Solution Processed Micro- and Nano-Bioarrays for Multiplexed Biosensing

Solution Processed Micro- and Nano-Bioarrays for MultiplexedBiosensingThis Feature article reports on solution dispensing methodologies which enable the realizationof multiplexed arrays at the micro- and nanoscale for relevant biosensing applications such asdrug screening or cellular chips.

Giuseppe Arrabito† and Bruno Pignataro*,‡

†Scuola Superiore di Catania, Via Valdisavoia 9, 95123, Catania, Italy‡Dipartimento di Chimica “S. Cannizzaro”, Universita degli Studi di Palermo, V. le delle Scienze, Parco d’Orleans II, 90128,Palermo, Italy

*S Supporting Information

G. Arrabito

Microarray technologies are nowadays relatively mature,their development starting with the onset of high-

density DNA arrays, then addressing with peptide and proteinmicroarrays, and now prefiguring perspectives in the field ofcellular arrays.1 Such high-throughput analytical tools areassociated with smaller usage of sample volumes, decrease inuse of reagents, rapid analyses, and increased sensitivity since, atthe microscale, the chemical species have a shorter distance todiffuse than in conventional macroscale reaction vessels.Undoubtedly, the great advantage of these devices is theirhighly parallel nature and ability to interrogate hundreds tothousands of different sensing molecules in one experiment.2

The area of bioarray technology fabrication evolves along threedifferent paths: patterning resolution, i.e., the decrease of the

printed feature size on the slide from micro- until nanoscale;multiplexing, i.e., the increase of the number and of the densityof tested biological targets (biomolecules or cells), and finallybiochip design, i.e., the careful development of smart platformsable to collect and compute real time biological information.As to the patterning resolution, the rapid realization and

characterization of micro- until nanoscale architectures with asingle molecule can be routinely achieved by nanolithographymethods.3,4 Accordingly, nanoarray devices5 permit a dramaticdecrease in the cost of the assay since, in comparison to micro-arrays, still a lower volume of reagents is required, responsetime is shorter, and higher sensitivity can be obtained up to thesubfemtomolar level.6

Moreover, multiplexing still constitutes a challenge formodern biological patterning methods. In principle, the higherthe biomolecular complexity of the array, the higher are thequantity and the quality (in term of density) of the attainablebiological information from a single experiment. In biomo-lecular assays, multiplexing is strictly dependent on resolution.As to the complexity of the arrays and the number of featuresincrease, reduction in size becomes more and more important,due to the fact that the area occupied by one array affects theamount of sample that can be used in a chip. In the following,we will consider examples of applications in which bioarraytechnology plays a fundamental role in important fields such as,for instance, biomolecular screening and cellular arrays. As tobiomolecular assays, thanks to the continuous miniaturizationefforts, they can be routinely performed in volumes of a fewmicroliters in high-throughput microtiter plates (i.e., 1536-wellmicroplates) executing more than 105 assays in a single screen,a great advantage with respect to perform the same reactionin conventional milliliter test tubes.7 However, the interesttoward further assay miniaturization, driven by the increase ofbiological interactions and the necessity to save time, sample, aswell as to increase sensitivity motivated the implementation ofrobotic liquid handling techniques able to dispense, in parallel,biological samples in the nanoliter scale. Currently employedrobotic systems suffer from several issues such as high costs,complexity of the instrumental setup, and reliability of the

Published: May 15, 2012

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extracted data.8 Consequently, simplifications of the exper-imental setup, costs reduction of the handling devices, andfurther miniaturization are all highly desiderated.As to single-cells chips, one has to consider the analysis of co-

operative patterning and geometrical effects on individual cellsurfaces. The complex biochemical and morphological scenarioof a cell determines a size requirement, i.e., different functionalproteins should be patterned together below the size of a singlecell. Since it is achieved, the response of the cell to suchmultiproteic subcellular dimensioned surfaces would be usableas a real-time detection of the biological function of thepattern9 into a single-cell.Finally, biochip design represents the last step in the

development of a mature array technology, dealing with liquidautomation and actuation via micro- and nanofluidic systemsalong with the optimization of the signal readout eventually viainterfacing with micro- electronic components and finally signalextraction and processing. A review from Seidel and Niessner(ref 10) reports on the current state-of-the-art automation foranalytical microarrays.

■ METHODS FOR MULTIPLEXINGThere exist several methods to produce patterns of biologicalmaterials on surfaces. However few of these are capable ofmultiplexing in a rapid, high-throughput, and efficient way.In general, biomolecular array fabrication can be categorized

into top-down and bottom-up approaches. While top-downdirectly or indirectly may generate from micro- to nanoscalestructures, bottom-up typically exploits intermolecular inter-actions to realize self-assembled or self-organized orderedstructures. Admittedly, bottom-up self-assembly methods arealso gradually gaining importance in biofabrication since theyresult in low-cost, solution based approaches allowing forprogrammed molecular architectures on 1-D, 2-D, and 3-Dscales.11 However, supramolecular organization attainable from“bottom-up” approaches is often difficult to extend from nano-to mesoscopic length scales or does not permit an accurateplacement of different desiderated structures on addressableregions of a surface. Top-down methods are currently the most-employed in the field of biomolecular array fabrication due tothe possibility to automate and precisely control the patterningprocess and the scaling on a large area.However, by considering multiplexing abilities, conventional

top-down fabrication techniques like photolithography, elec-tron beam lithography, and microcontact printing can patternat nanometer resolution on large areas, but the number ofdifferent materials printable in parallel is limited, realized viaindirect patterning approaches and requires clean rooms andexpensive instrumentation. Multiprotein patterning by suchtechniques has been recently reviewed by Ganesan et al.12

Notably, photolithography affected by the employment ofphotoresist developers and heat treatments might affect theactivity of delicate biomolecules. Great concerns reduce multi-plexing abilities by microcontact printing due to contaminationfrom the stamp material and the unprecise stamp/substratealignment.13 These limitations motivated the developmentof new, original unconventional micro- and nano- top-downfabrication methods with high flexibility and ambient conditionsoperation, as well as lower costs as droplet microdispensingmethods (namely, pin printing, inkjet printing, electrohydrody-namic, and pyroelectrodynamic printing) and nanotip printingtechniques (serial dip pen nanopatterning, 1D and 2D Dip PenNanopatterning, and hollow tip dispensing).

