Ion Mobility Spectrometry and Its Applications in Detection of Chemical Warfare Agents
Marko A.Ma¨kinen
University of Eastern Finland
Osmo A.Anttalainen
Environics Oy(Finland)
Mika E.T.Sillanpa¨a¨
Lappeenranta University of Technology and University of Eastern Finland
When fast detection of chemical warfare agents in the?eld
is required,the ion mobility spectrometer may be the only
suitable option.This article provides an essential survey
of the different ion mobility spectrometry detection tech-
nologies.(To listen to a podcast about this feature,please
go to the Analytical Chemistry multimedia page at pubs.
https://www.doczj.com/doc/641789967.html,/page/ancham/audio/index.html.)
The threat of weapons of mass destruction(WMDs),such as
chemical warfare agents(CWAs)and toxic industrial chemicals,is
of great concern worldwide.This menace makes it necessary to
detect the presence of such weapons in both military and civil
environs.Toxic chemicals are used in large quantities by industry,
and the information needed to synthesize them has become more
accessible through the Internet.In addition to providing protection
from chemical attacks,fast detection is needed to detect possible
chemical leaks which may occur during accidents,disposal,or
dumping.Many of these chemicals are hazardous to human health;
others may be in?ammable or pose environmental risks.
The purpose of this Feature is to highlight how ion mobility
spectrometry(IMS)technologies can be applied to the detection
and identi?cation of these chemicals.The properties of the most
common CWAs are described,and the principles,limitations,and
advantages of analytical IMS detection together with its future
prospects are discussed.
INTRODUCTION TO CHEMICAL WARFARE AGENTS
Toxic chemicals have great potential to in?ict signi?cant casualties, thus chemical weapons are classi?ed as WMDs.Furthermore,CWAs are easy to disguise and are practically imperceptible,thus making them easy to use against the public.CWAs are mostly dispersed as vapors or aerosols;the more volatile the agent
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the faster it evaporates and disperses.In addition,CWAs or their degradation products may linger as soil contaminants.Structures of common CWAs are shown in Figure1.Earlier articles have evaluated the different technologies for detection of CWAs.1,2 Classi?cation of CWAs.CWAs are divided into?ve different categories.3,4
i)Vesicating and blistering agents such as sulfur and nitrogen mustards cause extensive and irreversible tissue damage.Lewisite is the most infamous of the organoarsenic warfare agents.ii) Choking agents or pulmonary intoxicants are,for example,phos-gene,which does not detoxify naturally,has a cumulative effect, and may persist in sheltered areas or buildings for a long time and chlorine,which at moderate concentrations is a weak pulmonary irritant but in high concentrations is extremely lethal. Both are also indispensable industrial chemicals.iii)Nerve agents are organophosphonates,which are further divided into three subcategories:G agents(G denotes German origin),such as Tabun(GA),Sarin(GB),and Soman(GD);V agents(mainly VX; some variants exist);and Novichok agents.Tabun was initially used as a pesticide but has been utilized for military purposes. Sarin is an extremely volatile colorless liquid that has a mild aroma of rotting fruit if impure.Soman is more poisonous than the two aforementioned and has a camphor-like odor if impure.VX is an odorless liquid with an appearance similar to motor oil.Novichoks are considered to be the most hazardous agents ever made.5iv) Blood-born agents are cyanogens such as HCN and CNCl.They are distributed by the vascular system.v)Incapacitating agents are non-lethal CWAs.CS(tear gas)is widely used for various purposes.In addition,some nations are producing,stockpiling, and transporting large quantities of toxic industrial chemicals.
ION MOBILITY SPECTROMETRY
Principles.IMS involves both ionization of the sample and analysis of the ions formed at ambient temperature and at ambient or reduced pressure.This analytical method is used in various demanding applications including?eld or on-site detection of vapor phase species such as chemical weapons,explosives,and drugs. The?rst analytical device was introduced in the late1960s,6and CWA detectors were introduced in the1970s and early1980s.7 At atmospheric pressure,the ion-molecule reactions of CWAs result in ef?cient formation of stable and identi?able product ions, which spurred the widespread use of IMS for CWA detection.8 IMS is used more extensively than any other method in the detection of trace CWAs and explosives.9
Recently,appreciation of the IMS technique and its various applications has increased.10IMS instruments are widely used by military,security,customs,and border authorities.The majority of the commercial instruments are used for explosives detection,11and probably the most noticeable application is the walk-through portals in airports used to monitor for traces of explosives.12 In IMS,gas phase ions are created by ionizing neutral molecules using photon,corona,?ame,ESI,or radioactive ionization.Most of the instruments use radioactive ionization sources like63Ni or241Am because they are simple,convenient, and stable.The ions produced are separated by their different velocities through a drift gas in an electric?eld.IMS separation is based on ion mobility,with the relationship between ion velocity and electric?eld depending on the ion’s weight, charge,and shape.11The temperature,pressure,and molecular properties of the drift gas also play important roles.13To remove background interference and improve sensitivity and selectivity,additional reagent gases(dopants)can be used to create alternate reactant ions such as Br-,Cl-,NO3-,NO2-, or NH4+.14Thus,ion mobility spectrometers are similar to TOF mass spectrometers,although operation at ambient pressure has its own pros and cons.In case of CWAs,the instruments mainly operate in positive mode;i.e.,the ions formed from the samples are positively charged.However,some exceptions exist because agents with halogen atoms and low proton af?nity are unable to form stable positively charged ions.
Advantages and Limitations.There are many CWA detection techniques.For example,common laboratory methods and instru-ments such as GC,LC,CE,and MS,individually or in combination, may be used.On-site screening techniques include surface acoustic wave(SAW),electrochemical,and spectrophotometric sensors.All these techniques have their pros and cons;for example,SAW sensors can be small and portable but are sensitive to moisture and may suffer from de-wetting effects that reduce responsiveness.15Spectrophotometric techniques are based on color change reactions(detection papers or detection tubes)or emission lines(?ame photometric detection[FPD]).Color change experiments are easy to perform but need a relatively high amount of sample,can be time consuming,and give ambiguous results. FPD is fast and sensitive but produces false positives.Typical drawbacks to all these techniques are false positives and adsorp-tion of the agents onto instrument surfaces.2However,IMS also suffers from these disadvantages.
