Surface‐Enhanced Raman Spectroscopy: Concepts and Chemical Applications

Surface‐enhanced Raman scattering (SERS) has become a mature vibrational spectroscopic technique during the last decades and the number of applications in the chemical, material, and in particular life sciences is rapidly increasing. This Review explains the basic theory of SERS in a brief tutorial and—based on original results from recent research—summarizes fundamental aspects necessary for understanding SERS and provides examples for the preparation of plasmonic nanostructures for SERS. Chemical applications of SERS are the centerpiece of this Review. They cover a broad range of topics such as catalysis and spectroelectrochemistry, single‐molecule detection, and (bio)analytical chemistry.     

Peering into Biological Nanopore: A Practical Technology to Single‐Molecule Analysis

As a unique technique at the singe‐molecule level to explore the distribution and temporal order of events, nanopore technology has attracted increasing attention. In comparison to the previous applications in DNA sequencing, this Focus Review highlights the technical details of biological nanopores, especially α‐hemolysin, in the analysis of peptides and proteins. The instrument configurations, experimental interferences, and data analysis including the conformation of peptides and proteins and their interactions for single‐molecule detection are discussed.     

Single‐Molecule Sensors: Challenges and Opportunities for Quantitative Analysis

Measurement science has been converging to smaller and smaller samples, such that it is now possible to detect single molecules. This Review focuses on the next generation of analytical tools that combine single‐molecule detection with the ability to measure many single molecules simultaneously and/or process larger and more complex samples. Such single‐molecule sensors constitute a new type of quantitative analytical tool, as they perform analysis by molecular counting and thus potentially capture the heterogeneity of the sample. This Review outlines the advantages and potential of these new, quantitative single‐molecule sensors, the measurement challenges in making single‐molecule devices suitable for analysis, the inspiration biology provides for overcoming these challenges, and some of the solutions currently being explored.     

Assembling DNA through Affinity Binding to Achieve Ultrasensitive Protein Detection

Recent advances in DNA assembly and affinity binding have enabled exciting developments of nanosensors and ultrasensitive assays for specific proteins. 1 – 6 These sensors and assays share three main attractive features: 1 , 4 , 7 1) the detection of proteins can be accomplished by the detection of amplifiable DNA, thereby dramatically enhancing the sensitivity; 2) assembly of DNA is triggered by affinity binding of two or more probes to a single target molecule, thereby resulting in increased specificity; and 3) the assay is conducted in solution with no need for separation, thus making the assay attractive for potential point‐of‐care applications. We illustrate here the principle of assembling DNA through affinity binding, and we highlight novel applications to the detection of proteins.     

Ultrasensitive Detection and Binding Mechanism of Cocaine in an Aptamer‐based Single‐molecule Device

Aptamer serves as a potential candidate for the micro‐detection of cocaine due to its high specificity, high affinity and good stability. Although cocaine aptasensors have been extensively studied, the binding mechanism of cocaine‐aptamer interactions is still unknown, which limits the structural refinement in the design of an aptamer to improve the performance of cocaine aptasensors. Herein, we report a label‐free, ultrasensitive detection of single‐molecule cocaine‐aptamer interaction by using an electrical nanocircuit based on graphene‐molecule‐ graphene single‐molecule junctions (GMG‐SMJs). Real‐time recordings of cocaine‐aptamer interactions have exhibited distinct current oscillations before and after cocaine treatment, revealing the dynamic mechanism of the conformational changes of aptamer upon binding with cocaine. Further concentration‐dependent experiments have proved that these devices can act as a single‐molecule biosensor with at least a limit of detection as low as 1 nmol?L^–1. The method demonstrated in this work provides a novel strategy for shedding light on the interaction mechanism of biomolecules as well as constructing new types of aptasensors toward practical applications.     

Ortho‐Dichlorobenzene Doped with Terrylene—a Highly Photo‐Stable Single‐Molecule System Promising for Photonics Applications

The study of a new dye‐matrix system—quickly frozen ortho‐dichlorobenzene weakly doped with terrylene—via single‐molecule (SM) spectroscopy is presented. The spectral and photo‐physical properties, dynamics, and temperature broadening of SM spectra at low temperatures are discussed. The data reveal a broad inhomogeneous distribution, which indicates a high degree of matrix inhomogeneities, but at the same time, huge fluorescence emission rates and extraordinary SM spectral and photochemical stability with almost complete absence of blinking and bleaching. These unusual properties render the new system a promising candidate for applications in photonics, for example, for delivering single photons on demand.     