The success of a multiplexed microarray technology is notonly connected to the fabrication methodologies but also to thedevelopment of suitable substrate surfaces for biomoleculeimmobilization and to the detection methods.As far as substrate surfaces are concerned, soft supports

(i.e., polystryrene, nitrocellulose membrane), conventionallyused for biochemical analysis (immunoblot), are typically notcompatible with microarrays. This is due to the scarce bio-molecular density, the high spot spreading, and the low signal/noise ratio. Many groups make use of glass slides due to the lowfluorescence background and the possibility to get high protein-binding capacity by chemical functionalization via aminosilane,polylysine, or aldehydic groups.14 Also, conducting assays onsolid substrates requires biomolecular attachment to its surface.In this sense, several chemical reactions are reported for pep-tide immobilization on surfaces. Peptide arrays are typicallyfabricated through in situ peptides synthesis, are built up in alinear fashion, and can be selectively immobilized by the firstamino acid. Alternatively, peptides can be immobilized usingbioorthogonal thiazolidine ring formation via a glyoxylyl groupreaction with 1,2-amino thiols, 1,3 dipolar cycloaddition ofterminal alkynes with azides, and Diels−Alder productformation of benzoquinone with cyclopentadiene and nativechemical ligation. On the other side, protein immobilizationrequires more careful consideration,15 since it poses severalconsequences on the quality of the analysis influencing theinherent biological activity of the protein immobilized at thesolid surface, the accessibility to the ligands present in solutionand affecting reproducibility, selectivity, and device perform-ance. The immobilization process should maintain proteinactivity and allow the correct orientation and accessibility tobiomolecules for the correct analyte recognition at the solid−liquid interface. In particular, proteins can be immobilized byusing covalent or noncovalent immobilization. Covalentattachment can be executed by employing random nonspecificcross-linking approaches via chemically activated surfaces (e.g.,aldehyde, epoxy) or specific biomolecular affinity taginteractions (e.g., streptavidin−biotin, his-tag−nickel-chelates).The first ones can lead to poor preservation of proteinbiological activity, while the second ones allow proteins tomaintain a correct orientation. In noncovalent interactions,hydrophobic (nitrocellulose, polystryrene) or positivelycharged (polylysine) surfaces are typically employed. Thesesubstrates are currently used in ELISA or Western blotting. Inaddition, by employing polyacrylamide gel pads and agarosethin films, it is possible to generate 3D proteic gel matrixesfeatured with higher capacity in protein immobilization incomparison to a 2D surface.Importantly, when conducting a multiplexed analysis on a

multiprotein sample, different molecules often need to beanalyzed at the same time. Problems can arise when proteinsbind nonspecifically at surfaces. This can be significantlyreduced by employing polymers like polyethyleneglycolbecause of its resistance toward protein adsorption in aqueousmedia.Finally, carbon nanotubes are also currently being developed

as nanoscale building blocks for analytical devices becauseof their excellent mechanical, electrical, and electrochemicalproperties. They can be easily derivatized with differentfunctional groups for covalent attachment of biomolecules inorder to generate high-efficiency biosensing platforms.16

As far as detection methods are concerned, high-qualityassays are required to translate specific multiplex biomolecular

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interaction phenomena into observable and quantitativeparameters. Typically, this is achieved through measurementsof radioactivity, photon absorption, or photon emission. Albeitsuccessfully applied in high-throughput screening, radioactivemethods have become much less popular because of hazards inhandling radioactive materials and the possibility of non-radioactive alternatives. Photon emission is by far the dominantassay methodology due to its ability to deliver speed, accuracy,and sensitivity necessary for successful assays. This is achievedprimarily through fluorescence and chemiluminescence. Whilewith fluorescence the energy needed for producing excitedstates is gained through light absorption, with chemilumines-cence energy results from exothermic chemical reactions. Thisdifference fundamentally affects assay features and performance.Because of the high influx of photons in fluorescent assays,the background is high and sensitivity is lowered. Notably,by chemiluminescence light intensities are lower but thebackground is virtually absent since no photons need to beintroduced in the samples leading to higher sensitivities.However, to date there is a scarce number of chemilumines-cence assays along with a limited development for multiplexing.On the other side, thanks to the high number of differentfluorophores, multiplexing via fluorescence can be much moreeasily achieved allowing multibiomolecular process assays on amicroarray scale. Also, fluorescence methods always requirelabeling strategies which can pose synthetic challenges andmultiple label issues and may exhibit interference. For thisreason, label-free detection techniques (for example, surfaceplasmon resonance (SPR), atomic force microscopy (AFM),nanowires, ellipsometry) are starting to acquire more and moreimportance since they eliminate the need for secondaryreactants. For a review on this topic you may refer to Rayet al.17 Finally, colorimetric detection constitutes a label-freemethod whose use is increasing, especially in clinicalapplications. They are quite attractive because the associatedexperimental setups permit multiplex analysis at relatively lowcost. The principle of detection is based on the formation of asoluble or insoluble colored product leading to measurablespot/substrate optical contrast.In this Feature, we specifically focus on solution-processed

top-down methodologies for the fabrication of biological arrays.We point out their capabilities as multiplexing tools and giveinformation about their automation capabilities. In order to givea broader picture of this field, in the Supporting Information webriefly discuss the latest advancements of photolithography,microcontact printing methods, electrodeposition, scanningelectrochemical microscopy, and modified scanning atomicmicroscope tips for biological patterning in multiplexed format,as well. The presented examples of biomolecular patterning,self-assembled monolayer methodologies, and site specificimmobilization are frequently employed as fundamentalmeans to acquire accurate molecular-scale control of bio-material deposition on surfaces. Together with a description ofthe fabrication process for each different technique, we discussthe multiplex analytical abilities of the array devices providinginformation about the number of different analytes investigatedin parallel, the selectivity, and sensitivity of the bioassaysexecuted in an array format.

