AN01 White Paper

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Plasma-based Cleansing Techniques for Biopharmaceutical Research: An Introduction to and Explanation of the Technology

White Paper Introduction Plasma-based Cleansing Techniques for Biopharmaceutical Research: Recently, IonField Systems™ introducedAn Introduction the TipCharger™, to and a Explanation device that generates of the Technology a self-contained, low-temperature, atmospheric plasma for cleaning pipette tips used in automated life science laboratories. The patented technology uses a Author: Geoffrey Schwartz dielectric barrier discharge (or gap plasma)Special Thanks that tois Dr. generated Kurt H. Becker, Professorin small, and Director, well-like Department cavities of Physics and Engineering conforms Physics, to Stevens a standard Institute of Technology SBS footprint. Currently, the device is passiveRecently, in that IonField plasma Systems generation introduced isa device triggered that gener via- thedown insertion of biological of pipette and organic tips, contaminants making it on both easy to install and integrate with most automatedates liquid handlers. The result is the ionizationthe exterior and and molecular interior surfaces breakdown of the tips. of Unlike biological and organic contaminants ona bothself-contained, the exterior low-temperature, and interior atmospheric surfaces plasma of the tips.traditional Unlike solvent-based traditional pipet solvent-based tip wash methods, plasma pipette tip wash methods, plasma as a statefor cleaning of matter and sterilizing (neither pipet a gas tips norused ain liquid) automated generates as a no state solid of matter or liquid (neither waste a gas nor and a liquid)only generates life science laboratories. The patented technology uses a no solid or liquid waste and only small amounts of gas- small amounts of gaseous by-products.dielectric Unlike barrier traditional discharge solvent-based(or gap plasma) that tip is generatedwashing methods,eous by-products. plasma And, cleaning unlike traditional does not solvent-based simply dilute contaminants, but removesin small, them well-like at a molecular cavities and level,conforms allowing to a standard laboratories SBS tip washing to reuse methods, polypropylene plasma cleaning tips does and not simply reduce the number of tip boxes needed.footprint. This Currently,paper discusses the device isthe passive mechanism in that plasma and applicationdilute contaminants, behind but this removes revolutionary them at a molecular generation is triggered via the insertion of pipet tips, making level, allowing laboratories to reuse polypropylene tips cleaning method. it easily installed and integrated with most automated liquid and reduce the number of tip boxes needed. This paper handlers. The result is the ionization and molecular break- discusses the mechanism and application behind this revolutionary cleaning and sterilizing method.

Background and Early Studies In 1927, the term plasma was first adopted by Nobel Prize winning chemist Irving Langmuir to describe the properties of gases ionized by high voltage to form free electrons and positive ions in a neutral background gas. These ionized gases conduct electricity and are strongly affected by magnetic fields. Although quite common in nature – it is Background and early studies estimated that over 99% of all matter in the known universe is in the form of plasma – lightning and flames are the only two naturally occurring forms(negative on earth (Figure electrode). 1). However, through Further, advancements by constantlyin physics and chemistry and The term plasma was first adopted by Nobel Prize winning as well as the harnessing of alternatingsimultaneously currents, the breadth switching of man-made the polarityplasmas continues of each to widen. electrode, chemist Irving Langmuir in 1927 to describe the properties Most of the early studies on man-madeelectrons plasmas andwere performed charged using ions vessels continuously either under low-pressure move between or vacuum of gases ionized by high voltage to formfilled free with electrons inert gases andproviding a ready source of electrons and positive ions. Through the application of a high poles forming what is known as a dielectric barrier positive ions in a neutral background voltagegas. Theseto metal ionizedelectrodes within these vessels, electrons are accelerated towards the anode (positive electrode) and discharge. gases conduct electricity and are stronglyconversely, affected positive ions by move toward the cathode (negative electrode). Further, by constantly and simultaneously switching the polarity of each electrode, electrons and charged ions continuously move between poles forming what magnetic fields. Although common in nature, it is is known as a dielectric barrier discharge.In the Inpresence the presence of ofa agas, gas, free free electrons electrons and activatedand activated ions collide ions with estimated that over 99% of all matterother atoms in the and molecules known present in the gas field resulting in a sustained and steady-state plasma. These experi- collide with other universe is in the form of plasma,lightningments ultimatelyand flames led toare the development of neon signs, streetlights and fluorescent light bulbs (Figure 2). a t o m s a n d the only two naturally occurring forms on Earth. Through molecules present advancements in physics and chemistry as well as the in the gas field harnessing of alternating currents, the breadth of man- resulting in a made plasmas continues to widen. sustained and steady-state Most of the early studies on man-made plasmas were plasma. These performed using vessels either under low-pressure or Figure 1. Lightning: A Recognizable Formexperiments of Naturally- Figure 2. Man-Made Plasma Lights Up the Las Vegas Sky vacuum -filled with inert gases providingOccurring a ready Plasma source of ultimately led to Figure 1. Man-Made Plasma Lights Up electrons and positive ions. Through the application of a the development of the Las Vegas Sky high voltage to metal electrodes within these vessels, n e o n s i g n s , electrons are accelerated towards the anode (positive streetlights and fluorescent light bulbs (Figure 1). electrode) while positive ions move toward the cathode

