Name: bridging the bio-electronic interface with biofabrication
Uploaded: 2012-06-06T13:00:00
Duration: 16 min 38 s
Description: Watch this Scientific Journal Video about Bridging the Bio-Electronic Interface with Biofabrication at JoVE.com

We acknowledge funding from DTRA for support of this manuscript and from ONR, DTRA, and NSF for partial support of underlying research.

1. Alginat elektroaflejring <ol><li> Tilslut en strømforsyning til de brugerdefinerede fabrikerede elektroderne via patchkabler med krokodillenæb. En indiumtinoxid (ITO) omfattede objektglas vil fungere som anode (arbejdselektroden) og platinfolie vil tjene som katode (modelektrode). Placere elektroderne, så ITO overflade, der skal funktionaliseres imod modelektroden og er anbragt enten lodret dyppes i en opløsning eller horisontalt, således at aflejringen opløsningen indeholdt på overfladen. </li><li> Fremstilling af en alginat aflejring opløsning ved blanding af 1% alginat og 0,5% CaCO 3 (efter vægt) i destilleret vand og derefter autoklavering af opløsningen. Det anbefales, at kontinuerligt omrøre opløsningen, når den ikke er i brug. </li><li> Nedsænkes begge elektroder i belægningsopløsning. Den anvendte alginat kan fluorescensmærket med fluorescerende (Invitrogen), som pr Cheng et al. 14, for at tillade fluorescence billeddannelse af den resulterende film. </li><li> Anvende en konstant strømtæthed (3 A / m 2) i 2 minutter; spænding vil forskyde i området af 2-3 V. </li><li> Afbryde elektrode og fjerne ikke-deponeret opløsning. Forsigtigt skylles filmen med NaCl (0,145 M) for at fjerne overflødigt alginat. </li><li> Inkuber filmen kortvarigt (~ 1 min) i CaCl2 (0,1 M) for at styrke gelen. Skylning med NaCl (0,145 M) og inkuber i en ønsket opløsning suppleret med CaCl2 (1 mM). </li><li> Billedet med et fluorescens mikroskop (fig. 1B). </li></ol> 2. Samaflejring af Kommunikation Cellepopulationer i alginat <ol><li> Et signal-afsender cellekultur (W3110 vægt E. coli), dyrket i LB-medier, og et signal-modtager cellekultur (MDAI2 + pCT6-lsrR - ampr + pET-dsRed - KanR), dyrket i LB medium + 50 ug / ml af kanamycin og ampicillin, skal dyrkes natten over ennd re-inokuleret i vækst til en optisk densitet (ved 600 nm) af en. Justere optiske densitet af modtageren cellekulturen til 0,4-0,6 med LB før brug. </li><li> Fremstilling af en aflejring opløsning af 2% alginat og 1% CaCO 3, og blandes med hver celle kultur i et forhold på 1:1 til en slutkoncentration på 1% alginat, 0,5% CaCO 3, med cellerne fortyndet til en densitet på omkring halvdelen dyrkning densitet. </li><li> Anvende et objektglas mønstret med to ITO elektroder er indeholdt i en polydimethylsiloxan (PDMS) og (fremstillet ifølge Sylgard instruktioner og skæres til den ønskede størrelse) og en platin-modelektrode. Tilslut en ITO elektrode til en strømforsyning med platin som beskrevet i procedure 1 (Alginatbandager elektroaflejring). </li><li> Elektroderne dyppes ned i en belægningsopløsning indeholdende modtageren celler. Indstille strømforsyningen til en konstant strøm ved en densitet på 3 A / m 2, hvor overfladearealet dimension er defineret ved en enkelt elektrode, på hvilken deposition vil forekomme. </li><li> Anvender de nuværende i 2 minutter for at tillade samaflejring af cellerne i alginatmatrix. </li><li> Skylles filmen som beskrevet i trin 1.5. </li><li> Skifte den anodiske forbindelsen til det andet, tilgrænsende, ITO-elektrode. </li><li> Gentage aflejring procedure (trin 2.4 - 2.7), men denne gang indføres opløsningen indeholdende afsender celler. </li><li> Inkuber i 2-elektroden chip indeholdende samaflejrede celler og calcium-alginat natten over ved 37 ° C i phosphatbufret saltvand (PBS) suppleret med 10% LB-medier og 1 mM CaCl2. </li><li> Efter inkubering billede ved hjælp af et fluorescensmikroskop (figur 2B). </li></ol> 3. Chitosan elektroaflejring <ol><li> Tilslut