Open-access microfluidic patch-clamp array with raised lateral cell trapping sites

A novel open-access microfluidic patch-clamp array chip with lateral cell trapping sites raised above the bottom plane of the chip was developed by combining both a microscale soft-lithography and a macroscale polymer fabrication method. This paper demonstrates the capability of using such an open-access fluidic system for patch-clamp measurements. The surface of the open-access patch-clamp sites prepared by the macroscale hole patterning method of soft-state elastic polydimethylsiloxane (PDMS) is examined; the seal resistances are characterized and correlated with the aperture dimensions. Whole cell patch-clamp measurements are carried out with CHO cells expressing Kv2.1 ion channels. Kv2.1 ion channel blocker (TEA) dosage response is characterized and the binding activity is examined. The results demonstrate that the system is capable of performing whole cell measurements and drug profiling in a more efficient manner than the traditional patch-clamp set-up.     

Monitoring the absorption of organic vapors to a solid phase extraction medium applications to detection of trace volatile organic compounds by integration of solid phase absorbents with fiber optic Raman spectroscopy

By combining fiber optic Raman spectroscopy with a C-18 solid phase extraction medium, real time in situ detection of organic vapors is demonstrated. The response of the probe is fully reversible for benzene, trichloroethylene and carbon tetrachloride vapors. Because of the high degree of selectivity afforded by Raman spectroscopy, the composition of mixtures of the vapors can also be determined using the C-18 probe. The detection of ppm levels of benzene in water via headspace analysis using the C-18 probe is demonstrated.     

Miniaturised flow-through PLM coupled with spectrophotometric detection for copper(II) speciation in fungicides†

Construction of a novel miniaturised Permeation Liquid Membrane (mPLM) set-up is described. A hydrophobic Celgard 2500 membrane with copper(II) carrier separates the source and receiving solutions flowing in the channels on two sides of the membrane. The set-up consists of two half-cells with channel width of 2?mm and depth variable between 100 and 500?µm made of poly(dimethylsiloxane) (PDMS) using a moulding technology. In contrast to most previous constructions, the new mPLM operates in full flow-through mode, in both source and receiving solutions. This allows for on-line and real-time coupling with the detection system, which significantly reduces the time of analysis. In the present set-up a UV/Vis spectrophotometric detection was applied for Cu(II) transport. For this purpose a composition of the receiving solution was optimised to allow for both free metal ion buffering, which is necessary for maintaining the driving force of the transport, and spectrophtometric detection. The applied UV/Vis detection method is linear in the range 2?×?10^?6?M – 4?×?10^?5?M (R ^2?=?0.996) with a detection limit of 10^?6 M in the volume of ca 350?µL (including the receiving channels, cuvette and tubing). The relative standard deviation does not exceed 5%. The transport capabilities of the mPLM set-up are demonstrated using four commercial copper-based fungicides: Nordox 75?WG, Miedzian 50?WP, Curzate 49.5?WP and Mag 50?WP.     

基于拉曼光谱的聚酯过程酯化反应中清晰点的在线检测

现有的人工采样分析方法如折光指数法、色谱法等操作复杂,存在时间上的滞后和人为误差,无法实时评估聚酯生产中的酯化反应过程,确定反应的清晰点。为此,该文搭建了以1 064 nm为激光波长的在线拉曼分析系统,对对苯二甲酸(PTA)和1,3-丙二醇(PDO)生成聚对苯二甲酸丙二醇酯(PTT)的酯化反应过程进行光谱连续采集和处理。利用主成分分析法提取光谱信息,并结合酯化反应原理,利用位于1 604 cm~(-1)附近的苯环基团和位于1 720 cm~(-1)处的芳酯基团的拉曼特征峰面积比,建立了一种用于确定清晰点的定性分析方法。结果表明:拉曼光谱分析法可以准确反映聚酯过程酯化反应中的清晰点,且具有实时性好、操作方便、分析速度快的优势,可为聚酯生产的工艺控制和产品品质控制提供参考。     