1 www.ionfieldsystems.com Recent Developments: Atmospheric-Pressure ‘Cold’ Plasma Although originally reported by Werner von Siemens in 1857 as a method for generating ozone, the technology remained Recent Developments:underdeveloped Atmospheric-Pressure until modern materials ‘Cold’ Plasma allowed for the production of more effective andAN-01 efficient dielectric materials. Although originally reported by Werner von Siemens in 1857 as a method for generating ozone, the technology remained Over the past thirty years advancements in dielectric materials, namely ceramic coated electrodes, provide an insulating underdevelopedAtmospheric-Pressure until modern materials ‘Cold’ Plasma allowed for the production of more effective and efficient dielectric materials. layer that allows for stable discharges to occur. The result is the generation of atmospheric-pressure plasmas that form Over the past thirty years advancements in dielectric materials, Thenamely plasma ceramic ions andcoated metastable electrodes, atomic provide species an react insulating Althoughin the absence originally of reported inert by gases Werner or vona vacuum. Siemens in layer that allows1857 as fora method stable for discharges generating toozone, occur. plasma The technology result is the generationrapidly with biologicalsof atmospheric-pressure and organic solvents plasmas to facilitate that form in the absenceremainedThe of induction inert underdeveloped gases of or atmospheric a untilvacuum. modern materials plasma allowedbegins withtheir the breakdown capturing and eventual of free removal. electrons to charged dielectric plates. for the production of more effective and efficient dielectric The inductionmaterials.Through of atmospheric Over changes the past plasmain thirtypolarity, begins years these advancementswith electronsthe capturing in are Inof forced general,free electrons tothere mobilize are tofour charged prototypical between dielectric reactiona set of plates.mechanisms plates resulting in an electron Through changesdielectricavalanche in materials, polarity, (Figure i.e thesethe 3). development electronsBy drawing of are ceramic airforced in-between coated to mobilize that these between play plates a rolea set and in of the throughplates removal resulting the of electron material in an electron from field, a the components of air are avalanche electrodes,(Figurebombarded 3). enabled By with drawing the electrons, use ofair insulating in-between causing layers these the that allowfracturingplates andcontaminated throughand/or activationthe surface electron by plasmaof field, CO cleaning.the, H componentsO, Oxidation O , and (theof Nair .are The result is a ‘cold’ or for stable discharges to occur. The result is the generation addition of oxygen to a compound to2 form2 an oxide)2 of the 2 bombarded‘non-thermal’ with electrons, plasmacausing fieldthe fracturing where most and/or of activationthe input ofenergy CO2, isH 2channeledO, O2, and toN2 the. The electron result is component a ‘cold’ or of the plasma while ‘non-thermal’of atmospheric plasma field pressure where plasmas most thatof theform input in the energy absence is channeledcontaminant to theis the electron most common component mechanism. of the Reduction plasma while ofthe inert resulting gases or a vacuum.ions and neutral components remainof theat or contaminant near room (the temperature. removal of oxygen) In addition, is another highly energetic metastable the resulting ions and neutral components remain at or near room temperature. In addition, highly energetic metastable atomic species are generated. These species havepossible the appropriate route. Electron- number and ion- of induced electrons, decomposition but are displaced to higher atomic speciesThe inductionare generated. of atmospheric These species plasma have begins the with appropriate the processes number are of two electrons, other reaction but areroutes. displaced to higher shells and exhibitcapturingshells andhigh of freeexhibit reactivity electrons high and to reactivity chargedsome selectivity dielectric and some plates.in their selectivity reaction incapabilities. their reaction The plasma capabilities. ions and The metastable plasma ions and metastable atomic speciesThroughatomic react changes species rapidly in react polarity,with biologicalsrapidly these electrons with and biologicalsare organic forced solvents to andCommon organicto facilitate to solvents all their processes breakdownto isfacilitate the fact and that their eventual electron-driven breakdown removal. and eventual removal. mobilize between a set of plates resulting in an electron dissociation and ionization of the plasma constituents are avalanche (Figure 2). critical in the formation of reactive radicals and ions. In laymen’s terms, the various species and mechanisms of By drawing air in-between these plates and through the action provide for both electrophilic and nucleophilic attack of organics at the molecular level (Figure 3).