strømforsyningen til elektroderne via krokodillenæb. En guld belagt siliciumchip fungerer som katode (arbejdende elektrode), og en platinfolie vil tjene som anode (modelektrode). Placer guldelektroden overflade, så thpå den imod modelektroden og begge er placeret enten vertikalt at dyppes i en opløsning eller horisontalt, således at aflejringen opløsningen indeholdt på overfladen. </li><li> Forbered en chitosanopløsning ved at blande chitosan flager i vandet og langsomt tilsætning af 2 M HCl til at opløse polysaccharider (endelige pH 5,6), og sørg for at følge proceduren beskrevet af Meyer et al. 15. </li><li> Placere elektroderne til en chitosan-opløsning (0,8%), fuldstændigt neddyppet det ønskede område for aflejring. Chitosan anvendes, kan fluorescensmærket med 5 - (og-6)-carboxyrhodamin 6G succinimidylester (Invitrogen), som pr Wu et al 8, til at afbilde galvanisk filmen ved fluorescensmikroskopi.. </li><li> Anvende en konstant strømtæthed (4 A / m 2) i 2 minutter. Spændingen vil forskyde i området 2-3 V. Beregn strømtætheden som funktion af guld overfladeareal af den arbejdende elektrode udsættes for depositioN opløsning. </li><li> Skyl elektroden med DI-vand for at fjerne overflødigt chitosan. Chippen kan opbevares i vand eller PBS (10 mM, pH 7,0). </li><li> Billedet med et fluorescens mikroskop (fig. 3C). </li></ol> 4. Elektrokemisk Transduktion med en funktionaliseret chitosanfilm <ol><li> Codeposit chitosan og glucoseoxidase (GOx) fra en opløsning (1% chitosan, 680 U / ml GOx, pH 5,6) ved en strømtæthed på 4 A / m 2 på en mønstret elektrode ifølge Procedure 3 (Chitosan elektroaflejring). En chitosanfilm indesluttet i GOx vil blive genereret. </li><li> Fastgøre den behandlede elektroden til en tre-elektrodesystem som arbejdselektroden, en platintråd som modelektrode og Ag / AgCI som referenceelektroden, som beskrevet i figur 4A. </li><li> Elektroderne dyppes ned i en phosphatpufferopløsning (0,1 M, pH 7,0) indeholdende NaCl (0,1 M). </li><li> Elektrokemisk konjugat proteinet til chitosanfilm ved at anvende en konstant spænding (0,9 V) til 60s hjælp kronoamperometri 10. </li><li> Placere chip i phosphatpuffer (0,1 M, pH 7,0) og vaskes i 10 minutter på en orbitalryster at fjerne eventuelle uomsatte NaCI og ukonjugeret GOx. </li><li> Igen fastgøre de tre-elektrode som beskrevet i trin 4.2 og nedsænkes i en opløsning af 5 mM glucose. Ved hjælp af cyklisk voltammetri, potentialet feje i en positiv retning til 0,7 V. anvendes en kontrolfilm, der ikke indeholder glucoseoxidase som en sammenligning for mængden af oxidation ses i sweep (figur 4B). </li><li> Fjern elektroderne fra glucoseopløsning og skylles med phosphatpuffer (0,1 M, pH 7,0) og derefter placere elektroderne i et 10 ml bægerglas indeholdende 8 ml phosphatpuffer (0,1 M, pH 7,0). Forspænde GOx-funktionaliserede chip til 0,6 V for at tjene som arbejdselektroden (figur 4C). </li><li> Tilsættes portioner af glucose til bufferen (hver alikvot forøger glucosekoncentrationen ved4 mM). </li><li> Generere en standardkurve mellem steady state strøm og glucosekoncentrationen i GOx-funktionaliserede chitosanfilm. </li></ol> 5. Protein Funktionalisering Brug Enzymatisk Assembly <ol><li> Anvende et objektglas mønstret med en tilstødende guld og ITO-elektrode er indeholdt i en PDMS brønd. Forspænde guldelektrode med en katodisk potentiale til electrodeposit chitosan som vist tidligere. Skylles filmen kort i DI-vand og derefter PBS med pipette. </li><li> Tilsættes en opløsning af 3 uM blå fluorescens-mærkede &quot;AI-2 Synthase&quot; 16 (ved hjælp af en DyLight labelling kit) + 100 U / ml Tyrosinase i PBS. </li><li> Inkuberes i 1 time ved stuetemperatur, skylles filmen med PBS. </li><li> Anvende en anodisk potentiale til ITO-elektrode til codeposit en alginat belægningsopløsning indeholdende modtager celler (som fremstillet i trin 2.1-2.2). Følg trin 