■ MULTIPLEXING VIA PIN PRINTINGPin printing is currently among the most diffused patterningtechnique in biological applications. In particular, the parallelprinting with multiple heads to create DNA microarrays is one

of the most widely used methods to fabricate fundamentaltools for genomics18 and protein function determination.19 Theuniformity in spot formation depends on several parameters assample viscosity and pin and substrate surface properties. Spotdimensions depend on the printing speed, on the solutionsurface tension, and on the solution wettability of the substratesurface.20

Two pin printing typologies can be distinguished: solid pinprinting and split pin printing. In solid pin printing, the pin isloaded by the sample solution through immersion. Then,the pin contacts the substrate surface to dispense the sample.Albeit its simplicity, the printing procedure is affected by severaltime-consuming sample reloads needed to realize a singlemicroarray.In split pin printing, pins are loaded with a solution from a

well plate by capillary force action into the pins microchannels(diameter between 10 and 100 μm). Notably, Gosalia andDiamond21 employed split pin printing to realize a chemicalcompound microarray consisting of nanoliter droplets ofglycerol. By means of the addition of successive reagents toeach spot via aerosol deposition, they were able to profile thekinetics of protease screening reactions in single droplets(Figure 1A) reaching subnanomolar detection limits for humanplasmin cleavage of substrates like (CBZ-FR)2-R110. Nielsenet al.22 employed split pin printing to fabricate multiplexedantibodies arrays, having sensitivities comparable to standardELISA methods, and being able to monitor the activation,uptake, and signaling of ErbB receptor tyrosine kinases.They integrated these arrays with 96-well microtiter platesin order to identify and analyze small molecule inhibitors ofsignal transduction processes with high speed and precision(Figure 1B). These array could quantify purified antigens over a1 000-fold concentration range and down to 1 ng/mL.Duburcq et al.23 reported on the pin printing fabrication

of a peptide−protein microarray on glass slides onto whichglyoxylyl peptides were immobilized by site-specific α-oxosemicarbazone ligation and proteins by physisorption. By theemployment of an immunofluorescence assay, the microarraypermitted to achieve sensitive and specific serodetection ofmultiple antibodies directed against pathogens as different ashepatitis C virus, hepatitis B virus, human immunodeficiencyvirus, Epstein−Barr virus, and syphilis. Such peptide−proteinmicroarrays showed good specificities and sensitivities(0.01 mg/mL) for antibody detection. More recently, Lin et al.24

developed a reliable multiplexed peptide microarray-basedimmunoassay (peptide covered the primary sequences ofαS1-casein, αS2-casein, β-casein, κ-casein, β-lactoglobulin)fabricated by pin printing for large-scale epitope mapping offood allergens in milk. The epitopes identified via fluorescencedetection by the peptide microarray were consistent with thoseidentified by conventional analytical methods. By employingreplicate arrays of an immunolabeled serum pool, reproduci-bility was evaluated, while specificity and sensitivity weredetermined by employing serial dilution of the serum pool anda peptide inhibition assay.Notwithstanding pin printing is the most known array

fabrication methodology, it is a tedious and time-consumingprocess. Indeed, after several prints, pin tends to degrade.When a pin is used to print multiple solutions, it must bewashed and cleaned to avoid cross contamination. Indeed,because of the impact at contact, the pin structure deformation,and the clogging from contaminants, pin-based arrayingis prone to suffer from slide-to-slide inconsistency.20



Also, because of the required physical contact, it could bepossible to damage either the substrate or the deposited pro-teins.

■ INKJET PRINTING FOR MULTIPLE SOLUTIONSDISPENSING

Inkjet printing is a soft and robust micropatterning techniquedue to its rapidity and low cost nature, not requiring the use ofmasks, stamps, or other costly and time-consuming conven-tional processing equipment.25 It permits the dispensing offluid droplets by generating a pressure pulse within a confinedliquid, causing its ejection from a micrometric orifice (nozzle),the drop volume being affected by the nozzle size and typically

in the picoliter to the nanoliter range. It has the great advantageto allow precise and controlled parallel deposition of smallvolumes (typically from the picoliter to the nanoliter range) ofmultiple biological materials using independent jets (i.e.,independent controlled microchannels) on almost any possibletype of substrates (solid, gel, liquid surfaces) being a contactlesstool. Moreover, the hardware integration with automatedtranslation stages enables precise pattern placement andregistration for preparing multilayered patterns with differentbiomaterials. In contrast to thermal inkjet based on resistive orlaser heaters, piezoelectric systems employ piezoelectricactuators, such as lead zirconate titanate (PZT), to dispensefluids, thereby reducing possible damage to the biomaterial.26

Figure 1. Multiplexed biochemical assays by pin- and inkjet printing methods. (A) Profiling of protease activity with three fluorogenic substrates inmicroarrays fabricated by pin-printing. The blue fluorescence resulted from the cleavage of a thrombin substrate, and the red and green fluorescenceresulted from the cleavage of the BODIPY TR-X and FL casein substrates, respectively, by chymotrypsin. Spots with no color had no substrate.Reprinted with permission from ref 21. Copyright 2003 National Academy of Sciences, U.S.A. The inset shows the complete compartmentalizationof each reaction center. (B) On the top, multiple inhibition of EGFR and ErbB2 phosphorylation in cells treated with the small molecule PD 153035.At the bottom, a dual color ratiometric ratio graph showing the inhibition of the two proteins. The ratio of Cy3 to Cy5 fluorescence at each spot isproportional to the fraction of receptor that is phosphorylated at Y1068 (EGFR) or Y1248 (ErbB2). Reprinted with permission from ref 21.Copyright 2003 National Academy of Sciences, U.S.A. (C) On the left, the scheme of a two step multiplexed patterning via inkjet printing. Reprintedwith permission from ref 26. Copyright 2008 Elsevier. A mixed BAT/PEG thiol solution is first spotted on a gold-coated microscope slide. Astreptavidin layer is added and then two biotinylated proteins (b-HRP and b-BSA) are deposited in a second step and then antibodies are introducedsequentially. On the right, SPR image with a 3 × 3 patterned BAT/PEG array with streptavidin and biotinylated HRP layers. Scale bar is 300 μm.(D) On the left, images of drug-screening array by piezo inkjet printing made of alternating D-glucose (lines marked by arrows) and D-glucose/D-glucal rich spotted lines. On the right, gray scale pixel intensity distribution with Gaussian fits for the regions marked by rectangles 1 (solid line)and 2 (dashed line) in part enclosing representative D-glucose and D-glucose/D-glucal rich spots, respectively. B stands for background pixeldistribution, and F stands for foreground pixel distribution. Reprinted from ref 34. Copyright 2010 American Chemical Society.