Figure 3. Generation of Atmospheric Plasma and the Figure 4. Electron- and Ion-Driven Constituents Provide Multiple Subsequent Ionization/Activation of Room Air Opportunities for Oxidation and Reduction Reactions via Electrophilic Figure 3. Generation of Atmospheric Plasma and the Figure 3: Electron- andFigure ion-driven 4. Electron-constituents and provide Ion-Driven multiple Constituents Provide Multiple Figure 2: Generation of atmospheric plasma and the subsequent andopportunities Nucleophilic for Attack oxidation Against andDeoxyribonucleic reduction reactions Acid as an via Example ionization/activationSubsequent Ionization/Activation of room air. of Room Air electrophilic and nucleophilicOpportunities attack againstfor Oxidation Deoxyribonucleic and Reduction Reactions via Electrophilic Acid. and Nucleophilic Attack Against Deoxyribonucleic Acid as an Example

electron ﬁeld, the components of air are bombarded with Cold Plasma in the In general,electrons, there are causing four prototypical the fracturing and/orreaction activation mechanisms of CO2, that Automatedplay a role Laboratoryin the removal of material from a contaminated surface. OxidationH2O, O2, and(the N addition2. The result of oxygenis a ‘cold’ to or a compound ‘non-thermal’ to form an oxide) of the contaminant is the most common At present, most analytical methods used in the mechanism.plasmaIn Reduction general, ﬁeld where there of the most are contaminant of four the input prototypical energy (the isremoval channeled reaction of oxygen) mechanisms is another that possible play aroute. role in Electron- the removal and ion- of material from a contaminated to the electron component of the plasma while the resulting biopharmaceutical laboratory require at least one liquid induced decomposition processes are two other reaction routes. Common to all processes is the fact that electron-driven ionssurface. and neutralOxidation components (the remainaddition at orof nearoxygen room to a compoundtransfer operation, to form and nearly an oxide) 100% of of those the using contaminant viable is the most common dissociationtemperature.mechanism. and ionization Reduction of the plasma of theconstituents contaminant are critical (thecells inremoval the or formation intact of oxygen) organisms of reactive is call another for radicals the possible use and of ions. sterile, route. In laymen’s Electron- and ion- terms, the variousinduced species decomposition and mechanisms processes of action are twoprovide other for reactiondisposableboth electrophilic routes. pipette tips Commonand in nucleophilic order to to minimize all processesattack effects of organics is of the fact that electron-driven at the molecularIn addition, level highly (Figure energetic 4). metastable atomic species are cross-contamination. generated.duringdissociation and the ionization process, These of speciesthe plasma have theconstituents are critical in the formation of reactive radicals and ions. In laymen’s appropriateterms, the number various of species electrons, and but mechanisms are displaced to of actionExhaustive provide solvent-based for both electrophilic washing procedures and nucleophilic using attack of organics higherat the shellsmolecular and exhibit level high(Figure reactivity 4). and some organic or caustic materials have been the only alternative selectivity in their reaction capabilities. to the use of disposable pipette tips until IonField Systems

