Single Nucleobase Identification Using Biophysical Signatures from Nanoelectronic Quantum Tunneling

Nanoelectronic DNA sequencing can provide an important alternative to sequencing‐by‐synthesis by reducing sample preparation time, cost, and complexity as a high‐throughput next‐generation technique with accurate single‐molecule identification. However, sample noise and signature overlap continue to prevent high‐resolution and accurate sequencing results. Probing the molecular orbitals of chemically distinct DNA nucleobases offers a path for facile sequence identification, but molecular entropy (from nucleotide conformations) makes such identification difficult when relying only on the energies of lowest‐unoccupied and highest‐occupied molecular orbitals (LUMO and HOMO). Here, nine biophysical parameters are developed to better characterize molecular orbitals of individual nucleobases, intended for single‐molecule DNA sequencing using quantum tunneling of charges. For this analysis, theoretical models for quantum tunneling are combined with transition voltage spectroscopy to obtain measurable parameters unique to the molecule within an electronic junction. Scanning tunneling spectroscopy is then used to measure these nine biophysical parameters for DNA nucleotides, and a modified machine learning algorithm identified nucleobases. The new parameters significantly improve base calling over merely using LUMO and HOMO frontier orbital energies. Furthermore, high accuracies for identifying DNA nucleobases were observed at different pH conditions. These results have significant implications for developing a robust and accurate high‐throughput nanoelectronic DNA sequencing technique.     

Single Quantum Dot Analysis Enables Multiplexed Point Mutation Detection by Gap Ligase Chain Reaction

Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap‐LCR), the high sensitivity of single‐molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant‐specific ligation products are generated by Gap‐LCR and subsequently captured by QDs to form DNA–QD nanocomplexes that are detected by single‐molecule spectroscopy (SMS) through multi‐color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation‐free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre‐amplification.     

Quantitative enhanced Raman scattering of labeled DNA from gold and silver nanoparticles

Surface-enhanced resonance Raman scattering (SERRS) from silver nanoparticles using 514.5-nm excitation has been shown to offer huge potential for applications in highly sensitive multiplexed DNA assays. If the technique is to be applied to real biological samples and integrated with other methods, then the use of gold nanoparticles and longer wavelengths of excitation are desirable. The data presented here demonstrate that dye-labeled oligonucleotide sequences can be directly detected by SERRS using gold nanoparticles in a quantitative manner for the first time. The performance of gold and silver nanoparticles as SERRS substrates was assessed using 514.5-, 632.8-, and 785-nm excitation and a range of 13 commercially available dye-labeled oligonucleotides. The quantitative response allowed the limit of detection to be determined for each case and demonstrates that the technique is highly effective, sensitive, and versatile. The possibility of excitation at multiple wavelengths further enhances the multiplexing potential of the technique. The importance of effectively combining the optical properties of the nanoparticle and the dye label is demonstrated. For example, at 632.8-nm excitation, the dye BODIPY TR-X and gold nanoparticles make a strong SERRS combination with very little background fluorescence. This study allows the choice of nanoparticle and dye label for particular experimental setups, and significantly expands the applicability of enhanced Raman scattering for use in many disciplines.     

Aptatag‐Based Multiplexed Assay for Protein Detection by Surface‐Enhanced Raman Spectroscopy

Silver‐nanoparticle dimers held together by a Raman reporter, capped with DNA aptamers and stabilized by polyethylene glycol chains, can be used to develop a multiplexed heterogeneous bioassay for protein detection with high sensitivity and selectivity.     

Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection

We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.     

Multiphonon resonant Raman scattering (MRRS) of semiconductor nanomaterials for biodetection

Robust, rapid and sensitive detection of specific molecules at ultra low concentrations is becoming increasing important, especially for medical diagnosis, food safety and environmental protection. Various spectroscopic techniques have been developed to apply in analysis and assay fields, including fluorescent spectroscopy, infrared spectroscopy and surface-enhanced Raman spectroscopy (SERS), etc. Multiphonon resonant Raman scattering (MRRS) technique is usually used to investigate optical lattice vibrations and the interaction between phonon and electron in semiconductor nanomaterials. Recently, it exhibits fascinating prospects in biodetection due to its significant specific spectroscopic and material advantages. The multiple-order phonon lines are material intrinsic and thus can be used as a characteristic fingerprint signal to label the biomolecules. The strong anti-interference capacity to external environment allows an accurate detection. Narrow bandwidth makes the technique suitable for multiplexed analysis. Proper choice of nontoxic inorganic semiconductor materials facilitates in-vivo detection. This review focuses on the current theoretical and technical developments in MRRS of semiconductor nanomaterials and highlights their applications in bioanalysis. The basic principle, merits, challenges and future perspectives of MRRS detection technique are discussed.     