Probing Low‐Copy‐Number Proteins in a Single Living Cell

Single‐cell analysis techniques are essential for understanding the microheterogeneity and functions of cells. Low‐copy‐number proteins play important roles in cell functioning, but their measurement in single cells remains challenging. Herein, we report an approach, called plasmonic immunosandwich assay (PISA), for probing low‐copy‐number proteins in single cells. This approach combined in vivo immunoaffinity extraction and plasmon‐enhanced Raman scattering (PERS). Target proteins were specifically extracted from the cells by microprobes modified with monoclonal antibody or molecularly‐imprinted polymer (MIP), followed by labeling with Raman‐active nanotags. The PERS detection, with Raman intensity enhanced by 9 orders of magnitude, provided ultrasensitive detection at the single‐molecule level. Using this approach, we found that alkaline phosphatase and survivin were expressed in distinct levels in cancer and normal cells, and that extended culture passage resulted in reduced expression of survivin. We further developed acupuncture needle‐based PISA for probing low‐copy‐number proteins in living bodies.     

From Single Hydrogen Bonds to Extended Hydrogen‐Bond Wires: Low‐Dimensional Model Systems for Vibrational Spectroscopy of Associated Liquids

It is fair to say that if we ever wish to understand the anomalous properties of water, we need to study hydrogen bonds. Such a statement is based on statistical mechanics, which tells us how to calculate the structure and the thermodynamic properties of fluids and dense liquids from the forces between the particles. However, in the case of complex associated liquids, such calculations present a formidable—if not even insurmountable—challenge, which largely reflects our still‐limited understanding of the hydrogen‐bonding phenomenon itself. More experimental research on hydrogen‐bonded systems is required to develop a comprehensive, satisfactory theory for associated liquids. This Review gives an introduction to the latest experimental technique currently being used to study the ultrafast structural dynamics of hydrogen bonds, namely two‐dimensional infrared spectroscopy, and its applications to hydrogen‐bonded systems of systematically increasing complexity, starting from the single hydrogen bond of a diol to low‐dimensional extended networks of stereoselectively synthesized polyalcohols.     

Antibacterial Drug Release Electrochemically Stimulated by the Presence of Bacterial Cells – Theranostic Approach

A modified electrode with bioaffinity to E. coli bacterial cells was used to detect bacteria presence on its surface and to produce an electrical signal triggering antibacterial drug release from another coupled electrode. The Sense‐and‐Treat system was realized in two different versions and suggested as the first step to future theranostic applications where bacteria presence results in automatic antibacterial treatment.     

Single-molecule fluorescence detection in microfluidic channels—the Holy Grail in μTAS?

Both single-molecule detection (SMD) methods and miniaturization technologies have developed very rapidly over the last ten years. By merging these two techniques, it may be possible to achieve the optimal requirements for the analysis and manipulation of samples on a single molecule scale. While miniaturized structures and channels provide the interface required to handle small particles and molecules, SMD permits the discovery, localization, counting and identification of compounds. Widespread applications, across various bioscience/analytical science fields, such as DNA-analysis, cytometry and drug screening, are envisaged. In this review, the unique benefits of single fluorescent molecule detection in microfluidic channels are presented. Recent and possible future applications are discussed.Dedicated to the memory of Wilhelm Fresenius     

Rapid sampling of all‐atom peptides using a library‐based polymer‐growth approach

We adapted existing polymer growth strategies for equilibrium sampling of peptides described by modern atomistic forcefields with a simple uniform dielectric solvent. The main novel feature of our approach is the use of precalculated statistical libraries of molecular fragments. A molecule is sampled by combining fragment configurations—of single residues in this study—which are stored in the libraries. Ensembles generated from the independent libraries are reweighted to conform with the Boltzmann‐factor distribution of the forcefield describing the full molecule. In this way, high‐quality equilibrium sampling of small peptides (4–8 residues) typically requires less than one hour of single‐processor wallclock time and can be significantly faster than Langevin simulations. Furthermore, approximate, clash‐free ensembles can be generated for larger peptides (up to 32 residues in this study) in less than a minute of single‐processor computing. We discuss possible applications of our growth procedure to free energy calculation, fragment assembly protein‐structure prediction protocols, and to “multi‐resolution” sampling. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Electrochemistry on Paper‐based Analytical Devices: A Review