IMS as an analytical technique has several speci?c weaknesses. The primary drawbacks are its low resolving power,limited selectivity,16and the experimental nature of the technique.Matrix effects such as humidity,temperature,and the composition of the sample may in?uence the detector’s response.Ideally,IMS should be used in environments with controlled temperature,low humid-ity,and controlled amounts of dopants,thus requiring delicate engineering and parameter optimization for in-?eld use.The separation of ions is highly dependent on ion mobilities in a drift gas under the in?uence of an electric?eld,which may be affected by altering the polarizability and mass of the drift gas,thus changing the chemistry of ion-neutral molecule interactions. Selectivity could be improved by adjusting the electric?eld strength and drift tube pressure or temperature;other ions may also be examined.Nonetheless,high collision rates at atmospheric pressures could be advantageous in IMS.Based on the thermo-dynamic equilibrium between the ions and the neutral molecules, IMS is able to selectively ionize whole classes of compounds
with Figure1.Structures of some typical CWAs.
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collectively unique thermodynamic properties such as high proton af?nity.
Another drawback of IMS chemical interference in highly contaminated environments.Such interference may be reduced by using highly selective forms of ionization with additional reagent molecules.16However,if the measurements are made at high pressures in which gas-phase collisions may increase the frequency of the charge exchange reaction,the desired selectivity cannot be achieved.17False positives also may pose a problem. Some well-known examples of interferant cross-references in explosive trace detection include certain medications such as vasodilators containing nitroglycerin,certain ingredients in com-monly-used hand lotions,cardamom,and certain types of?re-?ghting foams.These problems may be ef?ciently solved with instrumental combinations like IM/MS,which could signi?cantly decrease the potential for false positive responses when screening for CWAs.18IM/MS has already been used to analyze CWAs in spiked food products.19
The bureaucracy involved in using radioactive sources,includ-ing complying with numerous ordinances and licensing require-ments,is also a limitation.Alternative,non-radioactive atmospheric pressure ionization methods include photoionization,laser ioniza-tion,corona discharge,and surface ionization.However,non-radioactive ionization sources suffer from limited lifetimes,aging, stability,and the need for power.
Even though IMS has the above-mentioned drawbacks,numer-ous advantages tip the balance in its favor.The main advantages of IMS are instrumental simplicity,small size,light weight, portability,reliability,ease of operation,real time monitoring capability,fast response,short analysis time,low power consump-tion,low operating cost,and high sensitivity,speci?cally for persistent CWAs.20
The analytical performance of IMS is superior to that of other CWA detection methods.Analysis and response times of IMS are very short,thus providing the basis for a real-time monitoring capability:the actual ion separation time in one scan is on the millisecond timescale,and total measurement time is only a few https://www.doczj.com/doc/641789967.html,mon agents are easy to ionize and to analyze,leading to relatively high reliability and https://www.doczj.com/doc/641789967.html,bining IMS with other analytical instruments such as GC and MS further increases the analytical power.These combinations provide a second dimension of separation,which increases selectivity and therefore reduces the number of false positives.An example is an analysis of TNT in which hand lotion can produce a false positive.Both substances produce equal signals in IMS and in MS but are distinct in a2D spectrum.21This IM/MS combination can also be used to analyze degradation products of CWAs from a complex aqueous mixture.22
Other advantages originate from user-friendliness and practical details.Instrumental simplicity and ease of use creates a large network of potential users by simplifying training.The small size and weight allows true portability.Easy maintenance and low power consumption together with robustness enable IMS usage in dif?cult environs such as those required for military applications.
TECHNIQUES IN CHEMICAL WARFARE AGENT DETECTION
Conventional IMS.A conventional ion mobility spectrometer consists of the reaction region,including the ion source and ion gate,and the drift region and detector.The sample compounds are ionized by proton transfer or electron capture reactions.The electronic gating grid(ion gate)introduces the ions into the drift region,where the ions travel along the electric?eld gradient.The ions are separated according to their velocities in the neutral, counter-?owing drift gas.Ions create a drift time related signal through collision and neutralization at the detector(a Faraday plate).The operating principle for a conventional IMS device is presented in Figure2.
Detection in conventional IMS is based on signal peak position and intensity.The selectivity is de?ned by resolving power,which in turn is de?ned by drift time per peak half width.In general, the length of the drift tube limits the selectivity,but advanced signal processing methods like FT may resolve this problem. Conventional IMS is dif?cult to miniaturize,so only a few manufacturers are fabricating handheld devices.Nevertheless,the majority of the instruments used for in-?eld detection are based on this conventional separation and detection technique.The current commercial manufacturers of conventional handheld IMS instruments for in-?eld detection include Smiths Detection,Bruker Daltonics,IUT GmbH,and GE Security.
Conventional IMS instruments have been widely used in CWA investigations.The early studies included pesticides,23organo-phosphorus compounds,24sarin,25various phosphorus esters including G nerve agents,26and methyl isocyanate.27More recently,research in Hill’s group has been especially active.They have analyzed the reduced mobility values of various CWA simulants and degradation products.28,29The combination of ESI and IMS has been used to characterize CWA degradation products,with detection limits<100ppb.30IMS combined with TOFMS has been used to study CWA simulants in
different Figure2.The operating principle for a conventional IMS instrument.