In situ Spectroscopic Insight into the Origin of the Enhanced Performance of Bimetallic Nanocatalysts towards the Oxygen Reduction Reaction (ORR)

It is vital to understand the oxygen reduction reaction (ORR) mechanism at the molecular level for the rational design and synthesis of high activity fuel‐cell catalysts. Surface enhanced Raman spectroscopy (SERS) is a powerful technique capable of detecting the bond vibrations of surface species in the low wavenumber range, however, using it to probe practical nanocatalysts remains extremely challenging. Herein, shell‐isolated nanoparticle‐enhanced Raman spectroscopy (SHINERS) was used to investigate ORR processes on the surface of bimetallic Pt₃Co nanocatalyst structures. Direct spectroscopic evidence of *OOH suggests that ORR undergoes an associative mechanism on Pt₃Co in both acidic and basic environments. Density functional theory (DFT) calculations show that the weak *O adsorption arise from electronic effect on the Pt₃Co surface accounts for enhanced ORR activity. This work shows SHINERS is a promising technique for the real‐time observation of catalytic processes.     

Laboratory Real‐Time and In Situ Monitoring of Mechanochemical Milling Reactions by Raman Spectroscopy

Mechanistic understanding of mechanochemical reactions is sparse and has been acquired mostly by stepwise ex situ analysis. We describe herein an unprecedented laboratory technique to monitor the course of mechanochemical transformations at the molecular level in situ and in real time by using Raman spectroscopy. The technique, in which translucent milling vessels are used that enable the collection of a Raman scattering signal from the sample as it is being milled, was validated on mechanochemical reactions to form coordination polymers and organic cocrystals. The technique enabled the assessment of the reaction dynamics and course under different reaction conditions as well as, for the first time, direct insight into the behavior of liquid additives during liquid‐assisted grinding.     

显微拉曼光谱用于研究大气单颗粒表面非均相反应

大气单颗粒表面的非均相反应研究因更接近大气实际条件,避免了堆积态研究中人为引入的误差,能够得到真实的反应过程与机理,获得反映大气实际条件的动力学参数.本研究建立了使用显微拉曼光谱研究大气单颗粒非均相反应的研究方法,并初步用于研究NO2与单颗粒CaCO3的非均相反应.研究结果表明显微拉曼光谱可同时获得颗粒物的化学组成和形貌变化,并能得到化学环境如相态的信息,对于研究反应过程很有帮助;而颗粒物沉降在基质上得到的拉曼光谱因不受形貌共振影响,有利于获得高质量的光谱.此外,将拉曼光谱研究单颗粒的方法与其他单颗粒非均相反应的研究方法进行了综合比较,表明显微拉曼光谱技术在单颗粒非均相反应研究中具有重要的特点和应用价值.     

Toward a glucose biosensor based on surface-enhanced Raman scattering

This work presents the first step toward a glucose biosensor using surface-enhanced Raman spectroscopy (SERS). Historically, glucose has been extremely difficult to detect by SERS because it has a small normal Raman cross section and adsorbs weakly or not at all to bare silver surfaces. In this paper, we report the first systematic study of the direct detection of glucose using SERS. Glucose is partitioned into an alkanethiol monolayer adsorbed on a silver film over nanosphere (AgFON) surface and thereby, it is preconcentrated within the 0-4 nm thick zone of electromagnetic field enhancement. The experiments presented herein utilize leave-one-out partial least-squares (LOO-PLS) analysis to demonstrate quantitative glucose detection both over a large (0-250 mM) and clinically relevant (0-25 mM) concentration range. The root-mean-squared error of prediction (RMSEP) of 1.8 mM (33.1 mg/dL) in the clinical study is near that desired for medical applications (1 mM, 18 mg/dL). Future studies will advance toward true in vivo, real time, minimally invasive sensing.     