2 www.ionﬁeldsystems.com Cold Plasma in the Automated Laboratory At present, most analytical methods used in the biopharmaceutical laboratory require at least one liquid transfer operation, and nearly 100% of those using viable cells or intact organisms call for the use of sterile, disposable pipet tips in order to minimize effects of cross-contamination. Exhaustive solvent-based washing procedures using organic or caustic materials have been the only alternative to the use of disposable pipet tips until IonField Sys- tems, in 2006, introduced cold plasma technology to the lab as a safe, reliable and practical cleaning method through the TipCharger System.

Conclusions Safe Since the arc-free dielectric barrier discharge plasma is Station’s cold-plasma field promotes the nearly produced by the TipCharger System at near room tempera- instantaneous conversion, volatilization and ultimate ture, it draws very little power. The induced plasma field removal of potential chemical or biological contami- is fully enclosed within a self-cleaning, inert chamber. nants from pipetting surfaces. Furthermore, these And all by-products of the cleaning process are effectively species are readily pipetted in and out of a pipet when trapped first by the TipCharger’s internal filtration system, the opening of the pipet is within the plasma field and secondly are vented to an external exhaust system. (Figures 5 and 6). This technique will actively Reliable cleanse the inner, wetted surfaces of liquid transfer With essentially no moving or serviceable parts, the devices such as cannulae, pin tools and disposable TipCharger Cleaning Station provides a robust pipet tips. And because plasma will flow easily solution for cleaning and sterilizing liquid transfer devices. into all microscopic surface imperfections and The unique combination of oxidizing free radicals and cavities, the process reduces tip-to-tip variations metastable atomic species formed within the Cleaning due to random surface properties.

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introduced cold plasma technology to the lab as a safe, reliable and practical cleaning method using the TipCharger system.

Summary

Safe Since the arc-free dielectric barrier discharge plasma is producedCold by Plasma the TipCharger in the Automated at near Laboratory room temperature, it draws veryAt present, little power.most analytical The induced methods usedplasma in the ﬁeld biopharmaceutical is fully laboratory require at least one liquid transfer operation, and nearly 100% of those using viable cells or intact organisms call for the use of sterile, disposable enclosed within a self-cleaning, inert chamber. All by- Figurepipet 5. Plasmatips in order Cleaning to minimize Action In effects and Around of cross-contamination. A Pipet Tip ExhaustiveFigure solvent-based 6. Artist’s washing Drawing procedures of Plasma Cleaning Action While Figure 5. Artist’s drawing of plasma cleaning action while tips productsusing of theorganic cleaning or caustic process materials are have effectively been the trappedonly alternative by to the Tipsuse ofAre disposable Inserted intopipet TipCharger tips until CleaningIonField SysStation- the TipCharger’stems, in 2006, internal introduced ﬁltration cold plasma system, technology and vented to the lab to as a safe,are reliable inserted and into practical the TipCharger. cleaning method through an externalthe TipChargerexhaust system. System.