Even though they were introduced less than a decade ago, electrochemical paper‐based devices (ePADs) have attracted widespread attention because of their inherent advantages in many applications. ePADs combine the advantages of microfluidic paper‐based devices (low cost, ease of use, equipment free pumping, etc.) for sample handling and processing with the advantages of sensitive and selective detection provided by electrochemistry. As a result, ePADs provide simplicity, portability, reproducibility, low cost and high selectivity and sensitivity for analytical measurements in a variety of applications ranging from clinical diagnostics to environmental sensing. Herein, recent advances in ePAD development and application are reviewed, focusing on electrode fabrication techniques and examples of applications specially focused on environmental monitoring, biological applications and clinical assays. Finally, a summary and prospective directions for ePAD research are also provided.     

Emerging Applications of Phase‐Change Materials (PCMs): Teaching an Old Dog New Tricks

The nebulous term phase‐change material (PCM) simply refers to any substance that has a large heat of fusion and a sharp melting point. PCMs have been used for many years in commercial applications, mainly for heat management purposes. However, these fascinating materials have recently been rediscovered and applied to a broad range of technologies, such as smart drug delivery, information storage, barcoding, and detection. With the hope of kindling interest in this incredibly versatile range of materials, this Review presents an array of aspects related to the compositions, preparations, and emerging applications of PCMs.     

Ultrasensitive fluorescence-based methods for nucleic acid detection: towards amplification-free genetic analysis

Real time PCR is the mainstay of current nucleic acid assays, underpinning applications in forensic science, point-of-care diagnostics and detection of bioterrorism agents. Despite its broad utility, the search for new tests continues, inspired by second and third generation DNA sequencing technologies and fuelled by progress in single molecule fluorescence spectroscopy, nanotechnology and microfabrication. These new methods promise the direct detection of nucleic acids without the need for enzymatic amplification. In this feature article, we provide a chemist's perspective on this multidisciplinary area, introducing the concepts of single molecule detection then focussing on the selection of labels and probe chemistry suitable for generating a signal detectable by ultrasensitive fluorescence spectroscopy. Finally, we discuss the further developments that are required to incorporate these detection platforms into integrated 'sample-in-answer-out' instruments, capable of detecting many target sequences in a matter of minutes.     

Noble‐Metal‐Free Materials for Surface‐Enhanced Raman Spectroscopy Detection

Surface‐enhanced Raman spectroscopy (SERS) is an attractive tool for the sensing of molecules in the fields of chemical and biochemical analysis as it enables the sensitive detection of molecular fingerprint information even at the single‐molecule level. In addition to traditional coinage metals in SERS analysis, recent research on noble‐metal‐free materials has also yielded highly sensitive SERS activity. This Minireview presents the recent development of noble‐metal‐free materials as SERS substrates and their potential applications, especially semiconductors and emerging graphene‐based nanostructures. Rather than providing an exhaustive review of this field, possible contributions from semiconductor substrates, characteristics of graphene enhanced Raman scattering, as well as effect factors such as surface plasmon resonance, structure and defects of the nanostructures that are considered essential for SERS activity are emphasized. The intention is to illustrate, through these examples, that the promise of noble‐metal‐free materials for enhancing detection sensitivity can further fuel the development of SERS‐related applications.     

Tetra(p‐tolyl)borate‐Functionalized Solvent Polymeric Membrane: A Facile and Sensitive Sensing Platform for Peroxidase and Peroxidase Mimetics

The determination of peroxidase activities is the basis for enzyme‐labeled bioaffinity assays, peroxidase‐mimicking DNAzymes‐ and nanoparticles‐based assays, and characterization of the catalytic functions of peroxidase mimetics. Here, a facile, sensitive, and cost‐effective solvent polymeric membrane‐based peroxidase detection platform is described that utilizes reaction intermediates with different pK_a values from those of substrates and final products. Several key but long‐debated intermediates in the peroxidative oxidation of o‐phenylenediamine (o‐PD) have been identified and their charge states have been estimated. By using a solvent polymeric membrane functionalized by an appropriate substituted tetraphenylborate as a receptor, those cationic intermediates could be transferred into the membrane from the aqueous phase to induce a large cationic potential response. Thus, the potentiometric indication of the o‐PD oxidation catalyzed by peroxidase or its mimetics can be fulfilled. Horseradish peroxidase has been detected with a detection limit at least two orders of magnitude lower than those obtained by spectrophotometric techniques and traditional membrane‐based methods. As an example of peroxidase mimetics, G‐quadruplex DNAzymes were probed by the intermediate‐sensitive membrane and a label‐free thrombin detection protocol was developed based on the catalytic activity of the thrombin‐binding G‐quadruplex aptamer.     

