Study of charge transfer contribution in Surface-Enhanced Raman scattering (SERS) based on indium oxide nanoparticle substrates.

Surface-enhanced Raman scattering (SERS) has outstanding merits in biochemical molecular analysis, and the development of new SERS substrates is the focus of research. Herein, In2O3 nanoparticles (NPs) were synthesized by a high temperature pyrolysis method with cubic phase and small particle size at 10 nm. The structures and properties of In2O3 NPs were characterized by X-ray powder diffraction (XRD), transmission electron microscope (TEM) and other characterization methods. Additionally, the SERS spectra of In2O3-MBA with the enhancement factor (EF) up to 1.22 × 104 is discussed. The results demonstrate that there is a charge transfer (CT) effect revealed between the adsorbed molecules of 4-mercaptobenzoic acid (4-MBA) and the substrates of In2O3 NPs, and it could be excited by long wavelength energy. Based on the In2O3 NPs, the study is beneficial to develop more potential semiconductor SERS substrates.

Digital microfluidics-based digital counting of single-cell copy number variation (dd-scCNV Seq).

Single-cell copy number variations (CNVs), major dynamic changes in humans, result in differential levels of gene expression and account for adaptive traits or underlying disease. Single-cell sequencing is needed to reveal these CNVs but has been hindered by single-cell whole-genome amplification (scWGA) bias, leading to inaccurate gene copy number counting. In addition, most of the current scWGA methods are labor intensive, time-consuming, and expensive with limited wide application. Here, we report a unique single-cell whole-genome library preparation approach based on digital microfluidics for digital counting of single-cell Copy Number Variation (dd-scCNV Seq). dd-scCNV Seq directly fragments the original single-cell DNA and uses these fragments as templates for amplification. These reduplicative fragments can be filtered computationally to generate the original partitioned unique identified fragments, thereby enabling digital counting of copy number variation. dd-scCNV Seq showed an increase in uniformity in the single-molecule data, leading to more accurate CNV patterns compared to other methods with low-depth sequencing. Benefiting from digital microfluidics, dd-scCNV Seq allows automated liquid handling, precise single-cell isolation, and high-efficiency and low-cost genome library preparation. dd-scCNV Seq will accelerate biological discovery by enabling accurate profiling of copy number variations at single-cell resolution.

Observation of tunable surface plasmon resonances and surface enhanced infrared absorption (SEIRA) based on indium tin oxide (ITO) nanoparticle substrates.

The application of surface enhanced infrared absorption (SEIRA) is severely restricted in many fields due to the SEIRA substrates are constructed mainly from expensive noble metals. Therefore, the development of new SEIRA substrates other than the noble metallic ones is very valuable. Here we introduced a new semiconductor SEIRA substrate, the indium tin oxide (ITO) nanoparticles (NPs), to study the SEIRA property. The results demonstrate that the ITO NPs show the SEIRA property and the enhancement is dependent to the doping ratio of the heteroatoms of tin. The ITO NPs with the 5% atomic doping ratio show the highest SEIRA enhancement factor (EF), which is about 24. The limit of detection (LOD) of the 1,1'-dicarboxyferrocene (dcFc) molecule was as low as 10-5 mol/L. The present study proves that the tin-doped indium oxide can be used as a new and inexpensive semiconductor SEIRA substrate. It also proves that the doped semiconductor NPs have strong potentials for being used as emerging SEIRA substrates.

Study of charge transfer effect in Surface-Enhanced Raman scattering (SERS) by using Antimony-doped tin oxide (ATO) nanoparticles as substrates with tunable optical band gaps and free charge carrier densities.

Surface-enhanced Raman scattering (SERS) has been applied in many fields, but still has the limitation of widespread applications on semiconductor substrates. In this work, a series of antimony-doped tin oxide (ATO) nanoparticles (NPs) have been synthesized by a hydrothermal method and were used as SERS substrates for the first time. Interestingly, a charge transfer (CT) effect was revealed between the probing molecules of 4-mercaptobenzoic acid (4-MBA) and the substrates of ATO NPs, which accounts for the SERS enhancement and shows dependence to the Sb ions doping ratios in ATO NPs. By considering the energy level diagram of the ATO-MBA complexes and the doping theory of semiconductors, this phenomenon is believed to connect to the variance of the optical band gap energy (Eg), which is accompanied with the changes of free charge carrier densities in conduction bands (CBs) of ATO NPs due to different doping contents. The study of the Eg- or free-charge-carrier-density-dependent property of the semiconductor-based SERS provides a new point of view for the development of new semiconductor SERS substrates and also contributes to the SERS CT mechanism.