tools and disposable pipette tips. Plasma will flow easily Conclusions into all microscopic surface imperfections and cavities, ReliableSafe reducing tip-to-tip variations due to random surface Since the arc-free dielectric barrier discharge plasma is Station’s cold-plasma field promotes the nearly With essentiallyproduced by the no TipCharger moving System or serviceable at near room parts, tempera the- instantaneousproperties. conversion, volatilization and ultimate TipChargerture, it cleaning draws very station little power. provides The ainduced robust plasma solution field for removal of potential chemical or biological contami- cleaningThe TipChargeris plasticfully System enclosed is disposable, protected within by multiple aand self-cleaning,patents metal and patents or inertpending.ceramic chamber. Copyright tips. 2009 The All Rights nants Reserved. from pipetting surfaces. Furthermore, these unique And combination all by-products of of the oxidizing cleaning process free are radicals effectively and speciesPractical are readily pipetted in and out of a pipet when trapped first by the TipCharger’s internal filtration system, the opening of the pipet is within the plasma field metastableand secondly atomic are species vented to formed an external within exhaust the system. cleaning (FiguresBy 5 cleaning and 6). This pipette technique tips on-linewill actively and in-process, integration station’s cold-plasma field promotes the nearly Reliable cleanseof the the inner, TipCharger wetted surfaces offers of liquid multipletransfer benefits to the devices such as cannulae, pin tools and disposable instantaneousWith essentially conversion, no moving volatilization or serviceable parts, and the ultimate automation process. Throughput is significantly increased, removalTipCharger of potential Cleaning chemical Station or provides biological a robust contaminants pipet tips. And because plasma will flow easily into allwhich microscopic is an surface important imperfections consideration and for labs operating at from pipettingsolution surfaces.for cleaning and sterilizing liquid transfer devices. The unique combination of oxidizing free radicals and cavities,high the volumesprocess reduces and tip-to-tip for business variations models based on per-test metastable atomic species formed within the Cleaning due topricing. random surface Direct properties. and indirect costs for tip and solvent The metastable species are readily pipetted in and out of a purchase, storage, use and disposal are dramatically pipette when the opening of the pipette tip is within the decreased when using the TipCharger. Plasma cleaning plasma field (Figures 4 and 5). does not simply dilute contaminants, but removes them at a molecular level, with the important benefit of eliminating contamination and carryover and improving overall process results.

In summary, integrating cold plasma cleaning technology within automated laboratories offers an opportunity to deliver better science as well as increase the efﬁciency and productivity of liquid handling processes.

Figure 5. Plasma Cleaning Action In and Around A Pipet Tip Figure 6. Artist’s Drawing of Plasma Cleaning Action While Tips Are Inserted into TipCharger Cleaning Station

Figure 4 Plasma Cleaning Action In and Around A Pipet Tip

This technique will actively cleanse the inner, wetted surfaces of liquid transfer devices such as cannulae, pin

3 www.ionﬁeldsystems.com Integrating the TipCharger into Automated Assays About the TipCharger™ Provides the Following Beneﬁts:

Confidence TipCharger cleans better than any other washing The TipCharger Cleaning System technology - in most applications the TipCharger will replaces existing wash stations and clean to background, so there is no difference between easily integrates with most existing plasma cleaning and a new tip. and new automation platforms. The system utilizes a low temperature, Cost Benefit TipCharger can save up to 98% on the cost of the atmospheric pressure plasma disposable tips and extends the life of fixed tips. process that cleans metal and plastic pipette tips and pin tools. Speed Incorporating the TipCharger System can result in a Treated surfaces are clean, dry and time savings of 10-30 seconds for every microplate have uniform surface properties. processed or rack of tips cleaned. The TipCharger cleaning process Convenience Clear away the clutter and save time: Integrating the reduces the incidence of micro- TipCharger System eliminates the need to store cases bubble formation and other random of new pipette tips and dispose of racks of hazardous surface effects that degrade liquid used tips. handling precision and accuracy, even with new disposable tips.

IonField Systems’ TipCharger improves the reproducibility of process results, shortens automation cycle times, reduces the number of lost runs, and eliminates environmental waste and liquid handling disposables. The overall result is increased conﬁdence in results and a more effective and productive laboratory operation.

TipCharger Plasma Cleaning Stations Available in 8, 96 and 384 channel versions About IonField Systems™

IonField Systems is an advanced USA and ROW Europe and Middle East technology company focused on the IonField Systems IonField Systems Limited development of state-of-the-art 3411 Silverside Road 7 Paynes Park, Hitchin pipette tip cleaning for life science Hanby Building Hertfordshire, SG5 1EH, laboratory research applications. Wilmington, DE 19810 USA UK IonField Systems provides on-site Call: +1.302.442.6901 Call: +44 (0) 1462 429 727 technical support services to assist Fax: +1.302.792.7407 Fax: +44 (0) 1462 429 798 laboratories in rapidly integrating Email: info@ionfieldsystems.com Email: info@ionfieldsystems.com the system into day-to-day www.ionfieldsystems.com www.ionfieldsystems.com operations.