In-Film Bioprocessing and Immunoanalysis withElectroaddressable Stimuli-Responsive PolysaccharidesBy Xiaohua Yang, Eunkyoung Kim, Yi Liu, Xiao-Wen Shi, Gary W. Rubloff,Reza Ghodssi, William E. Bentley, Zeev Pancer, and Gregory F. Payne*1. IntroductionTechnologies that allowed nucleic acids and proteins to be spatiallylocalized and analyzed on-chip enabled remarkable progress inbiosensing, genomics, and proteomics.Analogous efforts are underway to localize,culture, and analyze viable cells on-chip forapplications that range from performingfundamental studies in cell biology tomimicking the multi-organ metabolismof drugs. Three themes appear to beemerging for the on-chip cultivation ofcells. First, hydrogel films are generallypreferred for replicating biological micro-environments and preserving labile biolo-gical functions (e.g., to maintain cellviability).[1–7]Second, fabrication methodsfor patterning films often enlist convenient,spatiotemporally controllable stimuli. Forinstance, printing and photolithographicpatterning employ mechanical and opticalinputs,[6,8–14]while there are growingefforts to use electrical stimuli to performfunctions such as electroaddressing.[15–33]Finally, biological materials and mechan-isms may offer opportunities to ‘‘biofabri-cate’’ functional hydrogel films.[34–41]Forinstance, stimuli-responsive biologicalpolymers form hydrogels in response tomild stimuli, these hydrogel networks canbe reversibly formed/broken, and there isextensive biotechnological experience with biopolymer-basedhydrogels (e.g., gelatin and agarose).Recently, stimuli-responsive polysaccharides have beenobserved to be capable of electrodepositing at electrode surfacesin response to localized electrical signals.[42,43]In most cases, theseFULL PAPERwww.MaterialsViews.comwww.afm-journal.de[*] Prof. G. F. Payne, Dr. X. Yang, Dr. E. Kim, Dr. Y. Liu, Dr. X.-W. Shi,Prof. W. E. BentleyCenter for Biosystems ResearchUniversity of Maryland Biotechnology Institute5115 Plant Sciences BuildingCollege Park, MD 20742 (USA)E-mail: [email protected]. G. W. RubloffDepartment of Materials Science and EngineeringUniversity of MarylandCollege Park, MD 20742 (USA)DOI: 10.1002/adfm.200902092Prof. G. W. Rubloff, Prof. R. GhodssiInstitute for Systems ResearchUniversity of MarylandCollege Park, MD 20742 (USA)Prof. R. GhodssiDepartment of Electrical and Computer EngineeringUniversity of MarylandCollege Park, MD 20742 (USA)Prof. W. E. BentleyFischell Department of BioengineeringUniversity of MarylandCollege Park, MD 20742 (USA)Prof. Z. PancerCenter of Marine BiotechnologyUniversity of Maryland Biotechnology InstituteBaltimore, MD 21202 (USA)Advances in thin-film fabrication are integral to enhancing the power ofmicroelectronics while fabrication methods that allow the integration ofbiological molecules are enabling advances in bioelectronics. A thin-film-fabrication method that further extends the integration of biology withmicroelectronics by allowing living biological sy stems to be assembled,cultured, and analyzed on-chip with the aid of localized electrical signals isdescribed. Specifically, the blending of two stimuli-responsive film-formingpolysaccharides for electroaddressing is reported. The first, alginate, canelectrodeposit by undergoing a localized sol–gel transition in response toelectrode-imposed anodic signals. The second, agaros e, can be co-depositedwith alginate and forms a gel upon a temperature reduction.Electrodeposition of this dual polysaccharide network is observed to be asimple, rapid, and spa tially selective means for assembly. The bioprocessingcapabilities are examined by co-depositing a yeast clone engineered to displaya variable lymphocyte receptor protein on the cell surface. Resultsdemonstrate the in-film expansion and induc tion of this cell population.Analysis of the cells’ surface proteins is achieved by the electrophoreticdelivery of immunoreagents into the film. These results demonstrate a simpleand benign means to electroaddress hydrogel films for in-film bioprocessingand immunoanalysis.Adv. Funct. Mater. 2010,20,1645–1652ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim1645FULL PAPERpolysaccharides electrodeposit in response to electrochemicallyinduced pH gradients that neutralize the polymer. For instance,the aminopolysaccharide chitosan undergoes gel formation at thecathode surface in response to a localized high pH that results inthe conversion of its cationic ammonium groups into neutralamines.[44–46]Similarly, the acidic polysaccharides alginate[47,48]and hyaluronate[49,50]were observed to electrodeposit at the anodesurface in response to a localized low pH. An alternativemechanism for electrodepositing alginate films is illustrated inScheme 1.[51]In this case, insoluble CaCO3is suspended in asolution of sodium alginate. Anodic electrolysis reactions generatea pH gradient that causes the localized solubilization of Ca2þ,which then induces the formation of calcium alginate gels.An important feature of polysaccharide electrodeposition is thatit enables co-deposition—materials dissolved or suspended in thepolysaccharide solution can be incorporated into the electro-deposited films. One of the initial reports of co-deposition involvedthe entrapment the glucose oxidase and gold nanoparticles forbiosensor fabrication[52]and these studies were rapidly extended toother systems and enzymes[53–62]and to the co-deposition of arange of inorganic[21,63](e.g., carbon nanotubes[64–67]) and organicnanoparticles.[68]An alternative goal for co-deposition is togenerate composite surface coatings.[47,69–73]Two previousobservations are particularly relevant to the current study. First,it was recently reported that the stimuli-responsive film-formingpolysaccharide chitosan allowed the co-deposition of a secondpolysaccharide (heparin) that is unable to electrodeposit by itselfbut that confers distinct functions to the deposited film.[74,75]Second, viable bacterial cells were co-deposited in Ca2þ–alginatefilms (using the mechanism of Scheme 1) and these cells could begrown, induced, and released (by dissolving the films with sodiumcitrate that binds Ca2þ).[51]The goal of the work reported here is to extend the capabilities ofCa2þ–alginate film bioprocessing by allowing the film-entrappedcells to be probed with immunoreagents (i.e., antibodies) thatrecognize cell surface proteins. Immunoanalysis of cell surfaceantigens is integral to a variety