Enhanced charge-transfer induced by conduction band electrons in aluminum-doped zinc oxide/molecule/Ag sandwich structures observed by surface-enhanced Raman spectroscopy.

In the semiconductor/molecule/metal system, enhancing the efficiency of the charge-transfer (CT) plays a pivotal role in improving the sensitivity of semiconductor-based surface-enhanced Raman scattering (SERS). In this work, use of SERS for detection of an enhanced CT in a chemically-etched Al-doped ZnO (AZO), 4-mercaptopyridine (MPy) molecule, and Ag nanoparticles (NPs) (AZO/MPy/Ag) sandwich structure is reported. A series of CT routes are proposed in the energy level diagram of AZO/MPy/Ag assemblies under the excitation line at 633 nm. Very interestingly, for the first of its kind, a significant CT route from the conduction band (CB) of AZO to the lowest unoccupied molecular orbital (LUMO) of MPy molecule is detected. This route can remarkably improve the degree of CT in the AZO/MPy/Ag system by about 48% compared with that of the ZnO/MPy/Ag system. Furthermore, the uniquely enhanced CT route is also further confirmed by alternative probe molecules like p-aminothiophenol (PATP) and 4-mercaptobenzoic acid (MBA). The discovery of this extra CT route will inevitably play an irreplaceable role in SERS enhancement through its participating in the CT enhancement mechanism.

Surface Plasmon Resonance from Gallium-Doped Zinc Oxide Nanoparticles and Their Electromagnetic Enhancement Contribution to Surface-Enhanced Raman Scattering.

In recent years, semiconductor-based surface-enhanced Raman scattering (SERS) substrates have received considerable attention and led to a forefront in the SERS field. However, the lack of electromagnetic (EM) enhancement contributions highly precludes the development of semiconductive-substrate-based SERS. In this study, Ga-doped ZnO nanoparticles (NPs) were fabricated and employed as novel SERS substrates based on the EM enhancement contribution of surface plasmon resonance (SPR). The obtained Ga-doped ZnO NPs exhibited obvious SPR absorptions in the visible and near- and mid-infrared regions. SPR absorption can be readily tuned by changing the doping ratios of Ga3+ ions. The SERS spectra of Ga-doped ZnO/4-mercaptopyridine (MPy) were investigated at different excitation wavelengths of 488, 532, 633, and 785 nm. The spectral enhancement of Ga-doped ZnO substrates depended on the doping ratios, excitation wavelengths, and nearby SPR absorption. Ga-doped ZnO NPs with the highest free charge carrier density and the doping ratio of 5% showed the strongest SERS spectra. For the fixed doping ratio of 5%, the better is the match between excitation wavelengths and SPR absorption, the higher is the SERS spectral enhancement. This study showed the feasibility of EM contributions to SERS by using semiconductive substrates and can contribute to the development of the semiconductor-based EM mechanism.

Auto-Panning: a highly integrated and automated biopanning platform for peptide screening.

Biopanning, a common affinity selection approach in phage display, has evolved numerous ligands for diagnosis, imaging, delivery, and therapy applications. However, traditional biopanning has suffered from time-consuming processes, highly-repetitive procedures and labor-intensive manual operation. Herein, a highly integrated and automated biopanning platform (Auto-Panning) is proposed. Based on digital microfluidics (DMF), biopanning processes are integrated on a chip with highly reproducible, precise, automated liquid manipulation. Therefore, 3 rounds of Auto-Panning can be accomplished within 16 h, instead of nearly a week of complicated manual operations. Auto-Panning has been used to evolve a specific peptide against cancer biomarker EphA2 with excellent cellular penetrating ability and significant invasion suppression biofunction, successfully demonstrating the practicality of the platform. Overall, as an automated programmable molecular screening platform, Auto-Panning will further promote the discovery and applications of novel ligands.