Accordingly, for achieving spots with high resolution and well-defined morphology without the formation of undesirablesatellites, the drop rheological properties have to be optimizedby adding viscous, surfactant and biocompatible additives. Inthis sense we showed that additives like glycerol can increasefluid viscosity at a sufficient level in order to permit satellitesfree droplet formation along with the retaining of the biologicalactivity.27

Several papers report on the realization of multiplexed arraysvia piezodispensing nanoliter−picoliter droplets for realizingaccurate biomolecular assays involving DNA hybridization onfunctionalized surfaces28 and for characterizing interactionsinvolving delicate proteins.Antibodies have been one of the reagent of choice for the

preparation of protein microarrays by inkjet printing. Delehantyand Ligler reported on the employment of noncontact printerfor realizing an antibody microarray biosensor for multiplexdetecting protein and bacterial analytes (cholera toxin,staphylococcal enterotoxin B, ricin, and Bacillus globigii) bothindividually and in samples containing mixtures of them.Capture antibodies were biotynilated and immobilized asspots on the surface of an avidin-coated glass microscopeslide. The assay were executed under flow conditions usingfluorescent tracer antibodies which were able to bind toanalytes recognized by spotted antibodies.29 The limit ofdetection was of few nanograms of analyte per milliter or 104

cfu of bacterial cells per milliliter. Microarray assays requireminutes versus the hours need by conventional ELISA (timesneeded for incubation and multiple wash steps). More recently,Li et al. showed the fabrication of three-dimensional alginatehydrogel droplet microarrays to entrap antibody-coatedmicrobeads within spots inkjet printed on glass. Mass transportduring assays is greatly favored by 3D spot architectureand high gel porosity. The microarrays enabled multiplexedsandwich immunoassays to detect six proteins includingcytokines (TNF-alpha, IL-8, MIP/CCL4) and breast bio-markers (CEA and HER2) in buffer solutions and 10% serumby employing fluorescence sandwich assays with sensitivitydown to the pg/mL range.30

Hasenbank et al.26 showed multiprotein patterns by inkjetprinting for setting up a biosensor assay (Figure 1C). Themethod involved two sequential patterning steps. At first, thedeposition onto a gold-coated surface of a mixed thiol layer(BAT/PEG thiol solution) to provide oriented bindingcapabilities in a nonfouling background; second, the deposi-tion of multiple biotinylated proteins (b-HRP and b-BSA)(Figure 1C). Antibodies specific to each of the two proteinswere introduced in a development step in the SPR microscopein order to execute detection and highly specific binding of theantibodies to the immobilized proteins. In addition, Sukumaranet al. showed the advantage given by the combination ofenzyme encapsulation techniques in alginate and microarraymethods for an integrated screening platform for CYP450featured with nanomolar sensitivity detection31 by fluorescencemicroscopy. On the other hand, Lee et. al prepared aminiaturized 3D cell-culture array (the data analysis toxicologyassay chip or DataChip) for high-throughput toxicity screeningof drug candidates and their cytochrome P450-generatedmetabolites getting results comparable to those of 96-well plateassays.32 Together with heterophase assays, piezo inkjet dropletformation enables droplet microarray fabrication in order toconduct enzymatic assays in liquid droplets. On the basis of thisapproach, Mugherli et al. set up a robust enzymatic microarray

platform in which multiple reactions are conducted by piezodeposited nanoliter droplets maintained on a glass slide forthe in situ preparation of thousands of derivatives of pheny-lboronic acid and their successive accurate activity screening onthe NS3/4A protease of the hepatitis C virus.33 Recently, weapplied piezo-electric inkjet technology for drug screeningapplications.34 Picoliter drops containing a model substrate(D-glucose)/inhibitor (D-glucal) couple were serially inkjetprinted on a target enzymatic monolayer (glucose oxidase)linked to a glutaraldehyde activated silicon oxide solid surface(Figure 1D). The solid supported enzymatic surface was rinsedin water to remove the physisorbed layer finally resulting in acompact protein monolayer (height 3.6 nm) covalently linkedto the silicon oxide surface. It was possible to fabricate micro-arrays showing quality factors comparable to pin printingspotting along with high density, high throughput (10 spots/s),and a simple colorimetric detection that results in sensitivitysufficient for the discrimination between D-glucose (onlysubstrate) rich and D-glucose/D-glucal (substrate and inhibitor)spots.Finally, inkjet printing has also been recently applied for the

realization of a CNTs based immunosensor. CNTs were inkjetprinted on indium tin oxide (ITO) electrodes. Then captureanti-IgG antibodies were coupled through peptide bondformation to acidic functional groups on the nanotubes.Immunoassay were conducted via electrochemiluminescenceon luminophore [Ru(bpy)2PICH2]

2+] and IgG coated silicananoparticles. An excellent detection limit of 1.1 ± 0.1 pM ofIgG was found.16 Such results are promising for futuredevelopment of multiplexed assays.