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aerosol matrices;the main advantage of this combined technology is that it produces both2D and3D acquisition spectra,thus facilitating compound identi?cation.31,32With the same combina-tion,the identi?cation and quanti?cation of CWA degradation products in aquatic environments also becomes possible.22Alter-native,non-radioactive ionization techniques like chemical ioniza-tion,ESI and secondary electrospray ionization(SESI)have also been used to ionize CWA simulants,resulting in higher detection sensitivity.33
Other studies have investigated detection limits and reduced mobility values of substances present in hazardous military waste sites,34the problems of VX detection,35and explosively dis-seminated CWAs.36,37The response to phosgene has been determined by using membrane inlet IM/MS.38IM/MS has also been used to analyze lewisite and lewisite-mustard mixtures.39 The detection performance of corona discharge IMS has been investigated using CWAs and related compounds.40
A solid phase microextraction(SPME)sampling system coupled with IMS has been introduced as a fast and reliable analysis method for https://www.doczj.com/doc/641789967.html,ing this method,the precursor and byproducts of G
B and degradation products of VX present in soils have been analyzed with good reproducibility and detection at concentrations as low as10μg g-1.41SPME with pyrolysis GC/ IMS has been used to analyze tributylphosphate(a VX simu-lant).42Reduced mobility values and evaluated detection limits for various CWAs,including G-agents,mustard gas,and V-agents,have been studied with high-temperature SPME/ IMS.43
Aspiration IMS.Another type of IMS used to detect CWAs is aspiration IMS(AIMS).The ionization occurs at ambient conditions in the presence of a relatively high amount of water, which affects the kinetics of ionization reactions.The principle of operation of the AIMS instrument is depicted in Figure3.Ions travel through an orthogonal electric?eld in which they are de?ected to multiple channels located on the collecting electrode. Ions with a faster velocity collide earlier than slower ions;the detection in AIMS is based on characteristic signal patterns and utilizes pattern recognition methods.A Finnish company,Envi-ronics Oy,is a specialized manufacturer of the ChemPro handheld AIMS instrument.
AIMS has a direct interface with ambient air with?ow-through construction,allowing very fast response and recovery times.The instrument does not have a shutter grid and is typically operated without any membranes or dopants.The detection process is continuous;the?ow rate is1-2L min-1with detection levels of ppb or sub-ppb for CWAs.Furthermore,AIMS instruments differ from conventional devices by detecting the spatial distribution of the ion?ow.The polarity of the electric?eld is alternated continuously,thus ions of both polarities are separated and detected simultaneously.To improve selectivity, the electric?eld can be adjusted individually for each electrode pair.
The main advantages of this technology are that it has very high sensitivity and can potentially be miniaturized and mass produced.The devices are also simple and suf?ciently rugged for in-?eld use.Its sensitivity is easily adjusted because its ionization ef?ciency is?ow rate dependent.Furthermore,the detection limits approximately correspond to toxicity values;AIMS provides a sensitive and speci?c output for highly toxic chemical vapors.44The next generation,so-called“2nd order AIMS”(or ion focusing AIMS),uses rapidly-sweeping electrical?elds to create a large number of virtual channels.The ions are focused into the sample stream by means of an electric?eld or,more often, by controlled?ow streams.This construction improves detection by improving ion separation.
AIMS has been used to characterize the degradation products of soman and VX,45and the detection performance of AIMS devices has been investigated with CWAs and simulants.46Also, a miniaturized ion-focusing aspiration condenser-type IMS instru-ment has been used to detect various CWAs.47In addition,the toxic industrial chemicals ethyl parathion and toluene2,4-diiso-cyanate have been analyzed.48
Field Asymmetric IMS.The mechanism of?eld asymmetric IMS(FAIMS),also known as differential mobility spectrometry (DMS),is closely related to AIMS,but the ion separation process differs.In FAIMS,ions pushed by the gas?ow travel through a perpendicular electrical?eld.The electrical?eld is generated by an asymmetric AC dispersion voltage but also has a compensation voltage called the DC component.This radio-frequency(RF) region works as a?lter,and ions of both polarities are collected behind it.Ion mobility is a function of?eld strength,especially in the case of high?elds;hence,ion drifts caused by the asymmetric ?eld can be compensated for with a suitable compensation voltage. The advantages of this instrument are its small and simple construction and potentially high resolution.Detection is based on the signal peak position and intensity in the RF-voltage-Compensation Voltage(Vrf-CV)plane.The principle of FAIMS ion separation is shown in Figure4.The electric?eld is applied to the lower plate while the upper plate is grounded.Ion B reaches the detector while ions A and C stick to the plates.By adjusting the compensation voltage,different ions(A or C)will reach the detector.
Currently,technical development in the IMS?eld is mostly focused on FAIMS.One important feature is miniaturization:the fabrication and characterization of a micro-electro-mechanical-system(MEMS)radio-frequency ion mobility spectrometer
(rf-Figure3.The operating principle for an AIMS instrument.
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IMS)having high resolving power has been described.49-51The major commercial source of FAIMS instruments is Thermo Scienti?c.In addition,General Dynamics offers a commercial handheld FAIMS device.Owlstone manufactures miniaturized FAIMS microchips,as depicted in Figure 5.
FAIMS has been used to detect nerve and blister agent simulants,52various CWAs,53and organophosphorus com-pounds.54A comprehensive review including a summary of the basics together with a consideration of various applications of FAIMS has been recently published.55
FUTURE PROSPECTS
Relationship to MS.The combination of IMS with other detection techniques has become increasingly important.One grow-ing trend is to unite IMS with MS.In this combination,so called IM/MS,the ion mobility section serves as a pre-separation phase prior to MS analysis,enabling a second dimension of separation.IM/MS can separate conformers,isomers,or chiral molecules with the same molecular mass,thus improving analytical performance.56ESI IM/MS instrument parameters for the analysis of CWAs have been optimized.57Other combinations that are already used are GC or LC with IMS.Waters manufactures commercial Q-TOF instruments that can have an IMS section incorporated.
Recent developments with ambient pressure MS also come close to the principles and applications of IMS.These methods
include,for example,desorption electrospray ionization (DESI)58and direct analysis in real time (DART)59MS.Similarities include ionization at ambient pressure,sampling from surfaces,water-assisted ionization,and portability.When water is used as the solvent,the sprayed water molecules form hydronium ions in the presence of an electric ?eld.The sample molecules are bombarded by these hydronium ions;the ionization occurs via proton transfer from the hydronium ion to the sample molecule.These and various other atmospheric pressure ionization techniques could function as ion sources and sample introduction devices for compounds having low or nonexistent volatility.