2,3-2,6 af Metode 2 (samaflejring af cellepopulationer i alginat). </li><li> For at genereredet transmitterede signal (AI-2) enzymatisk efter skylning filmene tilsættes en opløsning af 500 uM S-adenosyl homocystein (SAH) i PBS suppleret med 10% LB medium og 1 mM CaCl2. Dække elektroderne for at forhindre fordampning af opløsningen og inkuberes natten over ved 37 ° C. Dette vil muliggøre en modtager celle respons ved at generere et rødt fluorescerende protein (dsRed). </li><li> Hosliggende elektroder kan afbildes med fluorescensmikroskopi ved at justere de filtre til at indfange den blå fluorescens af AI-2-syntase og rød fluorescens udtrykt ved de samaflejrede modtageren celler (figur 5B). </li></ol> 6. Repræsentative resultater Pålægges elektriske signaler kan skabe lokaliserede mikromiljøer (f.eks felter og gradienter) nær en elektrodes overflade, og disse stimuli kan udløse selvsamling af polysaccharider, såsom alginat, og chitosan at deponere en hydrogel film på elektrodeoverfladen. Fordi thans sol-gel-overgang forekommer ved elektrodeoverfladen, er den resulterende film electroaddressed med dens geometri matchende elektroden mønster (figur 1B, 3C). Biokompatible film, såsom alginat og chitosan tilvejebringe overflader, kan funktionaliseres med biologiske komponenter. Ved hjælp af alginat er unikke cellepopulationer er samaflejrede på forskellige adresser. Bevis for deres electroaddressment ses på samspillet mellem afsender og modtager cellepopulation. Molekylets autoinducer-2 (AI-2) diffunderer fra afsenderen cellerne og optages af modtagerenhederne celler, hvilket resulterer i ekspression af dsRed røde fluorescerende protein (figur 2A). I figur 2B er rød fluorescens kun observeret ved elektroden, hvor modtagerne er rettet. Amingrupperne stede på chitosan give det pH reaktionsevne kræves til elektroafsætning samt en overflade der er egnet til funktionalisering. Vianvendes disse unikke egenskaber ved elektrokemisk at konjugere biosensorer enzymet glucoseoxidase (GOx) til galvanisk chitosan film. Dette enzym derefter tilvejebringer evnen til påvisning af glucose gennem en enzymatisk reaktion (figur 4A) fremstilling hydrogenperoxid, som derefter kan elektrokemisk oxideret til frembringelse af en udgangsstrøm. På denne måde kan et kemisk signal transduceret til elektrisk. Figur 4B viser, at film, hvor GOx var elektrokemisk konjugeret frembringe en stærk anodisk signal i nærvær af glucose i modsætning til de film, der ikke indeholder GOx. Disse resultater indikerer GOx kan samles på en aflejret chitosan film og bibeholder katalytisk aktivitet. Endvidere Figur 4C viser en trinvis stigning i anodisk strøm produceret som reaktion på stigende glucosekoncentrationer. Standardkurven også til stede i Figur 4C viser, at trinnet-øges fortsatte i en næsten lineær FAShion afhængig af mængden af ​​glucose tilsat. Disse resultater viser, at enzymet bevarer også dens følsomhed over for stigende glucosekoncentrationer ved konjugation til chitosanfilm. Den nedre grænse for detektion blev ikke undersøgt her, som det tidligere er blevet karakteriseret for dette system i arbejdet Meyer et al. al. Vi har også påvist kovalent immobilisering af et enzym af interesse, konstrueret til at indeholde en brugerdefineret penta-tyrosin tag, at chitosan med en enzymatisk kontrolleret måde. Specifikt er denne proces medieret af enzymet tyrosinase. Som afbildet i ordningen i figur 5A (øvre), et enzym, AI-2 Synthase indbefatter en penta-tyrosin tag. Tyrosinase virker på tyrosin tag, oxiderende resterne har phenolgrupper til O-quinoner, som derefter kovalent binder til chitosan har aminer. Tegn på chitosanfilm funktionalisering med AI-2 syntase af tyrosinase samling observeres i figur 5B </stRong&gt;, hvor AI-2 syntase er fluorescensmærket blå. Idet AI-2 Synthase genererer AI-2 fra substratet S-adenosyl homocystein (SAH) på samme måde som afsenderen