Digital-WGS: Automated, highly efficient whole-genome sequencing of single cells by digital microfluidics.

Single-cell whole-genome sequencing (WGS) is critical for characterizing dynamic intercellular changes in DNA. Current sample preparation technologies for single-cell WGS are complex, expensive, and suffer from high amplification bias and errors. Here, we describe Digital-WGS, a sample preparation platform that streamlines high-performance single-cell WGS with automatic processing based on digital microfluidics. Using the method, we provide high single-cell capture efficiency for any amount and types of cells by a wetted hydrodynamic structure. The digital control of droplets in a closed hydrophobic interface enables the complete removal of exogenous DNA, sufficient cell lysis, and lossless amplicon recovery, achieving the low coefficient of variation and high coverage at multiple scales. The single-cell genomic variations profiling performs the excellent detection of copy number variants with the smallest bin of 150 kb and single-nucleotide variants with allele dropout rate of 5.2%, holding great promise for broader applications of single-cell genomics.

A Highly Sensitive, Accurate, and Automated Single-Cell RNA Sequencing Platform with Digital Microfluidics.

Single-cell RNA sequencing (scRNA-seq) is a powerful method in investigating single-cell heterogeneity to reveal rare cells, identify cell subpopulations, and construct a cell atlas. Conventional benchtop methods for scRNA-seq, including multistep operations, are labor intensive, reaction inefficient, contamination prone, and reagent consuming. Here we report a digital microfluidics-based single-cell RNA sequencing (digital-RNA-seq) for simple, efficient, and low-cost single-cell mRNA measurements. Digital-RNA-seq automates fluid handling as discrete droplets to sequentially perform protocols of scRNA-seq. To overcome the current problems of single-cell isolation in efficiency, integrity, selectivity, and flexibility, we propose a new strategy, passive dispensing method, relying on well-designed hydrophilic-hydrophobic microfeatures to rapidly generate single-cell subdroplets when a droplet of cell suspension is encountered. For sufficient cDNA generation and amplification, digital-RNA-seq uses nanoliter reaction volumes and hydrophobic reaction interfaces, achieving high sensitivity in gene detection. Additionally, the stable droplet handling and oil-closed reaction space featured in digital-RNA-seq ensure highly accurate measurement. We demonstrate the functionality of digital-RNA-seq by quantifying heterogeneity among single cells, where digital-RNA-seq shows excellent performance in rare transcript detection, cell type differentiation, and essential gene identification. With the advantages of automation, sensitivity, and accuracy, digital-RNA-seq represents a promising scRNA-seq platform for a wide variety of biological applications.

Stimuli-Responsive Microfluidic Interface Enables Highly Efficient Capture and Release of Circulating Fetal Cells for Non-Invasive Prenatal Testing.

Circulating fetal nucleated cells (CFCs) carrying whole genomic coding of the fetus in maternal blood have been pursued as ideal biomarkers for noninvasive prenatal testing (NIPT). However, a significant limitation is the need to enrich sufficient cells in quantity and purity for fetal genetic disorder diagnosis. This study for the first time demonstrates a stimuli-responsive ligand enabling interface on array patterned microfluidic chip (NIPT-Chip) for high efficient isolation and release of CFCs in untreated whole blood. Deterministic lateral displacement (DLD)-array was patterned in the chip to increase collision frequency between CFCs and surface-anchored antibody to achieve high efficient cell capture. More importantly, the stimuli-responsive interface enables gentle release of captured CFCs through a thiol exchange reaction for downstream gene analysis of NIPT. With the advantages of simple processing, efficient isolation, and gentle release, NIPT-Chip offers great potential for clinical translation of circulating fetal cell-based NIPT.

Centrifugal-Driven Droplet Generation Method with Minimal Waste for Single-Cell Whole Genome Amplification.