Non-destructive analysis of museum objects by fibre-optic Raman spectroscopy

Raman spectroscopy is a versatile technique that has frequently been applied for the investigation of art objects. By using mobile Raman instrumentation it is possible to investigate the artworks without the need for sampling. This work evaluates the use of a dedicated mobile spectrometer for the investigation of a range of museum objects in museums in Scotland, including antique Egyptian sarcophagi, a panel painting, painted surfaces on paper and textile, and the painted lid and soundboard of an early keyboard instrument. The investigations of these artefacts illustrate some analytical challenges that arise when analysing museum objects, including fluorescing varnish layers, ambient sunlight, large dimensions of artefacts and the need to handle fragile objects with care. Analysis of the musical instrument (the Mar virginals) was undertaken in the exhibition gallery, while on display, which meant that interaction with the public and health and safety issues had to be taken into account. Experimental set-up for the non-destructive Raman spectroscopic investigation of a textile banner in the National Museums of Scotland     

SERS decoding of micro gold shells moving in microfluidic systems

In this study, in situ surface‐enhanced Raman scattering (SERS) decoding was demonstrated in microfluidic chips using novel thin micro gold shells modified with Raman tags. The micro gold shells were fabricated using electroless gold plating on PMMA beads with diameter of 15 μm. These shells were sophisticatedly optimized to produce the maximum SERS intensity, which minimized the exposure time for quick and safe decoding. The shell surfaces produced well‐defined SERS spectra even at an extremely short exposure time, 1 ms, for a single micro gold shell combined with Raman tags such as 2‐naphthalenethiol and benzenethiol. The consecutive SERS spectra from a variety of combinations of Raman tags were successfully acquired from the micro gold shells moving in 25 μm deep and 75 μm wide channels on a glass microfluidic chip. The proposed functionalized micro gold shells exhibited the potential of an on‐chip microfluidic SERS decoding strategy for micro suspension array.     

Evaluation of the potential of Raman microspectroscopy for prediction of chemotherapeutic response to cisplatin in lung adenocarcinoma

The study of the interaction of anticancer drugs with mammalian cells in vitro is important to elucidate the mechanisms of action of the drug on its biological targets. In this context, Raman spectroscopy is a potential candidate for high throughput, non-invasive analysis. To explore this potential, the interaction of cis-diamminedichloroplatinum(II) (cisplatin) with a human lung adenocarcinoma cell line (A549) was investigated using Raman microspectroscopy. The results were correlated with parallel measurements from the MTT cytotoxicity assay, which yielded an IC(50) value of 1.2 ± 0.2 μM. To further confirm the spectral results, Raman spectra were also acquired from DNA extracted from A549 cells exposed to cisplatin and from unexposed controls. Partial least squares (PLS) multivariate regression and PLS Jackknifing were employed to highlight spectral regions which varied in a statistically significant manner with exposure to cisplatin and with the resultant changes in cellular physiology measured by the MTT assay. The results demonstrate the potential of the cellular Raman spectrum to non-invasively elucidate spectral changes that have their origin either in the biochemical interaction of external agents with the cell or its physiological response, allowing the prediction of the cellular response and the identification of the origin of the chemotherapeutic response at a molecular level in the cell.     

Casting New Physicochemical Light on the Fundamental Biological Processes in Single Living Cells by Using Raman Microspectroscopy

This Personal Account highlights the capabilities of spontaneous Raman microspectroscopy for studying fundamental biological processes in a single living cell. Raman microspectroscopy provides time‐ and space‐resolved vibrational Raman spectra that contain detailed information on the structure and dynamics of biomolecules in a cell. By using yeast as a model system, we have made great progress in the development of this methodology. The results that we have obtained so far are described herein with an emphasis placed on how three cellular processes, that is, cell‐division, respiration, and cell‐death, are traced and elucidated with the use of time‐ and space‐resolved structural information that is extracted from the Raman spectra. For cell‐division, compositional‐ and structural changes of various biomolecules are observed during the course of the whole cell cycle. For respiration, the redox state of mitochondrial cytochromes, which is inferred from the resonance Raman bands of cytochromes, is used to evaluate the respiration activity of a single cell, as well as that of isolated mitochondrial particles. Special reference is made to the “Raman spectroscopic signature of life”, which is a characteristic Raman band at 1602 cm^?1 that is found in yeast cells. This signature reflects the cellular metabolic activity and may serve as a measure of mitochondrial dysfunction. For cell‐death, “cross‐talk” between the mitochondria and the vacuole in a dying cell is suggested. DOI 10.1002/tcr.201200008     