celler, dets nærhed til samaflejrede modtager celler i nærvær af SAH også forårsager modtagerenhederne cellerne fluorescens reagerer ved at udtrykke dsRed (Figur 5A (lavere)). Rød fluorescens af modtageren celler (figur 5B) igen demonstrerer interaktion mellem adresser på grund af diffusion af AI-2 fra den ene til den anden, og yderligere viser, at enzymer immobiliseret til chitosanet bevarer aktivitet gang kovalent bundet. <img alt="Figur 1" src="/files/ftp_upload/4231/4231fig1.jpg" /> Figur 1. Alginat elektroaflejring. (A) Mekanisme af alginat elektroafsætning: Som en elektrode anodisk er forspændt, vandelektrolyse forekommer ved overfladen, hvilket genererer en lokaliseret lavt pH. Calciumcarbonatpartikler reagere med overskudaf protoner, som frigiver calciumkationer partiklerne opløses. I nærvær af alginat polymerkæder, bliver de ioner chelateret i en &quot;eggbox&quot; netværk, der danner et tværbundet hydrogel ved elektroden. Efterhånden som afstanden fra elektroden forøges, har alginat en større tendens til at forblive i opløsning på grund af den reducerede tilstedeværelsen af ​​calciumioner. (B) En L-formet mønster ITO-elektrode blev anvendt til electrodeposit alginat. En PDMS brønd blev fastgjort til elektroden til at indeholde en grøn fluorescens-mærket alginat (1%) og CaCO 3 (0,5%) belægningsopløsning. Efter elektroafsætning i 2 min. ved en strømtæthed på 3A / m 2, blev electroaddressed alginat hydrogelen afbildes ved fluorescensmikroskopi. <img alt="Figur 2" src="/files/ftp_upload/4231/4231fig2.jpg" /> Figur 2. Samaflejring af cellepopulationer. (A) reaktionsskema viser interaktionen mellem to E. coli-stammer: En population producerer autoinduceren-2 (AI-2), En signalmolekyle, og er betegnet &quot;AI-2 afsender.&quot; Den anden befolkning, kaldet &quot;AI-2 modtager,&quot; er en reporter af AI-2, efter modtagelsen af ​​AI-2 ved diffusion fra afsenderen, udtrykker det røde fluorescerende protein dsRed. (B) rød fluorescens billede af elektrodepar med AI-2 afsenderen population samaflejrede med alginat til venstre elektrode og AI-2 modtager population samaflejrede med alginat på højre elektrode. Forstørret billede viser dsRed udtryk for kun de AI-2 modtagere. <img alt="Figur 3" src="/files/ftp_upload/4231/4231fig3.jpg" /> Figur 3. Chitosan elektroaflejring. (A) reaktionsskema viser pH-afhængige elektroafsætning af chitosan. Elektrolyse af vand ved en katodisk forspændt elektrode forårsager en lokal høj pH (vist ved en lokaliseret farveændring af en pH-indikatorfarvestof nær katoden i mikrografi), som stimulerer sol-gel-overgang af chitosan i denne region. (B) aminer til stede på chitosan gav it pH-reagerende egenskaber. Over en pH-værdi på 6,3 (pKa for chitosan) aminerne deprotoneret, letter en overgang fra den protonerede opløselig form til uopløselige gelform. (C) et mønstret guldelektrode blev anvendt til electrodeposit chitosan. Elektroden, der er forbundet katodisk strømforsyningen, blev nedsænket i en grøn fluorescens-mærket chitosan (0,8%) belægningsopløsning. Efter elektroafsætning i 2 min. ved en strømtæthed på 4 A / m 2 blev electroaddressed chitosanfilm afbildes ved fluorescensmikroskopi. <img alt="Figur 4" src="/files/ftp_upload/4231/4231fig4.jpg" /> Figur 4. Elektrokemisk transduktion med en funktionaliseret chitosanfilm. (A) Skematisk viser opbygningen af ​​et tre-elektrodesystem. Funktionaliseret chitosan film tjener som arbejdselektroden, en platintråd som modelektrode og Ag / AgCI som referenceelektroden. Elektrokemisk transduktion af glucose fortsætter gennem the enzymatiske og elektrokemiske reaktioner vist, hvor produceret hydrogenperoxid kan være oxideret, og detekteres ved arbejdselektroden. (B) Cyclisk voltammagram (CV) på elektroden med en chitosan film indeholdende elektrokemisk konjugeret glucoseoxidase (GOx) udviser en stærk anodisk signal i en 5 mM glucose-opløsning. En