Currently,the smallest MS instrument with an external DESI source,the Mini 11,has dimensions of 22×12×18cm and weighs 5kg with batteries;60it is noticeably smaller than its predecessor,the Mini 10(shoebox-sized,32×22×19cm,10kg).61Even though the Mini 11is still fairly big when compared to handheld IMS instruments,it shows that the drive towards miniaturized mass spectrometers continues.This is also a challenge to standalone IMS devices because the analytical power of MS is superior.On the other hand,this trend should eventually produce a handheld combined IM/MS instrument.A recently published article de-scribes the design of a prototype ?eld-portable IM/MS instru-ment.62
Instrument Development.Any novel technology for CWA detection must ful?ll the requirements for the detection of CWAs,and the same instrument should be able to detect toxic industrial chemicals and potentially,yet to be determined non-traditional agents too.Further improvements in IMS will probably include lower detection limits,enhanced selectivity,faster operation,standardized libraries that enable comparisons between labora-tories,and overall enhanced instrument performance.The per-formance development is achievable with combinations of different measurement techniques using variations on IMS such as FAIMS with conventional drift or FAIMS with aspiration IMS.Also,instrument performance can be improved with the development of sampling and sample pre-treatment methods.On the other hand,the ongoing trend toward combining IMS with other analytical techniques like MS and GC will continue.Eventually IMS will be a standard part of a hybrid instrument.
Another area that will likely have an impact on IMS is the development of future technologies,presumably including ad-vanced manufacturing processes like nanotechnology,microme-chanics,and semiconductor technology applied to production of non-radioactive ionization systems and sensor structures,including those that are integrated with other types of gas sensors.All
these
Figure 4.The operating principle for a FAIMS
instrument.
Figure 5.Top)Micromachined FAIMS.Reprinted with permission from Miller,Eiceman,Nazarov &King (2000),copyright 2000Elsevi-er.50Bottom)FAIMS microchip placed on ?ngertip.Photo courtesy of Owlstone,used with permission.
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technologies will facilitate the manufacturing of remarkably small but complex systems leading to miniaturized“button-size”lab-on-a-chip personal detectors.However,a great deal of develop-mental research work is required for this.Presumably these instruments will become technically feasible before they are commercially sensible.This may be a limiting factor for new designs;thus near-or mid-future devices will be based on more traditional,relatively low investment manufacturing techniques.
From the user’s perspective,a low training burden and low lifetime costs are also considerations for instrument development. Any additional or complicated user interface is also a drawback. Therefore,current development is focusing on separating the actual measuring instrument and the user interface.Furthermore, forms of mobile communication provide novel possibilities such as networked sensor systems and personal information sources. When interfaces are separated from measurement devices,there has to be a central unit that collects and combines the information originating from real-time measurement devices.This contributes to the technical development of easy-to-use,multipurpose,and multifunctional detection devices.
There exist application-driven ideas to improve and develop IMS-based instruments even further.These include a disposable IMS analyzer for respiratory air quality monitoring as a separate device or attached to a personal gas mask.Continuous monitoring of stockpiled industrial chemicals and public infrastructures is possible by integrating IMS with ventilation systems.Another possibility is to use multiple parallel IMS instruments to produce an atmospheric pressure reactor chamber where reaction products are separated from reactants and enriched using ion mobility principles.
SUMMARY
Even though IMS technology has some disadvantages,the numerous advantages show that IMS is currently the favored state-of-the-art technology for detection of CWAs.The need for a rugged,easy-to-operate,sensitive,and reliable instrument for in-?eld use continues. In urban environments,the monitoring and detection of CWAs and toxic industrial chemicals play important roles in public safety.In addition,industrial methods that enable surveillance and fast warn-ings to detect leakage of hazardous chemicals or environmental pollutants are needed.In the future,IMS technology either alone or combined with other detection technologies will enable practical detection of CWAs and hazardous chemicals.
ACKNOWLEDGMENT
Funding by the Finnish Funding Agency for Technology and Innovation(TEKES)is acknowledged.Dr.Mary Metzler is thanked for language revision.
Osmo Anttalainen is a Vice President of Technology at Environics Oy and has been involved in IMS sensor development since1994.Marko Ma¨kinen is researcher focused in IMS,gas-phase chemistry,and environmental pollutants.Mika Sillanpa¨a¨is professor and head of the Laboratory of Applied Environmental Chemistry.His interests include environmental analysis and technologies in environmental engineering.Address correspondence to Ma¨ki-nen at Laboratory of Applied Environmental Chemistry,Department of Environmental Science,University of Eastern Finland,Patteristonkatu1, 50100Mikkeli,Finland;marko.makinen@uef.?;+358-40-3553713(phone); +358-15-3556363(fax).