■ ELECTROHYDRODYNAMIC ANDPYRO-ELECTROHYDRODYNAMIC PRINTING

Advancements in technologies for high-resolution noncontactliquid dispensing are constituted by methods like electro-hydrodynamic (E-jet)35 and pyroelectrohydrodynamic (pyroe-jet) printing.36 They enable submicrometer lateral resolutionbut have not been implemented for high-throughput andmultiplexed applications yet. Recently, a low cost, automatic<10 μm lateral resolution desktop system for E-jet printing hasalready been realized.37

Electrohydrodynamic printing has been introduced byRogers’ group for the preparation of high-resolution DNAarrays, with submicrometer lateral resolution. This techniqueuses electric fields to deliver inks to a substrate: a syringe pumpconnected to a glass capillary delivers ink to the cleaved endof the gold-coated capillary, that is the actual nozzle. Theapplication of a bias between the nozzle and a conductingsubstrate produces an electrostatic field (of the order of4 V μm−1) that causes mobile ions in the ink to concentratenear the surface of the meniscus at the nozzle. The repulsionbetween these ions causes a tangential stress deforming themeniscus into a conical shape (Taylor cone). At sufficientlyhigh electric fields, the electrostatic stress overcomes thecapillary tension at the apex of the liquid cone thus leading tothe formation of a droplet. Rogers et al. demonstrated thesuitability of the technique for the fabrication of functionalssDNA and dsDNA array,38 with lateral resolution down toabout 100 nm. Importantly, hybridization activity of the printedDNA molecules was shown with the example application ofDNA aptamer-based adenosine biosensing via fluorescencemicroscopy. In this regard, two different biotinylated strands[surf-ade-F] and [surf-no ade-F] (used as a control) with



Alexa546 modification on the 5′ end were serially e-jet printedon streptavidin-coated SiO2 substrates. Then, hybridizationwith two ssDNA occurred. The first is complementary to [surf-ade-F] and shows an adenosine aptamer on the 3′ while thesecond [ade-Q] is its 12-mer partial complement and shows aquencher label on its 3′ end. Hybridization quenched thefluorescence of the letters in comparison to the control. Byexposure with adenosine, aptamer strand [ade-Q] is releasedcausing an increased fluorescence signal (Figure 2A,B).Importantly, parallel multiplexing in E-jet printing is greatlyaffected by the electric field edge-effects caused by mutualinteraction between adjacent channels, even if many effortshave been spent to design multijet configurations to avoid suchproblems.39

To overcome these limitations, Ferraro et al. proposed achannel-less approach based on a pyroelectric printing thatexploits electric fields generated in polar dielectric crystals, suchas lithium niobate, that exhibit a pyroelectric effect,36 i.e., anincrease in temperature (the heat source can be a focused laserbeam or the hot tip of a soldering iron) generates a polarization

leading to uncompensated charges (i.e., an electric field) on thesurface of the niobate surface. When the niobate substrate isplaced near a second substrate that supports droplets or films ofa liquid, the pyroelectric effect in the niobate substrate initiateselectrohydrodynamic phenomena leading to droplet dispensing(Figure 2C). The printing occurs on a dielectric plate insertedbetween the crystal and the substrate which supports the ink.If compared to electrohydrodynamic, this new methodpotentially has higher speed, is electrode and nozzle-less,does not require electrodes or high-voltage power supplies, andpermits the achievement of high-resolution patterning, from afew micrometers to hundreds of nanometers (i.e., volumes oforder of attoliters), the resolution depending on the volume ofthe droplet reservoir. Although in its infancy, pyro e-jet hasalready shown the possibility to operate in a self-assembledspatially periodic multijets format (Figure 2D) in controlledconditions for multiplexed operations40 and also for auto-mated manipulation of dielectric microtargets onto which liquiddroplets are delivered, namely, the pyroelectric-adaptive-nanodispenser (PYRANA) microrobot.41 To date, no examples

Figure 2. Adenosine biosensor by e-jet printing and instrumental setup for pyro e-jet printing. (A) On the top, scheme for an adenosine biosensorvia electrohydrodynamic jet printing and on the bottom, fluorescence images of the letters printed with [surf-ade-F] and underlined with [surf-noade-F] as the control. The scale bar is 50 μm. (B) On the top, fluorescence images after hybridization with two complementary strands (ade-aptamerand ade-Q) and successive reaction with adenosine. On the bottom, images after hybridization with the same strands and subsequent reaction withcytidine. Figures reprinted from ref 38. Copyright 2008 American Chemical Society. (C) Scheme for a multijet system and visualization setup, and inpart D, optical pictures of different self-assembled liquid multijets obtained for PDMS polymeric liquid films at different thickness (7.2 μm on the leftand 6.2 μm on the right). Reprinted with permission from ref 40. Copyright 2011 The Royal Society of Chemistry.




of sensing applications are present in literature; however,we envisage this noncontact methodology as one of the bestcandidates to enable subpicoliter droplets dispensing in amultiplexed fashion.

■ ELECTROCHEMICAL TECHNIQUES FORMULTIPLEXING

Electrochemical-driven deposition methods can become anuseful approach for the realization of multiplexed biochips.Generally, they rely upon the application of an externalelectrical stimulus (electrical field) as the driving force formaterial deposition. In particular, two classes of techniques canbe considered as the best solution for biomolecular arrayfabrication: electrodeposition of array elements and scanningelectrochemical microscopy.In electrodeposition, ionic chemical species in the liquid

solution (metal or polymer ions) are moved by an electric fieldto an electrode. Electric current causes the reduction of ions tothe metal state leading to the formation of a solid-supportedfilm. Initially developed for applications in metallurgy andelectroplating, electrodeposition has been tentatively applied tothe fabrication of in situ array elements with applications inmultiplexed biosensing. Importantly, bioinks for electro-deposition need to be electrically conductive to be “printable”.In this sense, biomolecule-nanoparticle conjugates or mixturesrealized with biomolecules of interest and conductive polymers(chitosan, polypyrrole) are employed. Examples of proof-of-principle multiplex array fabrication are shown in theSupporting Information.On the other side, scanning electrochemical microscopy

(SECM) is a surface technique employing an ultramicroelec-trode (UME), i.e., an electrode of nanometer to submicrometerdimensions, which is immersed together with a solid sub-strate into an electrolyte solution containing an oxidizable (orreducible) chemical species. The UME is electrically biased inorder to generate a redox current (tip current). Then, byscanning the UME above the substrate, it is possible to gatherinformation about the surface reactivity and to generate apattern of metals, conductor polymers, or substrate etchedfeatures.42 SECM permits the generation of a pattern templateon a surface allowing biomolecular arrangement afterward.This results in an indirect-patterning methodology allowing forcovalent attachment of different biomolecules in a sequential,multisteps process. In the Supporting Information, applicationexamples of this approach are presented.