film ikke indeholder GOx tjente som en kontrol og viste ingen signal i den samme opløsning. (C) En standardkurve mellem anodisk strøm og glucosekoncentrationen viser en næsten lineær relation (hver alikvot forøget glucosekoncentrationen ved 4 mM og øgede også den aktuelle amplitude i den indsatte grafen i en trinvis måde). <img alt="Figur 5" src="/files/ftp_upload/4231/4231fig5.jpg" /> Figur 5. Protein funktionalisering med enzymatisk samling. (A, øvre) reaktionsskema viser tyrosin-tagged &quot;AI-2 Synthase&quot; er covalent bundet til en chitosan film af tyrosinase samling. De tyrosinrester bliver oxidteriseret til O-quinoner med tyrosinase virkning, og kan reagere med amingrupper på chitosan filmen under dannelse af en kovalent binding. (A, lavere) AI-2 Synthase genererer AI-2 fra et substrat (SAH); modtageren cellerne rapporterer den genererede AI-2 ved dsRed fluorescens ekspression. (B) fluorescensbilleder, der udviser en chitosan film på guld, funktionaliseret med blå-mærket AI-2-syntase. Hosliggende er AI-2 modtager celler samaflejrede med alginat på ITO. Efter tilsætning af den enzymatiske substrat til brønden og inkubationen udtrykke AI-2 modtager celler dsRed.

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan:+an+integrative+biomaterial+for+lab-on-a-chip+devices.">Chitosan: an integrative biomaterial for lab-on-a-chip devices.</a> Lab Chip. 10, 3026-3042 (2010).</li><li>Luo, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+assembly+of+a+metabolic+pathway+enzyme+in+a+pre-packaged+reusable+bioMEMS+device.">Programmable assembly of a metabolic pathway enzyme in a pre-packaged reusable bioMEMS device.</a> Lab Chip. 8, 420-430 (2008).</li><li>Spinks, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+"dual+mode"+actuation+in+chitosan/polyaniline/carbon+nanotube+fibers.">A novel "dual mode" actuation in chitosan/polyaniline/carbon nanotube fibers.</a> Sensor Actuat B-Chem. 121, 616-621 (2007).</li><li>Yi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+assembly+of+genetically+modified+viral+nanotemplates+via+nucleic+acid+hybridization.">Patterned assembly of genetically modified viral nanotemplates via nucleic acid hybridization.</a> Nano letters. 5, 1931-1936 (2005).</li><li>Kim, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox-cycling+and+H2O2+generation+by+fabricated+catecholic+films+in+the+absence+of+enzymes.">Redox-cycling and H2O2 generation by fabricated catecholic films in the absence of enzymes.</a> Biomacromolecules. 12, 880-888 (2011).</li><li>Xie, Y., Xu, B., Gao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+transdermal+delivery+of+model+drug+compounds+by+MEMS+microneedle+array.">Controlled transdermal delivery of model drug compounds by MEMS microneedle array.</a> Nanomedicine. 1, 184-190 (2005).</li><li>Odaci, D., Timur, S., Telefoncu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microbial+biosensor+based+on+bacterial+cells+immobilized+on+chitosan+matrix.">A microbial biosensor based on bacterial cells immobilized on chitosan matrix.</a> Bioelectrochemistry. 75, 77-82 (2009).</li><li>Selimoglu, S. 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Funct. Mater. 20, 1645-1652 (2010).</li><li>Cheong, M., Zhitomirsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrodeposition+of+alginic+acid+and+composite+films.">Electrodeposition of alginic acid and composite films.</a> Colloid Surface A. 328, 73-78 (2008).</li><li>Tsao, C. Y., Hooshangi, S., Wu, H. C., Valdes, J. J., Bentley, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+induction+of+recombinant+proteins+by+minimally+rewiring+native+quorum+sensing+regulon+of+E.+coli.">Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of E. coli.</a> Metab. Eng. 12, 291-297 (2010).</li></ol>

Production and Free Access to this article is sponsored by the United States Defense Threat Reduction Agency.

