm × 20 nm using 30 keV electron beam (EB) drawing on negative calixarene resist. In order to form such patterns, we studied the dependence on resist thickness of the dot size and the packing. We propose EB drawing on an extremely thin film for very highly packed dot-array formation. Our experimental results demonstrate the possibility of forming highly packed dot-array patterns with a pitch of 20 nm × 20 nm and a resist thickness of about 13 nm, which corresponds to about 1.6 Tbits in(-2).

Scanning electron microscope for in situ study of crystallization of Ge2Sb2Te5 in phase-change memory.

By introducing electrical connections into the chamber of a scanning electron microscope (SEM) via its holder assembly, it has become feasible to in situ observe and electrically characterize electronic devices. The in situ SEM was applied to investigate electric-pulse-induced behavior of Ge(2)Sb(2)Te(5) in a lateral phase-change memory cell. Randomly distributed nuclei with sizes from 20 to 80 nm were initiated at a low voltage pulse. Initially, grain growth depended strongly on pulse amplitude at around 60.3 nm/V and then a weak pulse amplitude dependence was observed at around 13.5 nm/V. Device resistance during crystallization dropped by two to three orders of magnitude with two falling steps, which probably resulted from amorphous to face-centered-cubic and subsequently to hexagonal transitions, respectively.