Composite‐Scattering Plasmonic Nanoprobes for Label‐Free,Quantitative Biomolecular Sensing

Biosensing based on localized surface plasmon resonance (LSPR) relies on concentrating light to a nanometeric spot and leads to a highly enhanced electromagnetic field near the metal nanostructure. Here, a design of plasmonic nanostructures based on rationally structured metal–dielectric combinations is presented, called composite scattering probes (CSPs), to generate an integrated multimodal biosensing platform featuring LSPR and surface‐enhanced Raman spectroscopy (SERS). Specifically, CSP configurations are proposed, which have several prominent resonance peaks enabling higher tunability and sensitivity for self‐referenced multiplexed analyte sensing. Using electron‐beam evaporation and thermal dewetting, large‐area, uniform, and tunable CSPs are fabricated, which are suitable for label‐free LSPR and SERS measurements. The CSP prototypes are used to demonstrate refractive index sensing and molecular analysis using albumin as a model analyte. By using partial least squares on recorded absorption profiles, differentiation of subtle changes in refractive index (as low as 0.001) in the CSP milieu is demonstrated. Additionally, CSPs facilitate complementary untargeted plasmon‐enhanced Raman measurements from the sample's compositional contributors. With further refinement, it is envisioned that the method may lead to a sensitive, versatile, and tunable platform for quantitative concentration determination and molecular fingerprinting, particularly where limited a priori information of the sample is available.     

Multiplexed microfluidic surface-enhanced Raman spectroscopy

Over the past few decades, surface-enhanced Raman spectroscopy (SERS) has garnered respect as an analytical technique with significant chemical and biological applications. SERS is important for the life sciences because it can provide trace level detection, a high level of structural information, and enhanced chemical detection. However, creating and successfully implementing a sensitive, reproducible, and robust SERS active substrate continues to be a challenging task. Herein, we report a novel method for SERS that is based upon using multiplexed microfluidics (MMFs) in a polydimethylsiloxane platform to perform parallel, high throughput, and sensitive detection/identification of single or various analytes under easily manipulated conditions. A facile passive pumping method is used to deliver Ag colloids and analytes into the channels where SERS measurements are done under nondestructive flowing conditions. With this approach, SERS signal reproducibility is found to be better than 7%. Utilizing a very high numerical aperture microscope objective with a confocal-based Raman spectrometer, high sensitivity is achieved. Moreover, the long working distance of this objective coupled with an appreciable channel depth obviates normal alignment issues expected with translational multiplexing. Rapid evaluation of the effects of anion activators and the type of colloid employed on SERS performance are used to demonstrate the efficiency and applicability of the MMF approach. SERS spectra of various pesticides were also obtained. Calibration curves of crystal violet (non-resonant enhanced) and Mitoxantrone (resonant enhanced) were generated, and the major SERS bands of these analytes were observable down to concentrations in the low nM and sub-pM ranges, respectively. While conventional random morphology colloids were used in most of these studies, unique cubic nanoparticles of silver were synthesized with different sizes and studied using visible wavelength optical extinction spectrometry, scanning electron microscopy, and the MMF-SERS approach.     

Photonic Crystal Hydrogel Enhanced Plasmonic Staining for Multiplexed Protein Analysis

Plasmonic nanoparticles are commonly used as optical transducers in sensing applications. The optical signals resulting from the interaction of analytes and plamsonic nanoparticles are influenced by surrounding physical structures where the nanoparticles are located. This paper proposes inverse opal photonic crystal hydrogel as 3D structure to improve Raman signals from plasmonic staining. By hybridization of the plasmonic nanoparticles and photonic crystal, surface‐enhanced Raman spectroscopy (SERS) analysis of multiplexed protein is realized. It benefits the Raman analysis by providing high‐density “hot spots” in 3D and extra enhancement of local electromagnetic field at the band edge of PhC with periodic refractive index distribution. The strong interaction of light and the hybrid 3D nanostructure offers new insights into plasmonic nanoparticle applications and biosensor design.     