■ NANOTIP PRINTING: GENERAL CONSIDERATIONS

Nanotip printing techniques are a class of techniques allowingone to achieve submicrometer resolution, high spot density,and complex arrays, being technologies developed based onthe atomic force microscopy (AFM) concept. These methodsare able either to perform an adding process of molecules to asubstrate and include dip pen nanolithography, bias-assistedatomic force microscopy nanolithography, hollow tips, andnanoshaving.43 In spite of dip pen nanolithography, the othertechniques mentioned above have been to date only tentativelyused for multiplexing and more details are given in theSupporting Information.Introduced by Mirkin et al. in 1999, dip-pen nanolithography

(DPN)44 permits to fabricate nano- and micro-scale patterns inambient conditions and recently high-throughput capabilities.The method is based on the ink transportation from an

ink-coated AFM tip to a receiving surface. Two possible deposi-tion mechanisms exist, i.e., the diffusive and the liquid ones. Inthe diffusive regime, the tip is dipped into the ink solution withsubsequent solvent evaporation leading to molecules coatingthe DPN tip to be deposited on the surface substrate by thenaturally occurring water meniscus. The ink deposition rate isthen a direct function of the molecular diffusion rate, which isby itself quite slow (tip−surface contact times of 1−10 s arenecessary to realize micrometer size spots) and molecular-specific, as well. These features complicate the implementationof multiplexed-patterning.45,46 On the other side, by consider-ing liquid inks, the deposition mechanism is more rapid thandiffusive case since these are directly transferred through theliquid without additional menisci. Tip−surface contact times of0.01−0.1 s are employed to get micrometer size spots as afunction of the surface tension occurring between the liquidand the tip, the surface tension between liquid and the surface,and the viscosity of the liquid.47 Importantly, unlike thediffusive mechanism, multiplexed depositions can be easilyrealized, the deposition rates being mainly governed by theliquid properties with respect to molecular diffusion thusfacilitating multiple realization of multiple features on themicroscale resolution. Since conventional fluorescence detec-tors have a resolution down to 1 μm, this method is alreadyable to fulfill the needs of accessible and easy-implementablemultiplexed biological assays in array format.48

To date, successful control in liquid ink printing has beenpossible by using additives such as glycerol, agarose, andtricine to influence viscosity, wettability, and the ink-surfaceinteractions.49 With the benefit of a solid knowledge platformabout liquid DPN printing mechanism, we can anticipate theliquid-ink deposition to be the prime method of choice forenabling multipatterning biological molecules rapidly and atlow cost.

■ NANO-PEN ARRAYS FOR MULTIPLEXEDPATTERNING

Single tip writing is a slow and serial approach with extremelylow throughput, not suitable for bioanalytical applications. Afundamental advancement is then constituted by the parallelwriting of different inks in a single step by employing arrays ofpens. Throughput in DPN can be enhanced via the realizationof devices as 1D or 2D pen arrays for simultaneously writingink on large areas and a surface as large as 1 cm2 has alreadybeen patterned by employing 2D array of 55 000 pens.50 Then,multiple inking on such pens arrays becomes a crucialparameter. The conventional inking process for single pensinvolves pen soaking in an ink solution for a few seconds andsubsequent blow drying with N2. This method can inducevariability in pen arrays inking due to inhomogeneous solventdrying depending on the duration and the angle of the N2stream as well as the soaking process. An efficient methodshould deliver well-defined amounts of ink to each pen.However, challenges are associated with inking each pen withdifferent inks without cross-contamination and assuring uni-form ink coating for different inks on different pens. To date,two possible approaches for multiple pen inking on 1D-2Dpens arrays have been investigated via inkjet printing ormicrofluidic inkwells. The inkjet printing approach has beenshown by Wang et al.51 to be an easy method to deliverindependent inks to each pen or to several pens within a 1D or2D pen array (see Figure 3A,B). They showed the feasibilityby inking cantilevers within a 7-pen array with different



fluorophore-labeled phospholipids in a lipidic carrier of1,2-dioleonyl-sn-glycero-3-phosphocholine (DOPC). Theinked pens array was then used to pattern four different inksin arrays of squares (10 μm wide) and made of 300 nm parallelline features. Albeit simple and controllable, this method hasnot been used so far because of issues in controlling inkspreading at each pen surfaces and the cross contaminationbetween adjacent pens. On the other side, microfluidic inking isan established and more diffused strategy to precisely confine atthe same time different inks at separation scales of the sameorder of distance between pens especially in a 1D pens arrays.This is achieved by inkwell loading chips, microfluidic devicesetched out of silicon wafers using a deep reactive ion etchprocess that creates nearly vertical side walls. Fluid actuationoccurs by open-channel capillary flow from macro-reservoirsto individual microchannels and ultimately to microwellswhere tips are inked (see Figure 3C).52 Since the microfluidicselements are in open configuration, they are relatively clog-free,

easy to fabricate and, in principle, it is possible to reuse them byappropriate cleaning procedures. Issues derive from solventevaporation, this problem may be solved by the employment ofhigh-boiling or hygroscopic cosolvents.

■ 1D PEN ARRAYS FOR MULTIPLEXED BIOSENSINGVIA LIQUID INKS

Multiplexing has been extensively shown in several papersvia employment of liquid inks loaded on 1D pen arrays. Janget al.53 showed multifeatures hydrogel and phospholipidpatterning with precise registration, printing four differentdye-labeled hydrogel inks (PEG-DMA) within a small area(50 × 50 μm2) on hexamethyldisilazane (HMDS) spin-coatedsilicon substrates. Rhodamine, FITC, Alexa347, and a mixtureof rhodamine and FITC were used as red, green, blue, andorange dyes, respectively. The volume mixture ratio betweendyes and PEG-DMA solutions was balanced to exhibit similarfluorescence intensity at each dye-labeled ink. pH sensors are of