Our procedures demonstrate the electrodeposition and functionalization of biopolymer films, a process we term biofabrication. Through functionalization with cells and biomolecules we create biological surfaces capable of interacting with each other and the electrode address they are assembled upon. The first step, electrodeposition, takes place through the triggered self-assembly of biopolymers, alginate and chitosan in our studies, in response to an electrical signal. As stated earlier a pH gradient is generated which can be controlled by the current density and deposition time, providing additional control over the film dimensions and properties 6,17. We have found that a variety of current density and deposition time combinations can be used for the electrodes indicated in Table 1. While use of other electrodes is feasible, adjustments to the procedure would be necessary. Compared with other techniques of film formation the process of electrodeposition is simple, rapid and reagentless. There is no need for an extensive repertoire of expensive equipment and laborious preparations. Importantly, the process can withstand minor experimental deviations and can be easily started over if a problem occurs. 
Chitosan is capable of responding to a high cathodic pH gradient due to important functional properties conferred to it by a high content of primary amines. At high pH (greater than its pKa of ~6.3) the amines are deprotonated and chitosan becomes insoluble, allowing for film formation. Following deposition, the films will remain attached to the electrode. However, the ability exists to delaminate them if desired. The films will remain stable as long as the pH of the solution does not drop below the pKa. Acidic solutions protonate the amines and the subsequent electrostatic repulsions swell the gel until it dissolves 18. That is, the assembly/disassembly process is reversible on demand and allows for removal of deposited films and reuse of electrodes. Conveniently, the pH range at which the sol-gel transition takes place is close to that in which most biological components function optimally. This makes the process ideal for the retention of functionality during assembly6.
Alginate film formation is facilitated by the anodic electrolysis of water as well as the presence of calcium carbonate 7. The localized low pH at the anode solubilizes the calcium carbonate leading to the release calcium cations. These ions are chelated by alginate, forming a crosslinked network on the electrode surface. Alginate films are notably reversible by competition for calcium ions from other chelating compounds such as citrate or EDTA, which can be used to dissolve the films, allowing for the re-use of the underlying electrodes. Thus, alginate films are relatively fragile when subjected to physiological conditions because calcium ions are easily scavenged from the gel matrix, weakening its structure and promoting film delamination or redissolution. To overcome this limitation, we have included an incubation step for the film in 1 M CaCl2 to strengthen the gel. Additionally, we recommend that the film's incubation solution (cell media, etc.) be supplemented with CaCl2 at a concentration of 500 &mu;M-3 mM.
The second major procedure is the functionalization of the deposited film with relevant biological components. This can be achieved in two ways, the first being electrochemical conjugation, a strategy that allows for rapid, reagentless assembly of proteins with exceptional spatial control 10. However, functionalization in this manner is limited by the diffusion of Cl- ions through the film to the electrode as well as the diffusion of HOCl, the generated reactive intermediate, back out into solution. The ability of electrochemically active molecules to pass through the film allows for the transduction of chemical and biological signals into easy-to-read electrical signals 15. We have shown tyrosinase-mediated coupling as a second strategy for enzyme functionalization to chitosan, demonstrated by covalently attaching AI-2 Synthase. This strategy allows the functionalization process to be controlled and selective - dependent on a specific reagent, tyrosinase, which acts discriminately on proteins containing a tyrosine tag 9. 