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Bioinformatics Analysis of Next-Generation Sequencing Data – Epigenome and Chromatin Interactome 要点： Enhancers are marked by multiple modifications Characteristic histone methylation patterns at active genes 涉及的相关技术： NGS Epigenetics CHIP-Seq 3C NGS(Next-Generation Sequencing)的原理： 最近市面上出现了很多新一代测序仪产品，例如美国Ｒｏｃｈｅ Ａｐｐｌｉｅｄ Ｓｃｉｅｎｃｅ公司的４５４基因组测序仪、美国Ｉｌｌｕｍｉｎａ公司和英国Ｓｏｌｅｘａ ｔｅｃｈｎｏｌｏｇｙ公司合作开发的Ｉｌｌｕｍｉｎａ测序仪、美国Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ公司的ＳＯＬｉＤ测序仪、Ｄｏｖｅｒ／Ｈａｒｖａｒｄ公司的Ｐｏｌｏｎａｔｏｒ测序仪以及美国Ｈｅｌｉｃｏｓ公司的ＨｅｌｉＳｃｏｐｅ单分子测序仪。所有这些新型测序仪都使用了一种新的测序策略——循环芯片测序法（ｃｙｃｌｉｃ－ａｒｒａｙ ｓｅｑｕｅｎｃｉｎｇ），也可将其称为“新一代测序技术或者第二代测序技术”。所谓循环芯片测序法，简言之就是对布满ＤＮＡ样品的芯片重复进行基于ＤＮＡ的聚合酶反应（模板变性、引物退火杂交及延伸）以及荧光序列读取反应。２００５年，有两篇论文曾对这种方法做出过详细介绍。与传统测序法相比，循环芯片测序法具有操作更简易、费用更低廉的优势，于是很快就获得了广泛的应用。 传统的Ｓａｎｇｅｒ测序法及新一代ＤＮＡ测序技术工作流程图
感受现代科技 【学习目标】 1、知识：感受现代科技给人类生活带来的新变化，认识科技与生活，科技发展与社会发展的关系，懂得“科学技术是第一生产力”的道理。 2、能力与情感：感悟现代科技的神奇与力量，理解科技是社会发展的强大推力，激发学生 对科技重要性的认识，增强学生对科学的兴趣，培养学生热爱科学的精神。 3、过程与方法：依据教学内容和学生的认识规律设置了“课前预习”、“课堂助学”、“课堂巩固”、“课后拓学”、“教学反思”五个模块的教学整合，运用多媒体等教学手段，采用自主体验、 探究活动、案例情境等方法来完成教学目标。 【学习重点、难点】 领略现代科技的神奇与力量，理解“科技是第一生产力”。 【学习过程】 一、预习初探： （一）快快行动，书外的知识真有趣： 1、生活体验：观察生活，请你说说我们身边有哪些科技产品？例举实例说说这些科技产品给我们的生活带来哪些新变化？ 2、想象天地：展现你的想象天份，想象你准备发明一样科技产品，使你的未来生活更美好。 3、图片收集：上网收集有关科技产品的图片，准备创办科技小展览，领略现代科技的神 奇与力量。 （二）阅读课本，书本的知识真寻味： 4、我们现在的生活与科技________________。丰足的衣食，舒适的住行，千百年来一直是人类_________________。 5、科学技术是________________的强大推力，是________生产力。______________已成为当代经济发展的火车头。 6、________________是人类文明的标志。科学技术的进步为人类创造了巨大的 ______________和_________________。
illumina 转录组测序简明实验流程一、实验基本流程图 mRNA Library Construction
二、mRNA建库流程 1.材料准备 1.2. 1.3.
2.样品准备和QC 选择质量合格的Total RNA作为mRNA测序的建库起始样品，其质量要求通过Agilent 2100 BioAnalyzer检测结果RIN≥7，28S和18S的RNA 的比值大于或等于1.5：1，起始量的要求范围是0.1∽1ug。用QUBIT RNA ASSAY KIT对起始的Total RNA进行准确定量。 3.建库实验步骤 3.1.mRNA纯化和片段化 3.1.1.mRNA纯化 纯化原理是用带有Oligod(T)的Beads对Total RNA中mRNA进行纯化。 3.1.2.mRNA片段化 3.2.1st Strand cDNA 合成 3.3.2nd Strand cDNA 合成 根据下表制备反应体系，然后在PCR仪上运行Program3，然后将第2链cDNA合成产物用144uL AMPure XP Beads进行纯化，最后用60μL的Nuclease free water进行重悬，取出 55.5μL以备下一步使用;
3.4.Perform End Repair/dA-tail 3.5.Adaptor Ligation 根据下表制备反应体系，然后在PCR仪上运行Program5、Program6，然后100uL AMPure XP Beads进行纯化后用52.5μL的Resuspension Buffer进行重悬，再用50uL AMPure XP Beads 3.6.PCR扩增 根据下表制备反应体系，然后在PCR仪上运行Program7，然后再45μL用AMPure XP Beads 进行纯化，最后用23μL的Resuspension Buffer进行重悬，取出20μL以备下一步使用；
AFLP分子标记实验 扩增片段长度多态性 Amplified fragment length polymorphism（AFLP 是在随机扩增多态性（RAPD和限制性片段长度多态性（RFLP技术上发展起来的DNA多态性检测技术,具有RFLP技术高重复性和RAPD技术简便快捷的特点,不需象RFLP 分析一样必须制备探针，且与RAPD标记一样对基因组多态性的检测不需要知道其基因组的序列特征，同时弥补了 RAPD技术重复性差的缺陷。同其他以PCR为基础的标记技术相比，AFLP技术能同时检测到大量的位点和多态性标记。此技术已经成功地用于遗传多样性研究，种质资源鉴定方面的研究，构建遗传图谱等。 其基本原理是：以PCR（聚合酶链式反应为基础,结合了 RFLP、RAPD的分子标记技术。把DNA进行限制性内切酶酶切，然后选择特定的片段进行PCR扩增（在所有的限制性片段两端加上带有特定序列的’接头”用与接头互补的但3-端有几个随机选择的核苷酸的引物进行特异PCR扩增,只有那些与3-端严格配对的片段才能得到扩增，再在有高分辨力的测序胶上分开这些扩增产物，用放射性法、荧光法或银染染色法均可检测之。 一、实验材料 采用青稞叶片提取总DNA 实验设备 1. 美国贝克曼库尔特CEQ8000毛细管电泳系统, 2. 美国贝克曼库尔特台式冷冻离心机， 3. 美国MJ公司PCR仪,