Direct Readout of Single Nucleobase Variations in an Oligonucleotide

Direct, low‐cost, label‐free, and enzyme‐free identification of single nucleobase is a great challenge for genomic studies. Here, this study reports that wild‐type aerolysin can directly identify the difference of four types of single nucleobase (adenine, thymine, cytosine, and guanine) in a free DNA oligomer while avoiding the operations of additional DNA immobilization, adapter incorporation, and the use of the processing enzyme. The nanoconfined space of aerolysin enables DNA molecules to be limited in the narrow pore. Moreover, aerolysin exhibits an unexpected capability of detecting DNA oligomers at the femtomolar concentration. In the future, by virtue of the high sensitivity of aerolysin and its high capture ability for DNA oligomers, aerolysin will play an important role in the studies of single nucleobase variations and open up new avenues for a broad range of nucleic‐acid‐based sensing and disease diagnosis.     

Single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions

A sensitive method for the analysis of single nucleotide polymorphisms (SNPs) in genomic DNA that utilizes nanoparticle-enhanced surface plasmon resonance imaging (SPRI) measurements of surface enzymatic ligation reactions on DNA microarrays is demonstrated. SNP identification was achieved by using sequence-specific surface reactions of the enzyme Taq DNA ligase, and the presence of ligation products on the DNA microarray elements was detected using SPRI through the hybridization adsorption of complementary oligonucleotides attached to gold nanoparticles. The use of gold nanoparticles increases the sensitivity of the SPRI so that single bases in oligonucleotides can be successfully identified at a concentration of 1 pM. This sensitivity is amply sufficient for performing multiplexed SNP genotyping by using multiple PCR amplicons and should also allow for the direct detection and identification of SNP sequences from 1 pM unamplified genomic DNA samples with this array-based and label-free SPRI methodology. As a first example of SNP genotyping, three different human genomic DNA samples were screened for a possible point mutation in the BRCA1 gene that is associated with breast cancer.     

Universal Imaging of Full Strain Tensor in 2D Crystals with Third‐Harmonic Generation

Quantitatively mapping and monitoring the strain distribution in 2D materials is essential for their physical understanding and function engineering. Optical characterization methods are always appealing due to unique noninvasion and high‐throughput advantages. However, all currently available optical spectroscopic techniques have application limitation, e.g., photoluminescence spectroscopy is for direct‐bandgap semiconducting materials, Raman spectroscopy is for ones with Raman‐active and strain‐sensitive phonon modes, and second‐harmonic generation spectroscopy is only for noncentrosymmetric ones. Here, a universal methodology to measure the full strain tensor in any 2D crystalline material by polarization‐dependent third‐harmonic generation is reported. This technique utilizes the third‐order nonlinear optical response being a universal property in 2D crystals and the nonlinear susceptibility has a one‐to‐one correspondence to strain tensor via a photoelastic tensor. The photoelastic tensor of both a noncentrosymmetric D_3h WS₂ monolayer and a centrosymmetric D_3d WS₂ bilayer is successfully determined, and the strain tensor distribution in homogenously strained and randomly strained monolayer WS₂ is further mapped. In addition, an atlas of photoelastic tensors to monitor the strain distribution in 2D materials belonging to all 32 crystallographic point groups is provided. This universal characterization on strain tensor should facilitate new functionality designs and accelerate device applications in 2D‐materials‐based electronic, optoelectronic, and photovoltaic devices.     

Optical fiber probe for biomedical Raman spectroscopy

In vitro experiments have demonstrated the ability of Raman spectroscopy to diagnose a wide variety of diseases. Recent in vivo investigations performed with optical fiber probes were promising but generally limited to easily accessible organs, often requiring relatively long collection times. We have implemented an optical design strategy to utilize system throughput fully by characterizing the Raman distribution from tissue. This scheme optimizes collection efficiency, minimizes noise, and has resulted in small-diameter, highly efficient Raman probes that are capable of collecting high-quality data in 1 s. Performance has been tested through simulations and experiments with tissue models and several in vitro tissue types, demonstrating that this new design can advance Raman spectroscopy as a clinically practical technique.     

Encoded Multichromophore Response for Simultaneous Label‐Free Detection

The self‐assembly of molecularly precise nanostructures is widely expected to form the basis of future high‐speed integrated circuits, but the technologies suitable for such circuits are not well understood. In this work, DNA self‐assembly is used to create molecular logic circuits that can selectively identify specific biomolecules in solution by encoding the optical response of near‐field coupled arrangements of chromophores. The resulting circuits can detect label‐free, femtomole quantities of multiple proteins, DNA oligomers, and small fragments of RNA in solution via ensemble optical measurements. This method, which is capable of creating multiple logic‐gate–sensor pairs on a 2 × 80 × 80‐nm DNA grid, is a step toward more sophisticated nanoscale logic circuits capable of interfacing computers with biological processes.     