We show the usefulness and biocompatibility of multi-address systems by replicating natural pathways on a chip. First we organized two cell populations (i.e. "senders" and "receivers") at distinct addresses, and showed that they interacted across adjacent electrodes to deliver AI-2 and generate a fluorescence response. This concept has also been demonstrated by Cheng et al. in a microfluidic chip 14. We also mimicked the interaction, but instead used an enzyme to synthesize AI-2 for delivery. In this way, a synthetic intracellular pathway, AI-2 synthesis, was replicated through biofabrication and functioned much as it would in solution.
In both cases, assembly of multiple addresses presents the challenge of avoiding non-specific binding between addresses because each deposition solution must be introduced to the entire electrode array, even though electrodeposition is only intended at one address. Gentle yet thorough washing can remove the majority of residual solution from non-biased electrodes; the use of flow in microfluidic channels may further minimize non-specificity. Particularly for the adjacent biofabrication of chitosan and alginate addresses, we recommend depositing the chitosan film first, following this with biofunctionalization steps, and after this, electrodepositing alginate. Although we have not done so here, we have found that blocking the chitosan film with inert proteins (such as milk, BSA, etc.) greatly diminishes non-specific binding of unwanted molecules to chitosan's aminated surface.
We have found utility in establishing patterned electrodes, often found in bioMEMS devices, as the "blueprints" for a complex arrangement of cells and biomolecules. The uses of electrodeposited chitosan in bioMEMS devices can go well beyond the examples mentioned here 19. Chitosan can be deposited on various microscale geometries - such as in microchannels and on non-planar surfaces 20,15. The films can also be modified with other polymers and a variety of proteins, DNA, nanoparticles, and redox-active molecules for novel properties 21,22,23. In bioMEMS devices, chitosan films have been used for drug delivery, redox and small molecule detection, biocatalysis, and cell studies 20,23,24,25. Similarly, alginate is widely used as a cell-entrapment matrix and has been explored for reversible fluidic containment of cell populations and in-film immunoanalysis 26,27,28. Composite films for tissue engineering applications have been fabricated using alginate electrodeposition, with components such as with hydroxyapatite for orthopedic implants 29. 
In our demonstrations of biofabrication, we have shown both the interactions between biological components and across the bio-electronic interface to be equally applicable; this brings into reach the prospect of integrating all varieties of interactions for sophisticated performance in on-chip signal transmission. Accordingly, biofabrication may facilitate the fabrication of devices with reduced "minimum feature sizes" as a direct follow-on to rapid developments in microfabrication, as often motivated by consumer electronics. That is, next next-generation devices might in fact include labile biological components that offer nature's exquisite assembly and recognition capabilities at even smaller length scales than man-made systems. We envision near-term applications in analytical instrumentation, environmental sensors, and even biocompatible implantable devices.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the component</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Power Supply</td>
 <td>Keithley</td>
 <td>SourceMeter 2400</td>
 </tr>
 <tr>
 <td>Three electrode potentiostat</td>
 <td>CH Instruments</td>
 <td>Potentiostat/Galvanostat 600D </td>
 </tr>
 <tr>
 <td>RE-5B Ag/AgCl Reference Electrode with Flexible Connector </td>
 <td>BASi</td>
 <td>MF-2052</td>
 </tr>
 <tr>
 <td>Gold coated silicon wafer, 500um Si, 12nM Cr, 120nM Au, SiO2 for insulation</td>
 <td> custom fabricated</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Indium Tin oxide coated glass slide, rectangular, 8-12 ohm resist</td>
 <td>Sigma-Aldrich</td>
 <td>578274 </td>
 </tr>
 <tr>
 <td>Platinum sheet/foil (0.002 in)</td>
 <td>Surepure Chemetals</td>
 <td>1897</td>
 </tr>
 <tr>
 <td>Slim Line 2" Alligator Clips </td>
 <td>RadioShack</td>
 <td>270-346 </td>
 </tr>
 <tr>
 <td>Multi-Stacking Banana Plug Patch Cord</td>
 <td>TSElectronic</td>
 <td>B-36-02 B-24-02</td>
 </tr>
 <tr>
 <td>SYLGARD 184 silicone elastomer kit</td>
 <td>Dow Corning</td>
 <td>NC9020938 From Fischer</td>
 </tr>
 <tr>
 <td>Fluorescecence stereomicroscope</td>
 <td>Olympus</td>
 <td>MVX10 MacroView</td>
 </tr>
 <tr>
 <td>cellSens Standard </td>
 <td>Olympus</td>
 <td>version 1.3</td>
 </tr>
 </tbody>
</table>
Table 1. Electrodeposition and fluorescence visualization equipment.