4. 安玛西亚电泳仪等。 三、实验试剂 1. 试剂:请使用高质量产品，推荐日本东洋坊TOYOBO公司的相关产品 DNA提取试剂盒； EcoRI酶,Msel酶,T4连接酶试剂盒； Taq 酶,dNTP, PCR reactio n buffer; 琼脂糖电泳试剂：琼脂糖，无毒GeneFinder核酸染料替代传统EB染料;超纯水（18.2M ? ? cm 2. 其他实验需要物品 微量移液枪（一套及相应尺寸Tip头,PCR管，冰浴等。 四、实验流程 1、总DNA提取 使用DNA提取试剂盒提取植物基因组DNA,通过紫外分光光度计检测或用标准品跑胶检测。一般来说，100ng的基因组DNA作为反应模板是足够的。 2、EcoR1酶消化（20ul体系/样品 EcoR1 1ul
下一代测序（NGS）彻底改变了基因组学研究领域，使全基因组测序比以往任何时候都更有效率。然而，典型的NGS工作流程是鲜有革新的，因为它面临许多手动操作步骤和来自成本、通量以及结果变异性的诸多挑战。传统的样品制备和数据分析方法非常耗时，并更易出错。 针对这些挑战，自动化技术为此提供了相应的解决方案，并通过减少样品间变异提高了最终数据的精准度。然而，为您的NGS工作流程选择适合的自动化设备是一个复杂的过程。为了给您的实验室配备最佳的自动化整合系统，首先要对以下的四个因素进行评估，然后再作出决定： ? 自动化将如何影响您的实验流程？ ? 您可选的自动化方案有哪些？ ? 需要多少培训？ ? 您的自动化解决方案是否需要扩展，以满足未来的需求？
Ilumina文库构建杂交选择和靶向捕获簇扩增和测序Ilumina文库构建杂交选择和靶向捕获簇扩增和测序
图3，高通量PCR纯化自动化工作流程 您是否在纯化时使用真空泵和离心过滤？图3描述了一个中高通 进一步加速：靶标富集技术 量的自动化工作流程图。 基于磁珠技术的靶标富集方法，比如SureSelect靶标富集 试剂盒和SureSelect人全外显子试剂盒，能使您仅对感兴 趣的基因片段测序，提高了几个数量级的实验效率。这些操 作很容易实现和高度扩展，凸显出Bravo自动化液体处理 平台的速度和精度的优势。
如果您的实验室需要更高的通量，您可考虑增加一个更全面的自动化系统。安捷伦BenchCel 微孔板工作站是一个灵活的、可扩展的、通量可媲美大型系统的紧凑桌面式平台。由市面上最灵活和调度高效的安捷伦VWorks 软件控制，BenchCel 工作站可用于复杂的和简单的应用流程，比起传统的手动操作方法能提供更长的无人值守时间和更大的通量。 对于超高通量、高生产率的实验室，您可能需要放弃桌面型系统。安捷伦独立的BioCel 自动化系统基于一个易于定位的直驱机械臂(DDR)。这种高度灵活的系统能与安捷伦其它自动化模块，或第三方设备组合形成定制系统，以满足您实验室的需要。 自动化方案的选择 想一想您实验室的整个工作流程。有各种不同的自动化解决方案——从垂直移液工作站到BenchCel 工作站再到BioCel 系统，可以满足不同的通量需求。自动化将提高实验数据的准确性和一致性——您是否需要一个完全无人值守的自动化解决方案？安捷伦拥有一系列的自动化设备可供选择，以适应不同实验室的需要。安捷伦的Bravo 自动液体处理平台具有宽量程的高精度移液性能、兼容不同类型微孔板的灵活性以及独特的开放式设计，易于整合到其他的自动化工作流程中。结合Bravo 自动化液体处理平台可以大大减少样品制备和检查下一代测序文库质量所花费的时间，并通过减少样本间变异提高了数据质量。 图4. 桌面型选择： A ．Bravo 自动化液体处理平台 B ．BenchCel 微孔板操作工作站独立的高通量系统：C. BioCel 系统 A B C
第十四课感受现代科技 项目一现代科技在身边 教师寄语：今日沟通于昔日之最大差异：在于科技的介入，已超越时间、空间，甚至权利与阶级的围墙。 学习目标： ●情感态度价值观：感受现代科技发展带来的新变化，培养学生热爱科学的情感和品质。 ●能力：培养学生观察、收集、整理、归纳信息的能力。 ●知识：了解科技对社会发展具有推动作用，理解科学技术是第一生产力。 重难点：科技是社会发展的强大推动力 学习过程： 一、课前预习（提前预习课本，顺便做以下小题，相信你能完成!） 1.我们现在的生活与科技_________。 2.___________________是第一生产力。 3._______________已成为当代经济发展的火车头。 4.科学技术是_______________的标志。 5.科技是社会发展的______________。 二、合作探究，共同进步（合作有助于提高学习效率，要努力哦！） 知识点一：现代科技给生活带来新变化 1：（见教材P58页-59页）图片1：液晶彩色电视图片2：利用网络学习 图片3：山里人用上了手机图片4：磁悬浮列车 说一说：观察生活，列举实例，说说现代科技给我们的生活带来哪些新的变化？ 2：阅读教材（P59页教材正文），说说现代科技的发展给人类的生活产生了怎样的影响？（学生讨论交流） 。3：（见教材P59页-60页）想象和推测一下，随着科技的发展，5年、10年、20年、50年后人们的生活可能是怎样的？ 。 知识点二：科技——社会发展的强大推力 1：（见教材P61页），材料中的数字变化说明了什么？（学生讨论交流） 上述材料中的数字变化说明：“”。 2：为什么说“科学技术是第一生产力”？ ① ② 实践证明：高新技术及其产业已经成为当代经济发展的火车头。 3：（见教材P62页）说一说：网络学校的发展将会给人们的学习方式带来了哪些变化？ 4、①科学技术的进步使精神文明建设有了新的载体. 说一说：在思想文化传播的载体方面，你知道有哪些新的传播手段呢？ ②科学技术的进步丰富了人们的 . 5、科学技术的进步为人类创造了巨大的财富和财富 三、课堂小结 通过学习我学会：