High-quality whole-genome amplification (WGA) of individual cells is the primary step for characterizing the genetic information on single cells in biology and medicine. As the most popular single-cell WGA method, multiple displacement amplification (MDA) is often plagued by the nonuniform amplification. The droplet MDA has been an innovative tool to solve this dilemma by mitigating the amplification bias and increasing the genomic coverage. Despite these advantages, the time-consuming droplet generation process, the waste of small volume samples and the difficulty of parallel operation for multiple single-cell samples remain major obstacles. Herein, we introduce a centrifugal-driven droplet generation method for rapid and convenient generation of uniform droplets from a relatively small volume sample (5 µL) in 60s with more than 98% sample utilization. We have performed quantitative digital droplet PCR using this method, demonstrating its capability of amplifying nucleic acids at the single-molecule level. Single-cell centrifugal-driven droplet MDA (cd-MDA) has also been conducted for single-cell sequencing, achieving uniform amplification and broad genomic coverage. With the single-molecule sensitivity, minimum sample waste, high genomic coverage, and excellent sequencing evenness, this centrifugal-driven droplet generation method is promising for convenient and scalable use in digital PCR and single-cell whole-genome research.

A simple strategy to improve the sensitivity of probe molecules on SERS substrates.

In this work, we report a simple strategy to improve the detection sensitivity as well as the spectral quality of probe molecules on surface-enhanced Raman scattering (SERS) substrates. On normal SERS substrates, due to the decreased absorption capacity and changes in the molecule orientations, SERS signals disappear when the analyte molecule concentrations reach a limit value. To solve this problem, the molecular template reagent (MTR) technique, a simple strategy based on SERS surface selection rules, is considered. By choosing the best MTR according to different samples, the effect of adjusting the molecular orientations of samples can be studied. In this process, 1-butanethiol, 1-hexanethiol, 1-octanethiol, 1-decanethiol, and 1-dodecanethiol, which are MTRs, are used to adjust the orientations of probe molecules under optimized conditions. The use of the MTR technique indicated that the limit of detection (LOD) of the probe molecules of p-aminobenzenethiol and 4-mercaptobenzoic acid on noble metal substrates showed an increase of one order of magnitude over the LOD of the pure probing molecule systems. Hence, the proposed method introduces a way to detect the molecules with an improved sensitivity at extremely low concentrations. The study corresponds to a proof-of-concept study of MTR-assisted SERS for SERS-based applications in ultrasensitive analyses.

Identification of native charge-transfer status of p-aminothiolphenol adsorbed on noble metallic substrates by surface-enhanced infrared absorption (SEIRA) spectroscopy.

p-Aminothiophenol (PATP) is a preferred molecule in research on surface-enhanced Raman scattering (SERS) because of its unique characteristics of high spectral activity, easily induced charge-transfer (CT), and sensitivity to molecular structural changes. However, some aspects are still unclear, such as the initial steady state of PATP on noble metallic substrates without strong additional excitation with incident and/or induced electromagnetic radiation. Information about the initial steady state, especially the intrinsic CT state, is of great importance to elucidate the dynamic processes of CT and/or molecular structural changes under additional excitation. To investigate the native state of an adsorbed molecule, a suitable probe method that does not disrupt the native state of the whole system, including both molecules and substrates, is required. SERS is not applied in this context because of its use of high-energy visible and near-infrared light. Herein, a low-energy probe method, surface-enhanced infrared-absorption (SEIRA) spectroscopy, is employed as a suitable method for studying the native adsorption state of PATP on silver nanoisland films. The molecular structure and adsorption state were investigated. The intrinsic CT state received particular attention by analyzing the CT-related vibration of B2 modes. Using Fourier transform infrared (FTIR), transmission SEIRA and reflection SEIRA spectroscopy, we explained why the relative intensities of some bands were different under different conditions. A quasi-standing orientation of PATP adsorbed on the substrates was also confirmed. More importantly, we demonstrated that there is no perceptible CT between PATP and silver nanoisland films; in contrast, CT generally occurs in a disruptive manner in SERS. Density functional theory (DFT) calculations and the selection rules for infrared (IR) transmission and reflection-absorption spectroscopy were used to analyze the spectra throughout the paper. SEIRA proved to be an effective technique to explore the native adsorption state of molecules without the excessive external disturbance induced by excitation. The results are very important in providing insight into molecules in surface-interface chemistry, enhanced spectroscopy and photoelectronics.

A reagent-assisted method in SERS detection of methyl salicylate.