Figure 3. 1D pens arrays for multiplexed biosensing. (A) Scheme for addressable inking of pen arrays by inkjet printing and (B) inking of a 1D penarray with four different inks (DOPC, dansyl, fluorescein, rhodamine) and corresponding multiplexed patterns written on glass. Reprinted withpermission from ref 51. Copyright 2008 John Wiley & Sons, Inc. (C) Optical image of a 1D cantilever tip array overlaid with a fluorescence image ofa 12-microchannel “Inkwell” containing solutions of four different dye-labeled IgG antibodies. Adapted with permission from Macmillan PublishersLtd. Nature Methods, ref 52, Copyright 2010. (D) Scheme for the fabrication of a combinatorial pH indicators array (OG, FL, and NF, see text forthe chemical names) by direct-writing parallel DPN on glass substrates with a fluorescence image of OG square-, FL triangle-, and NF cross-likestructures. Reprinted with permission from ref 54. Copyright 2011 The Royal Society of Chemistry. (E) Fluorescence images of T-cells activated byfunctional proteins bound to phospholipid multilayer patterns via streptavidin. Reprinted with permission from ref 9. Copyright 2008 John Wiley &Sons, Inc. (F) Proof-of-principle for coculture of 3T3 fibroblasts on fibronectin patterns (red spots) with C2C12 myoblasts on laminin patterns onlaminin (green spots). Distance between patterns is 66 μm. Reprinted with permission from ref 56. Copyright 2010 Elsevier.




course of fundamental importance for several biologicalapplications, and Martinez-Otero et al.54 showed the possibilityof realizing miniaturized combined arrays of three differentpH indicators. In this case, the authors reported acidity detec-tion on the nanoscale by employing different amino-reactiveof Oregon Greens 514 (OG), fluorescein (FL), and 5-carboxynaphthofluorescein (NF) covalently immobilized onthe amino group of a functionalized glass support (N-(6-aminohexyl)-3-aminopropyltrimethoxysilane SAM on the sur-face), which show different protonation states with distinctemissive properties and complementary pKa values. Thecombined use of these compounds allows for an accurate pHmonitoring within the pH 3.0−9.0 range (see Figure 3D) suchthat univocal pH values over this large range can be determinedfor any solution by measuring the response of the multiplexedarray of these three fluorophores. Recently, Irvine et al.46

optimized 1D pen arrays to fabricate a prostate specific antigen(PSA) immunoassay on nitrocellulose with fluorescent detec-tion: the achieved limits of detection are comparable with acommercial available ELISA kit and the spot density in thearrays is at least 2 orders of magnitude higher than thoseobtained via pin- or inkjet printing.55 Protein patterning viaDPN techniques at feature sizes smaller than a single cell haveshown great potential applications for use as tools to controlcellular activity at the single cell level. Sekula et al. employed1D pen arrays to execute simultaneous patterning of multi-component micro- and nano-structured supported DOPC lipidmembranes and multilayers containing various amounts ofbiotin and/or nitrilotriacetic acid functional groups. Adsorptionof functionalized proteins based on streptavidin or histidineaffinity tags permits to generate model peripheral membranebound proteins usable as high-efficiency substrates for celladhesion.9 In Figure 3E, a fluorescence image of cells adheredand activated in fluorescein-doped lipid patterns containing 5%biotinylated lipid is reported. The blue fluorescence indicatesthe nucleus of the cells by DAPI (4′-6-diamidino-2-phenyl-indole) staining and the red fluorescence shows CD69(cluster of differentiation 69, a human transmembrane C-typelectinprotein encoded by the CD69 gene) expression detectedby subsequent staining with a-CD69-PE and a-PE-TRITCantibodies. More recently, by employing DPN techniques, J. M.Collins and S. Nettikadan were able to generate subcellularpatterns of fibronectin and laminin to show56 the bindingdynamics of two different cell types (3T3 fibroblasts andC2C12 myoblasts) with respect to fibronectin and laminin witha spatial control at the single cell level in order to realize asingle cell coculture (Figure 3F). The authors showedattachment statistics for both cell types: about 70% of bindingareas have cells attached and 80% of those are constituted bysingle cells.

■ 2D PEN ARRAYS FOR HIGH-THROUGHPUTPATTERNING

Intense research activities on parallel pens tried to increasewriting throughput. First investigations were conducted at IBMand Stanford in the known IBM’s “millipede” that lead to therealization of a 32 × 32 probes array with each cantileverassigned to read and write on a 100 × 100 μm area. As to thelithography applications, Quate and co-workers57 developed acurrent-induced lithography in which each pen was independ-ently actuated for delivering material on the surface. Since theindependent control of the on/off state of each cantileverrequires actuation of the force applied to, and height of, the

cantilever above the surface, this approach finally resulted in aquite challenging operative principle. On the other side,patterning in conventional DPN occurs whenever a coatedtip is held in contact with a surface, i.e., pens are not actuatedindependently but are simultaneously brought into contact withthe surface and scanned together, which allows the duplicationof a single pattern a number of times equal to the number ofprobes in the array.This solution-defined passive probe array results in an easier

implementation of parallel-pen DPN58 that importantly can beintegrated with standard AFM systems. In fact, efforts in thissense led Mirkin and co-workers to the realization of 2Dcantilevers arrays,50 a bidimensional array of 55 000 with a penspacing of 90 and 20 μm in the x and y directions, respectively(Figure 4A). Importantly, the plane of the array of pens has tobe as parallel as possible to the plane of the receiving substratein order that all the pens exert the same amount of force onthe surface leading to uniform spot sizes across the array inextended periods of writing time. The procedure to align a penarray with respect to the surface is defined as “leveling”. Whilebeing accomplished quite easily via optical methods for 1D penarrays, the opacity of Si and Si3N4 cantilevers complicates thealignment of a 2D cantilever array on a surface. In addition, alsoif a single feature parallel patterning on 1 cm2 is possible, greathurdles come from multiplexing via inkwell devices since theoccupied area for a chip containing the ink reservoir should belarger than 0.5 m2. However, by employing inkjet printingtechniques, it is possible, in principle, to coat individual pensalso within 2D pen arrays with different materials.51 Suchdifficulties motivated researchers to develop 2D pen arraysthat could be more suitable for multiplexing, as polymer-penlithography (PPL) and hard-tip soft spring devices.