《交通规划》课程教学大纲 课程编号：E13D3330 课程中文名称：交通规划 课程英文名称：Transportation Planning 开课学期：秋季 学分/学时：2学分/32学时 先修课程：管理运筹学，概率与数理统计，交通工程学 建议后续课程：城市规划，交通管理与控制 适用专业/开课对象：交通运输类专业/3年级本科生 团队负责人：唐铁桥责任教授：执笔人：唐铁桥核准院长： 一、课程的性质、目的和任务 本课程授课对象为交通工程专业本科生，是该专业学生的必修专业课。通过本课程的学习，应该掌握交通规划的基础知识、常用方法与模型。课程具体内容包括：交通规划问题分析的一般方法，建模理论，交通规划过程与发展历史，交通调查、出行产生、分布、方式划分与交通分配的理论与技术实践，交通网络平衡与网络设计理论等，从而在交通规划与政策方面掌握宽广的知识和实际的操作技能。 本课程是一间理论和实践意义均很强的课程，课堂讲授要尽量做到理论联系实际，模型及其求解尽量结合实例，深入浅出，使学生掌握将交通规划模型应用于实际的基本方法。此外，考虑到西方在该领域内的研究水平，讲授时要多参考国外相关研究成果，多介绍专业术语的英文表达方法以及相关外文刊物。课程主要培养学生交通规划的基本知识、能力和技能。 二、课程内容、基本要求及学时分配 各章内容、要点、学时分配。适当详细，每章有一段描述。 第一章绪论（2学时） 1. 交通规划的基本概念、分类、内容、过程、发展历史、及研究展望。 2. 交通规划的基本概念、重要性、内容、过程、发展历史以及交通规划中存在的问题等。
第二章交通调查与数据分析（4学时） 1. 交通调查的概要、目的、作用和内容等；流量、密度和速度调查；交通延误和OD调查；交通调查抽样；交通调查新技术。 2. 交通中的基本概念，交通流量、速度和密度的调查方法，调查问卷设计与实施，调查抽样，调查结果的统计处理等。 第三章交通需求预测（4学时） 1. 交通发生与吸引的概念；出行率调查；发生与吸引交通量的预测；生成交通量预测、发生与吸引交通量预测。 2. 掌握交通分布的概念；分布交通量预测；分布交通量的概念，增长系数法及其算法。 3. 交通方式划分的概念；交通方式划分过程；交通方式划分模型。 第四章道路交通网络分析（4学时） 1. 交通网络计算机表示方法、邻接矩阵等 2. 交通阻抗函数、交叉口延误等。 第五章城市综合交通规划（2学时） 1. 综合交通规划的任务、内容；城市发展战略规划的基本内容和步骤 2. 城市中长期交通体系规划的内容、目标以及城市近期治理规划的目标与内容 第六章城市道路网规划（2学时） 城市路网、交叉口、横断面规划及评价方法。 第七章城市公共交通规划（2学时） 城市公共交通规划目标任务、规划方法、原则及技术指标。 第八章停车设施规划（2学时） 停车差设施规划目标、流程、方法和原则。 第九章城市交通管理规划（2学时） 城市交通管理规划目标、管理模式和管理策略。 第十章公路网规划（2学时） 公路网交通调查与需求预测、方案设计与优化。 第十一章交通规划的综合评价方法（2学时） 1. 交通综合评价的地位、作用及评价流程和指标。 2. 几种常见的评价方法。 第十二章案例教学（2学时）
#流程大放送#微生物基因组Denovo测序分析 知因无限 一介绍 微生物基因组De novo测序分析也叫微生物基因组从头测序分析，指不依赖于任何参考序列信息就可对某个微生物进行分析的测序分析技术，用生物信息学的方法进行序列拼接获得该物种的基因组序列图谱，然后进行注释等后续一系列的分析。微生物Denovo基因组测序及分析技术可以应用于医药卫生等领域。 二技术应用领域 1、基因组图谱的系统性构建 例子：过去几个月，肠病毒D68令数百名美国儿童患病。华盛顿大学的研究人员测序和分析了肠病毒D68（EV-D68）的基因组，这一成果将发表在新一期的Emerging Infectious Diseases杂志上。（Genome Sequence of Enterovirus D68 from St. Louis, Missouri, USA）肠病毒D68（EV-D68）能在儿童中引起严重的呼吸道疾病。其基因组序列可以“帮助人们开发更好的诊断测试，”共同作者Gregory Storch说。“有助于解释病毒感染为什么会造成严重的疾病，以及EV-D68为什么比过去传播得更广。”（来自于生物通的报道） 2、微生物致病性和耐药性位点检测及相关基因功能研究 例子：根据分泌蛋白、毒力因子、致病岛、必需基因等结果去探讨所测物种致病性和耐药性。 3、微生物的比较基因组分析，确定各个近缘微生物中的系统发育关系 二基本分析流程图
三可能的结果展示图 示例图1 微生物基因组的功能注释
示例图2 微生物基因组的系统进化关系 注：以上图片和文字来自参考文献21。 六参考文献 [1] Hong-Bin Shen, and Kuo-Chen Chou, "Virus-mPLoc: a fusion classifier for viral protein subcellular location prediction by incorporating multiple sites", Journal of Biomolecular Structure & Dynamics, 2010, 28: 175-86. [2]Hong-Bin Shen and Kuo-Chen Chou, "Virus-PLoc: A fusion classifier for predicting the subcellular localization of viral proteins within host and virus-infected cells.", Biopolymers. 2007, 85, 233-240. [3] Ren Zhang and Yan Lin, (2009) DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes. Nucleic Acids Research 37, D455-D458. [4] The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics. 2007 May 23;8(1):172. [5] The Pfam protein families database: M. Punta, P.C. Coggill, R.Y. Eberhardt, J. Mistry, J. Tate, C. Boursnell, N. Pang, K. Forslund, G. Ceric, J. Clements, A. Heger, L. Holm, E.L.L. Sonnhammer, S.R. Eddy, A. Bateman, R. D. Finn Nucleic Acids Research (2014) Database Issue 42:D222-D230. [6] Clustal W and Clustal X version 2.0.(2007 Nov 01) Bioinformatics (Oxford, England) 23 (21) :2947-8.PMID: 17846036. [7] Felsenstein, J. 2004. PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle. [8] Li et al (2010). De novo assembly of human genomes with massively parallel short read