Fast DNA sequencing with a graphene-based nanochannel device

Devices in which a single strand of DNA is threaded through a nanopore could be used to efficiently sequence DNA. However, various issues will have to be resolved to make this approach practical, including controlling the DNA translocation rate, suppressing stochastic nucleobase motions, and resolving the signal overlap between different nucleobases. Here, we demonstrate theoretically the feasibility of DNA sequencing using a fluidic nanochannel functionalized with a graphene nanoribbon. This approach involves deciphering the changes that occur in the conductance of the nanoribbon as a result of its interactions with the nucleobases via π-π stacking. We show that as a DNA strand passes through the nanochannel, the distinct conductance characteristics of the nanoribbon (calculated using a method based on density functional theory coupled to non-equilibrium Green function theory) allow the different nucleobases to be distinguished using a data-mining technique and a two-dimensional transient autocorrelation analysis. This fast and reliable DNA sequencing device should be experimentally feasible in the near future.     

利用拉曼光谱鉴别优化处理宝玉石

通过对一些宝玉石及其优化处理样品的测试,证明拉曼光谱在检测优化处理宝玉石中具有无可替代的独特优势。拉曼光谱可以检测红外光谱无法透过的闷镶翡翠样品,并可以清楚地检测出充填处理翡翠中的环氧树脂峰;可以明显地将绿松石的上蜡和注胶区分开来,相比于红外光谱,两者的区别更为明显;还可以精确地定位祖母绿裂隙中的充填物质,明确地判定祖母绿是否经充胶或染色处理。拉曼光谱在珠宝检测中的作用越来越重要,将会成为珠宝检测的重要手段之一。     

Efficient characterization for protein crystals using confocal Raman spectroscopy

Confocal Raman spectroscopy was applied to the characterization of various states emerging in the screening of protein crystallization. Four main characterized states, namely single crystals, microcrystals, precipitates, and clear drops without solid materials, appear in a droplet for crystallization; the first three states should be critically distinguished and characterized because of the limitations of visual observation under an optical microscope. Using lysozyme and other proteins, crystallization was performed by the hanging drop vapor diffusion technique and was monitored through an automated confocal Raman system. Prior to the spectroscopic analysis, an optical microscope with a charge-coupled device (CCD) camera and associated image processing software were used to rapidly identify the XY locations to be measured spectroscopically by focusing the laser beam on a test sample. Instead of the current image analysis by optical microscopy, confocal Raman spectroscopy with a high spatial resolution was used to identify the state of protein crystallization. Such real-time Raman monitoring also distinguished real protein crystals from pseudo-protein crystals emerging in a crystallization droplet.     

Multiplexed imaging by means of optically generated Kronecker products: 1. The basic concept

Atechnique for multiplexed imaging is described. By using mask combinations, this technique requires far fewer encoding masks to generate the same number of masking operations than do more traditional approaches. It is also theoretically capable of higher optical throughput efficiencies. The method is readily adaptable to new image geometries and to applications in multispectral imaging.     

Reagentless identification of single bacterial spores in aqueous solution by confocal laser tweezers Raman spectroscopy

We demonstrate that optical trapping combined with confocal Raman spectroscopy using a single laser source is a powerful tool for the rapid identification of micrometer-sized particles in an aqueous environment. Optical trapping immobilizes the particle while maintaining it in the center of the laser beam path and within the laser focus, thus maximizing the collection of its Raman signals. The single particle is completely isolated from other particles and substrate surfaces, therefore eliminating any unwanted background signals and ensuring that information is collected only from the selected, individual particle. In this work, an inverted confocal Raman microscope is combined with optical trapping to probe and analyze bacterial spores in solution. Rapid, reagentless detection and identification of bacterial spores with no false positives from a complex mixed sample containing polystyrene and silica beads in aqueous suspension is demonstrated. In addition, the technique is used to analyze the relative concentration of each type of particle in the mixture. Our results show the feasibility for incorporating this technique in combination with a flow cytometric-type scheme in which the intrinsic Raman signatures of the particles are used instead of or in addition to fluorescent labels to identify cells, bacteria, and particles in a wide range of applications.