With the explosive application of methyl salicylate (MS) molecules in food and cosmetics, the further detection of MS molecules becomes particularly important. Here we investigated the detection of MS molecules based on surface-enhanced Raman scattering (SERS) in a novel molecule/assistant/metal system constructed with MS, 4,4'-(hexafluoroisopropylidene) bis (benzoic acid) and Ag nanoparticles (AgNPs). The minimum detection concentration is 10-4 M. To explore the function of assisted reagent, we also referred another system without assistant molecules. The result demonstrates that SERS signals were not acquired, which proves that the assistant molecules are critical for the capture of MS molecules. Two possible mechanisms of MS/assistant/AgNPs system were speculated through two patterns of hydrogen bonds. The linker molecules acted as the role of the bridge between metallic substrates and target molecules through the molecular recognition. This strategy is very beneficial to the expanding of MS detection techniques and other hydrogen bond based coupling detections with SERS.

Controlling the orientation of probe molecules on surface-enhanced Raman scattering substrates: A novel strategy to improve sensitivity.

Typically, the surface-enhanced Raman scattering (SERS) technique is employed in precious metallic substrates with spontaneously adsorbed probing molecules to acquire signals. Preferred chemical conditions including aggregate status, hydrophilic/hydrophobic surroundings, and smart linkers were created to enable the detection of targets in very low concentrations (lower than 1.0 × 10-7 M). Although the sensitivity of SERS is applicable to certain areas, it is not satisfied in several cases that require obtaining good resolved signals involving extremely few surface molecules. Thus, further improvements in the sensitivity based on existing SERS techniques pose a challenge and is desirable for all aspects of analytical chemistry. In this study, a novel strategy was employed by constructing a molecular template (MT) on the SERS substrates with spontaneously adsorbed probe molecules to improve the detection sensitivity of probe molecules. The proposed MT-assisted SERS technique differed from previous methods as it provides a completely new method for improving the limit of detection (LOD) of SERS by controlling molecular orientations. The surface selection rules of SERS spectra were first introduced as an effective strategy to improve the detection sensitivity, and this was extremely beneficial with respect to analytical applications. The use of the MT-assisted SERS technique indicated that the LOD of probe molecules of p-aminobenzenethiol (8.0 × 10-9 M) and 4-mercaptobenzoic (1.0 × 10-7 M) acids on noble metallic substrates exhibited nearly one order of magnitudes. Hence, the proposed method paves a way to detect the molecules under improved sensitivity at extreme low concentrations. The study corresponded to a proof-of-concept study of MT-assisted SERS for SERS-based applications in ultra-sensitive analysis.

Graded nanowell arrays: a fine plasmonic "library" with an adjustable spectral range.

We present an effective approach for fabricating graded plasmonic arrays based on ordered micro-/nanostructures with a geometric gradient. Ag nanowell arrays with graded geometric parameters were fabricated and systematically investigated. The order of the graded plasmonic arrays is generated by colloidal lithography, while the geometric gradient is the result of inclined reactive ion etching. The surface plasmon resonance (SPR) peaks were measured at different positions, which move gradually along the Ag nanowell arrays with a geometric gradient. Such micro-/nanostructure arrays with graded and integrated SPR peaks can work as a fine plasmonic "library" (FPL), and the spectral range can be controlled using a "coarse adjustment knob" (lattice constant) and a "fine adjustment knob" (pore diameter). Additionally, the spectral resolution of the FPL is high, which benefits from the high value of the full height/full width at half-maximum and the small step size of the wavelength shift (0.5 nm). Meanwhile, the FPL could be effectively applied as a well-defined model to verify the plasmonic enhancement in surface enhanced Raman scattering. As the FPL is an integrated optical material with graded individual SPR peaks, it can not only be a theoretical model for fundamental research, but also has great potential in high-throughput screening of optical materials, multiplex sensors, etc.

[Preparation of Au Nanoparticles with Different Morphologies and Study of Their Property as Surface Enhanced Raman Scattering Substrates].