<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Chitosan, medium molecular weight</td>
 <td>Sigma-Aldrich</td>
 <td>448877</td>
 </tr>
 <tr>
 <td>Hydrochloric Acid, ARISTAR. ACS, NF, FCC Grade </td>
 <td>VWR</td>
 <td>BDH3030</td>
 </tr>
 <tr>
 <td>Sodium Hydroxide, Solution. 10.00N</td>
 <td>VWR</td>
 <td>VW3247</td>
 </tr>
 <tr>
 <td>Alginic acid, sodium salt</td>
 <td>Sigma-Aldrich</td>
 <td>180947</td>
 </tr>
 <tr>
 <td>Multifex-MM Precipitated Calcium Carbonate, 70nm particles</td>
 <td>Speciality Minerals Inc.</td>
 <td>100-3630-3</td>
 </tr>
 </tbody>
</table>
Table 2. Chitosan and alginate solution reagents.
<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Calcium chloride, dihydrate</td>
 <td>J.T. Baker</td>
 <td>0504 </td>
 </tr>
 <tr>
 <td>Sodium Chloride, Certified ACS crystalline</td>
 <td>Fischer Scientific</td>
 <td>S271</td>
 </tr>
 <tr>
 <td>Potassium Phosphate Monobasic, anhydrous</td>
 <td>Sigma-Aldrich</td>
 <td>P9791</td>
 </tr>
 <tr>
 <td>Potassium Phosphate Dibasic, anhydrous</td>
 <td>Sigma- Aldrich</td>
 <td>P3786</td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline</td>
 <td>Sigma- 
 Aldrich</td>
 <td>P4417</td>
 </tr>
 </tbody>
</table>
Table 3. Other solution components and buffer reagents.
<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company/Source</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Glucose oxidase from aspergillus niger</td>
 <td>Sigma-Aldrich</td>
 <td>G2133 </td>
 </tr>
 <tr>
 <td>Tyrosinase from mushroom</td>
 <td>Sigma-Aldrich</td>
 <td>T3824</td>
 </tr>
 <tr>
 <td>LB broth, Miller (granulated)</td>
 <td>Fischer Scientific</td>
 <td>BP9723-2</td>
 </tr>
 <tr>
 <td>"AI2-Synthase" (HGLPT)</td>
 <td>Lab stock 16 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>W3110 wildtype cells</td>
 <td>Lab stock 30 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MDAI2 + pCT6-lsrR-ampr + pET-dsRed-kanr cells </td>
 <td>Lab stock 30 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FluoroSpheres: 1&mu;m diameter, Ex/Em: 505/515</td>
 <td>Invitrogen</td>
 <td>F8765</td>
 </tr>
 <tr>
 <td>5-(and-6)-carboxyrhodamine 6G succinimidyl ester, Ex/Em: 525/560</td>
 <td>Invitrogen</td>
 <td>C-6157 </td>
 </tr>
 <tr>
 <td>DyLight antibody labeling kit, 405</td>
 <td>Thermo Scientific</td>
 <td>PI-53020</td>
 </tr>
 </tbody>
</table>
Table 4. Enzymes, cells, and other functionalization reagents.

bridging the bio-electronic interface with biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

Watch this Scientific Journal Video about Bridging the Bio-Electronic Interface with Biofabrication at JoVE.com

Bridging the Bio-Electronic Interface with Biofabrication

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Bridging Bio-elektroniske grænseflade med Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

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