Fluorogenic Ag+–Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining

Silver staining, which exploits the special bioaffinity and the chromogenic reduction of silver ions, is an indispensable visualization method in biology. It is a most popular method for in‐gel protein detection. However, it is limited by run‐to‐run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) analysis; limitations that are largely attributed to the tricky chromogenic visualization. Herein, we reported a novel water‐soluble fluorogenic Ag⁺ probe, the sensing mechanism of which is based on an aggregation‐induced emission (AIE) process driven by tetrazolate‐Ag⁺ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains.     

Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC‐ESI‐MS/MS

A new validated method for the quantitation of the abnormal phospholipid phosphatidylethanol (PEth)—a biomarker for ethanol uptake—has been developed by LC‐ESI‐MS/MS following miniaturised organic solvent extraction and reversed phase chromatography with phosphatidylbutanol (PBut) as internal standard. PEth homologues with two fatty acid substituents—PEth 18 : 1/18 : 1, PEth 16 : 0/16 : 0—were determined in post‐mortem blood collected from heavy drinkers at autopsy and also in whole blood samples from a volunteer after a single 60 g‐dose of ethanol. Furthermore, PEth 18 : 1/16 : 0 or its isobaric isomer PEth—16 : 0/18 : 1 was detected. In comparison to previous high‐performance liquid chromatography (HPLC) methods with evaporative light scattering detection (ELSD), the LC‐MS/MS‐method is more sensitive—with a limit of detection below 20 ng/ml—and more selective for single PEth homologues, while ELSD has been used for detection of the sum of PEth homologues with approximately 10 times less sensitivity. LC‐MS/MS enables monitoring of PEth homologues as biomarkers for harmful and prolonged alcohol consumption as with HPLC/ELSD earlier, where PEth is measurable in blood only after more than 50 g ethanol daily intake for more than 2 weeks. Because of its higher sensitivity, there is a potential to detect single heavy drinking by LC‐MS/MS, when PEth is formed in very low concentrations. This opens a new field of application of PEth to uncover single or multiple heavy drinking at a lower frequency and with a larger window of detection in blood than before by HPLC/ELSD or by use of other direct markers, e.g. ethyl glucuronide or ethyl sulfate. Copyright © 2009 John Wiley & Sons, Ltd.     

全内反射荧光成像技术及其在单分子检测中的研究进展

全内反射荧光显微镜技术是当今最灵敏的生物成像和检测方法之一,可以直接探测单个荧光分子。这种方法已成功地用于生命科学、化学、物理学等研究领域,获得了常规方法无法得到的重要信息。本文介绍了全内反射荧光显微镜的工作原理和实验技术,总结了近年来这种单分子检测方法在生命科学、化学等领域的重要应用,并对其发展前景进行了展望。     

A Rapid,Amplification‐Free,and Sensitive Diagnostic Assay for Single‐Step Multiplexed Fluorescence Detection of MicroRNA

The importance of microRNA (miRNA) dysregulation for the development and progression of diseases and the discovery of stable miRNAs in peripheral blood have made these short‐sequence nucleic acids next‐generation biomarkers. Here we present a fully homogeneous multiplexed miRNA FRET assay that combines careful biophotonic design with various RNA hybridization and ligation steps. The single‐step, single‐temperature, and amplification‐free assay provides a unique combination of performance parameters compared to state‐of‐the‐art miRNA detection technologies. Precise multiplexed quantification of miRNA‐20a, ‐20b, and ‐21 at concentrations between 0.05 and 0.5 nM in a single 150 μL sample and detection limits between 0.2 and 0.9 nM in 7.5 μL serum samples demonstrate the feasibility of both high‐throughput and point‐of‐care clinical diagnostics.