Raman microspectroscopy of live cells under autophagy-inducing conditions

The role of autophagy in numerous physiological responses triggered by a variety of mechanisms both in states of health and disease has raised considerable interest in this cellular process. However, the current analytical tools to study autophagy are either invasive or require genetic manipulation. Raman microspectroscopy is a potentially quantitative analytical method that has been shown to be useful for the label-free, non-destructive analysis of living biological cells and tissues. We present in this study initial efforts to study autophagy using Raman spectroscopy. The response of adherent mouse and human cancer cells to starvation conditions (glutamine deprivation and amino acid deprivation) was probed by Raman spectroscopy and compared to fluorescence microscopy results using autophagy-specific markers. We also demonstrate the capability of Raman spectroscopy to monitor the recovery dynamics of starved cells and to probe the heterogeneity in the response to starvation that can arise in cell populations. Finally, this work suggests that the 718 cm(-1) Raman line associated with phospholipids may be a useful spectral marker indicative of an autophagic response to starvation stimuli. Overall, this study establishes the utility of Raman spectroscopy to non-invasively detect biologically relevant changes in live cells exposed to conditions known to trigger autophagy.     

Distinguishing Individual DNA Bases in a Network by Non-Resonant Tip-Enhanced Raman Scattering

The importance of identifying DNA bases at the single-molecule level is well recognized for many biological applications. Although such identification can be achieved by electrical measurements using special setups, it is still not possible to identify single bases in real space by optical means owing to the diffraction limit. Herein, we demonstrate the outstanding ability of scanning tunneling microscope (STM)-controlled non-resonant tip-enhanced Raman scattering (TERS) to unambiguously distinguish two individual complementary DNA bases (adenine and thymine) with a spatial resolution down to 0.9 nm. The distinct Raman fingerprints identified for the two molecules allow to differentiate in real space individual DNA bases in coupled base pairs. The demonstrated ability of non-resonant Raman scattering with super-high spatial resolution will significantly extend the applicability of TERS, opening up new routes for single-molecule DNA sequencing.     

Distinguishing Individual DNA Bases in a Network by Non‐Resonant Tip‐Enhanced Raman Scattering

The importance of identifying DNA bases at the single‐molecule level is well recognized for many biological applications. Although such identification can be achieved by electrical measurements using special setups, it is still not possible to identify single bases in real space by optical means owing to the diffraction limit. Herein, we demonstrate the outstanding ability of scanning tunneling microscope (STM)‐controlled non‐resonant tip‐enhanced Raman scattering (TERS) to unambiguously distinguish two individual complementary DNA bases (adenine and thymine) with a spatial resolution down to 0.9 nm. The distinct Raman fingerprints identified for the two molecules allow to differentiate in real space individual DNA bases in coupled base pairs. The demonstrated ability of non‐resonant Raman scattering with super‐high spatial resolution will significantly extend the applicability of TERS, opening up new routes for single‐molecule DNA sequencing.     

Studies of particle drying using non-invasive Raman spectrometry and particle size analysis

The evaporation of methanol from needle-shaped particles of cellobiose octaacetate (COA) has been studied directly in a jacketed vacuum drier using in situ measurements by Raman spectrometry. A design of experiments (DoE) approach was used to investigate the effects of three parameters (method of agitation, % solvent loss on drying and jacket temperature), with the intention of minimising the drying time and extent of particle attrition. Drying curves based on Raman signals for methanol and COA in the spectra of the wet particles indicated the end of drying and revealed three stages in the drying process that could be used to monitor the progress of solvent removal in real time. Off-line particle size measurements based on laser diffraction were made to obtain information on the extent of attrition, to compare with the trends revealed by the Raman drying curves. The study demonstrated that non-invasive Raman spectrometry can be used to study the progress of drying during agitation of particles in a vacuum drier, allowing optimisation of operating conditions to minimise attrition and reduce drying times. Although a correlation between particle size and off-line Raman measurements of COA was demonstrated, it was not possible to derive equivalent information from the in situ Raman spectra owing to the greater effects of particle motion or bulk density variations of the particles in the drier.     