Different shapes of gold nanoparticles (NPs) have different enhancement effect in Surface Enhanced Raman Scattering (SERS). The polyhedral Au NPs have multiple angular structures, which show stronger enhancement effect than Au nanoplatelets. In recent years, the research on synthesis and properties of polyhedral Au NPs has attracted much attention. In this study the enhancement effect of the Au NPs in SERS was observed in Au NPs with the shape of dodecahedron, icosahedron, triangular plate and spherosome. Triangular Au NP films were prepared through a chemical reduction method using sodium borohydride as the reductant. As for synthesizing icosahedral Au NPs, Poly (vinyl pyrrolidone) is used as a capping agent and diethylene glycol is used as a reducing agent. Dodecahedral Au NPs were synthesized using icosahedral Au NPs as seeds. SERS spectra were detected for these three Au NPs as well as the traditional colloidal Au by using 4-mercaptopyridine and 4-mercaptobenzoic as probing molecules. Triangular Au NP films, icosahedral Au NPs and dodecahedral Au NPs were with average diameters of about 130, 100 and 120 nm. UV/Vis spectroscopy indicated that these three Au NPs had typical absorbance bands at 589, 598 and 544 nm. The results show that the Au polyhedron has better enhancement ability than the Triangular NPs.

Detection of physiological potassium ions level in human serum by Raman scattering spectroscopy.

Potassium ions play pivotal roles in biological systems. A simple and effective Raman scattering-based protocol for the detection of physiological potassium ions level in human serum has been developed. We employed sodium cobaltinitrite, which can react with potassium to form potassium-sodium cobaltinitrite. Combining hydrophobic surface and the Raman scattering for potassium detection, highly reproducible Raman spectra of the potassium-sodium cobaltinitrite can be obtained with highly sensitivity. The characteristic Raman peak of potassium-sodium cobaltinitrite was used to distinguish it from other kinds of matter, and its intensity was used to monitor the amounts of potassium ions in human serum. Because of the contribution of the hydrophobic surface aggregation to the Raman, this protocol has great potential for practical detection of the potassium in human serum and has extended the measuring potential use in the detection of potassium in human serum by spectrum method.

[Surface-Enhanced Raman Scattering Study on Photocatalysis of PATP When Adsorbed on Ag/TiO2 Nanotubes].

Surface-enhanced Raman scattering (SERS) is spectroscopic technique with ultra-sensitivity and high selectivity and has attracted great attention because of the potential applications in various fields. P-aminothiophenol (PATP) is often used as SERS probe molecule because it is easy to adsorb on SERS substrates and produce high-quality SERS signals. TiO2 is extensively used as photocatalyst although its photocatalytic efficiency is still needed to be improved. Noble metal-modified TiO2 is one of current important techniques for maximizing the efficiency of photocatalytic efficiency. In this article, a kind of bifunctional SERS substrates, Ag/TiO2 nanotubes, with photocatalysis property were prepared, the TiO2 NTs were prepared by anodic oxidation and noble metal Ag nanoparticles were deposited on the surface of TiO2 NTs by photoreduction method. The photocatalysis of PATP on Ag/TiO2 NTs and on Ag mirror substrates were studied. The SERS signals of PATP were decreased with the ultraviolet irradiation time, however, on Ag mirror substrates, SERS intensity of PATP was slightly changed, which indicated the photocatalysis reaction of PATP on Ag/TiO2 NTs substrates. The kinetics analysis results indicate that the kinetics of the photocatalysis follows the first order of the dynamical reaction.

Experimental and density functional theory study of Raman and SERS spectra of 5-amino-2-mercaptobenzimidazole.

Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS) and density functional theory (DFT) simulations were employed to study 5-amino-2-mercaptobenzimidazole (5-A-2MBI) molecules. Ag colloids were used as SERS substrates which were prepared by using hydroxylamine hydrochloride as reducing agent. Raman vibration modes and SERS characteristic peaks of 5-A-2MBI were assigned with the aid of DFT calculations. The molecular electrostatic potential (MEP) of 5-A-2MBI was used to discuss the possible adsorption behavior of 5-A-2MBI on Ag colloids. The spectral analysis showed that 5-A-2MBI molecules were slightly titled via the sulfur atoms adhering to the surfaces of Ag substrates. The obtained SERS spectral intensity decreased when lowering the 5-A-2MBI concentrations. A final detection limit on the concentration of 5×10(-7) mol · L(-1) was gained. SERS proved to be a simple, fast and reliable method for the detection and characterization of 5-A-2MBI molecules.