一 AFLP 分子标记实验 扩增片段长度多态性 Amplified fragment length polymorphism(AFLP 是在随机扩增多态性 (RAPD 和限制性片段长度多态性 (RFLP 技术上发展起来的 DNA 多态性检测技术, 具有 RFLP 技术高重复性和 RAPD 技术简便快捷的特点, 不需象 RFLP 分析一样必须制备探针, 且与 RAPD 标记一样对基因组多态性的检测不需要知道其基因组的序列特征,同时弥补了 RAPD 技术重复性差的缺陷。同其他以 PCR 为基础的标记技术相比, AFLP 技术能同时检测到大量的位点和多态性标记。此技术已经成功地用于遗传多样性研究, 种质资源鉴定方面的研究,构建遗传图谱等。 其基本原理是:以 PCR(聚合酶链式反应为基础, 结合了 RFLP 、 RAPD 的分子标记技术。把 DNA 进行限制性内切酶酶切,然后选择特定的片段进行 PCR 扩增(在所有的限制性片段两端加上带有特定序列的“ 接头” , 用与接头互补的但 3-端有几个随机选择的核苷酸的引物进行特异 PCR 扩增, 只有那些与 3-端严格配对的片段才能得到扩增 , 再在有高分辨力的测序胶上分开这些扩增产物,用放射性法、荧光法或银染染色法均可检测之。 一、实验材料 采用青稞叶片提取总 DNA 。 二、实验设备 1. 美国贝克曼库尔特 CEQ8000毛细管电泳系统, 2. 美国贝克曼库尔特台式冷冻离心机,
3. 美国 MJ 公司 PCR 仪, 4. 安玛西亚电泳仪等。 三、实验试剂 1. 试剂:请使用高质量产品,推荐日本东洋坊 TOYOBO 公司的相关产品。 DNA 提取试剂盒; EcoRI 酶,MseI 酶,T4连接酶试剂盒; Taq 酶,dNTP, PCR reaction buffer; 琼脂糖电泳试剂:琼脂糖,无毒 GeneFinder 核酸染料替代传统 EB 染料; 超纯水(18.2M ? ·cm 2.其他实验需要物品 微量移液枪(一套及相应尺寸 Tip 头,PCR 管,冰浴等。 四、实验流程 1、总 DNA 提取 使用 DNA 提取试剂盒提取植物基因组 DNA,通过紫外分光光度计检测或用标准品跑胶检测。一般来说, 100ng 的基因组 DNA 作为反应模板是足够的。 2、 EcoR1酶消化 (20ul 体系 /样品
2013年，单细胞测序技术开始成为科研界主流关注的焦点。 前言 2013年，单细胞测序技术（single-cell sequencing）荣膺《自然-方法》年度技术。单细胞测序技术有助于我们剖析细胞的异质性。它可以揭示肿瘤细胞基因组中发生的突变及结构性变异，而这些突变和变异往往有着极高的突变率。有了这些信息，我们就可以描述肿瘤细胞的克隆结构，并追踪疾病的进展及扩散范围。本文将介绍2013年单细胞测序技术在人类早期发育、癌症以及神经科学研究等几个重点领域的最新应用成果。 1. 单细胞测序技术简介 本节将概述如何获得一个单细胞的基因组及转录组。 单细胞基因组及转录组测序所需要的测序样本量要比单细胞中本身所含有的基因组及转录组分子高出好多个数量级，所以这对核酸扩增技术（amplification technology）也是一大考验。面对如此微量的分子，任何降解、样品损失、或者污染都会对测序质量带来非常严重的影响。而且多重扩增又容易带来试验误差，比如基因组或转录组覆盖不均一、背景噪声以及定量不准确等问题。 最近所取得的技术进步有望部分解决上述问题，使单细胞测序技术能
够走进更多的实验室，解决更多领域的科学问题。比较罕见的细胞、异质性的样本、与遗传嵌合或突变相关的表型、不能人工培养的微生物，这些都是单细胞测序技术能够一展所长的研究平台。使用单细胞测序技术能够发现克隆突变（clonal mutation）、隐藏的细胞类型，或者在大块组织样品研究工作中被“稀释”或平均掉的转录特征。 1.1 选择恰当的细胞 说到分离单细胞，显微操作（micromanipulation）无疑是一项非常精确的技术，而且利用毛细管（microcapillary）可以直接吸取细胞内容物，但是这项操作也需要耗费大量人力。很多组织解离之后都能够制成单细胞悬液，这种单细胞悬液很容易操作，而且可以用细胞分选器（cellsorter），根据细胞表面表达的特异性分子标志物对细胞进行分类富集操作。这种策略也被用来分离非常微量的循环肿瘤细胞。 1.2 单细胞转录组策略 现在有很多单细胞RNA测序操作流程可供选择，不过不管采用何种策略，首先都需要通过逆转录反应，利用RNA合成出cDNA。然后才会有所区别，比如有一些方法是对整个转录子进行测序，有一些方法只针对转录子的5'和3'端进行测序。不论采用何种方法，目的都只有一个，那就是捕获原始的RNA分子，然后均一的、准确地对其进行扩增。核酸的捕获效率主要受到逆转录反应的影响，不过我们可
走进都市农业感受现代科技 --"大东农业观光园"基地实践活动 设计者：大兴区第九小学窦雪征 指导教师：大兴区教师进修学校柏东河 ( 2008-05-27 13:40:19 ) 一、环境分析 我国是个农业大国。当前，在农业科技领域，中国不断缩小与发达国家的差距，农业科技部门在生物技术、高新技术、基础研究方面均取得较大进展，植物细胞和组织培养、花药培养、单倍体育种及其应用研究处于国际先进地位。但是，作为国家希望的小学生却对我国农业的了解知之甚少，甚至有的学生只认识餐桌上的蔬菜，田地里面的蔬菜就不认识了。而且由于社会环境的影响，学生对于农业生产的认识非常极端，认为农业生产只是辛苦的体力劳动，对其间蕴含的高科技文化知识基本不了解。 大兴区作为北京市的一个远郊区县，拥有着得天独厚的农业教育资源，在我们的身边就有向"大东农业观光园"这样的一个很好的农业教育基地。在大东农业观光园里面充分体现了现代农业。它目前拥有现代化日光温室34个，塑料大棚34个，露地80亩，为观光采摘、实验、示范、生产提供了场所。农业观光园里引进福建有机茶叶树，通过土壤改质能使其在北方正常生长；蔬菜嫁接的试验技术；大棚中的科学技术，如：所有地面具备节能、日光温室跨度大、内设供热、微喷、保湿、补光等多项功能；另外园区内设有科普知识长廊，为学生了解我国农业的历史文化和发展，创造了很好的条件。此基地实践活动适合小学高年级学生，他们可以在基地里面，进行参观、体验、学习了解高科技农业技术。通过基地的实践活动，可以增长学生现代农业知识，提高劳动能力，体验劳作的艰辛，培养学生良好的道德品质。可以很好的促进学生学习方式的变革，使学生直接从事实践性主题活动，实现学生自主学习和直接体验为主的学习方式，从而不