Determination of fructose using immobilized glucose isomerase in an on-line analyzer

The determination of fructose using a continuous analyzer based on analyte conversion in enzyme reactors followed by amperometric oxygen measurement is described. Two experimental setups were compared, allowing determinations in the ranges 0–180 and 0–25 mM fructose. In the former, fructose was continuously dialyzed versus a buffer stream conducting fructose to an enzyme reactor. This reactor contained two immobilized enzyme preparations, one with immobiized glucose isomerase (E.C. 5.3.1.5) that isomerized fructose to glucose and another that subsequently oxidized the former glucose by immobilized glucose oxidase (E.C. 1.1.3.4) with the consumption of dissolved oxygen. In the latter set-up, fructose was first isomerized in a glucose isomerase reactor, then glucose was continuously dialyzed and oxidized by glucose oxidase as above. This set-up was run in continuous operation for 1000 measurement cycles with a total decrease in response less than 15%.     

Preparation of Surface-enhanced Raman Scattering(SERS)-active Optical Fiber Sensor by Laser-induced Ag Deposition and Its Application in Bioidentification of Biotin/Avidin

A surface-enhanced Raman scattering(SERS) optical fiber sensor was prepared by the laser-induced deposition of Ag nanoparticle membrane on a silica optical fiber tip, which was applied to the real time SERS spectral monitoring on the biorecognition of biotin/avidin. The bioidentification of biotin/avidin was carried out through a indirect method, in which the bioidentification is based on the SERS response signal of a labeled dye(Atto610) after its fluorescence has been quenched totally by the deposited Ag nanoparticle membrane. By SERS monitoring the bioidentification process of biotin/avidin, it has been found that this recognition process is finished in 40 min. The lowest detection concentration of biotin is 1.0×10^-7 mg/mL. This research is promising in the application of immunoassays on line and in vivo.     

DNA-nanotube artificial ion channels

There is considerable interest in developing chemical devices that mimic the function of biological ion channels. We recently described such a device, which consisted of a single conically shaped gold nanotube embedded within a polymeric membrane. This device mimicked one of the key functions of voltage-gated ion channels: the ability to strongly rectify the ionic current flowing through it. The data obtained were interpreted using a simple electrostatic model. While the details are still being debated, it is clear that ion-current-rectification in biological ion channels is more complicated and involves physical movement of an ionically charged portion of the channel in response to a change in the transmembrane potential. We report here artificial ion channels that rectify the ion current flowing through them via this "electromechanical" mechanism. These artificial channels are also based on conical gold nanotubes, but with the critical electromechanical response provided by single-stranded DNA molecules attached to the nanotube walls.     

SE(R)RS devices fabricated by a laser electrodispersion method

A novel laser electrodispersion (LE) technique was employed to deposit gold nanoparticles onto Si and SiO(x) surfaces. The LE technique combines laser ablation with cascade fission of liquid metal micro-drops, which results in the formation of nanoparticles upon rapid cooling. The shape and the size distribution of the Au nanoparticles prepared by LE depend on the nature of the support. Gold nanoparticles were also deposited in the channels of microreactors fabricated by wet etching of Si and used as SE(R)RS sensors. The influence of the nanoparticle surface density as well as of the nature of the substrate on the Raman response was studied. At an appropriate surface density of the deposited nanoparticles a significant enhancement of Raman signal was observed showing the possibility to create efficient SERS substrates. Application of microfluidic devices in surface enhanced Raman spectroscopy (SERS) in continuous-flow mode with sensor regeneration is described.