■ 2D PEN ARRAYS FOR HIGH-THORUGHPUTMULTIPLEXED BIOSENSING

PPL is a significant evolution for multiple 2D patterning inwhich a soft elastomeric polymer (polydimethylsiloxane) isused to deliver inks onto a surface by controlling the movementof the pen array with a scanning probe microscope. A typicalpolymer pen array contains thousands of pyramid-shaped tipsmade with a master prepared by conventional photolithographyand subsequent wet chemical etching. These pyramids areconnected by a thin PDMS backing layer (thickness around50−100 nm) that is adhered to a glass support. The same moldused to fabricate the array can be used as an inkwell that can beaddressed and filled with different inks by employing inkjetprinting, so achieving perfect registry between the pens on thearray and the inkwells.59 A proof-of-principle multiplexed PPLenabled printing in a high-throughput and low cost manner hasbeen shown by Zheng et al.60 The pyramid-shaped wells inthe Si mold were first filled with three different dye-labeledproteins inks by inkjet printing, i.e., yellow, TRITC-conjugatedantimouse IgG (TRITC = tetramethylrhodamine isothiocya-nate); green, Alexa Fluor 488-conjugated antiprostate specificantigen (anti- PSA); red, Alexa Fluor 647-conjugatedanticholera toxin β (anti-CTβ). By fluorescence microscopy,authors showed that the inks have been properly addressedwith the inkjet printer and then printed on the surface(Figure 4B). Moreover generated PSA arrays have been labeledby their corresponding dye-labeled antibody, i.e., anti-PSAselectively binded onto the PSA regions with an undetectablebackground. This means that PPL-patterned proteins maintaintheir structural integrity. From these initial results, one can



consider 2D pens are a key tool for developing high-throughputand miniaturized assays to be conducted on a very large testarea (order of cm2) and requiring a few femtoliters volumes ofanalytes. Recently, an impressive evolution versus a versatilecantilever-free tip-based nanopatterning method has beenshown by Shim et al.61 There, an array of hard silicon tips ismounted onto an elastomeric backing. The approach, termedhard-tip, soft-spring lithography, can overcome the throughputissues of cantilever-based scanning probe systems and theresolution limits imposed by elastomeric stamps and tips, beingable to deposit molecules on a surface reaching a sub-50 nmlateral resolution over centimeter-scale areas (Figure 4C).However, achieving multiplexing with this device is still matterof research.

■ OUTLOOK AND CONCLUSIONSIn conclusion, we summarize the information provided in thisFeature paper in Table 1, pointing out the main features of eachfabrication technique, considering the attainable lateral spot sizeand times for a multiplexed array of dimensions suitable for onetypical analysis, the ability of multiplexing (i.e., the number of

different spots written in parallel), the sensitivity that is possibleto attain by employing optical detection methods and thepossibility of automating the preparation of the array. Alongwith this information, we provide some interesting applicationsin which the array devices play a fundamental role.Robust and low-cost fabrication of multiplexed micro- and

nano-arrays allow for the generation of new and unprecedentedcomplex platforms enabling biological information collectingtogether with low reagent consumption, shorter response times,and higher sensitivities. Conventional techniques such asphotolithography, electron beam lithography, and microcontactprinting are time-consuming, require clean rooms access, andare serial and often indirect approaches. As often indicatedby research papers, great issues specifically derive from thedifficult alignment procedures, thus becoming a not successfulapproach for long-term applications in multiplexing. On theother side, newer techniques based on liquid dispensingmethodologies such as inkjet printing and high-throughputdip pen nanolithography enable easy biological patterning inambient conditions, in a parallel manner with the direct writing

Figure 4. High-throughput biomolecular patterning via 2D pen arrays. (A) Optical image of a part of the 2D pens array. The inset is a SEM image ofthe same pens array at a different viewing angle. Reprinted with permission from ref 50. Copyright 2006 John Wiley & Sons, Inc. (B) Multiplexedprotein patterning via polymer pen lithography. (B.1) Scheme for multiple proteins PPL patterning. Fluorescence images of a Si mold inked withthree proteins by inkjet (B.2); a polymer pen array dipping inside the Si mold (B.3); and multiplexed proteins arrays made by PPL (B.4). Reprintedwith permission from ref 60. Copyright 2009 John Wiley & Sons, Inc. (C) Scheme of a hard-tip soft-spring tip array supported by a transparent,PDMS backing layer that provides mechanical flexibility to each tip and alleviates alignment issues. Adapted with permission from ref 61. Copyright2011 Macmillan Publishers Ltd.




approaches that do not require access to clean rooms or costlylaboratory equipment.Droplet dispensing techniques, namely pin- and inkjet-

printing, can easily permit the dispensing of different chemicaland biological systems in parallel with good automationabilities; these techniques are employed as an experimentalinvestigation tool for biological screening at the microscale. Inorder to go down to the micrometer range, electrohydrody-namic or pyroelectrohydrodynamic effects have to be coupled.However, the implementation of multiplexing is still a matter ofresearch.Electrochemical-based methods, such as electrodeposition

and scanning electrochemical microscopy, are possiblecandidates for multiplex array fabrication. In particular, electro-deposition can permit the direct pattern of different bio-molecular systems in a sequential scheme by using a mixture ora conjugate with a conductive material. On the other side,scanning electrochemical microscopy only allows for therealization of patterned surface chemistries (via desorptionof degradation of self-assembled monolayers) onto whichmolecular arrangement takes place afterward. Albeit multi-plexing is possible, the low throughput and the lack inautomation make them an unpractical approach.Scanning probe lithography based techniques (i.e., nano

pens) represent a more promising and low-cost patterningapproach that can enable multiplexing in ambient conditionswith nanoscale resolution and high registration accuracy.In order to become competitive toward droplet dispensing,throughput has to be enhanced. In this sense the intenseresearch activity in the field of 1D and, recently, 2D pen arraysduring the last 4 years resulted in methods which enablemultiplexed arrays fabrication with good throughput but stilllacking in automation.

As far as it concerns future applications, multiplexed arraymethodologies will constitute a key tool for enabling extractionof complex and meaningful biological data from an ultra rapidtotal analysis on a single biological sample. In this sense, afterthe completion of the Human Genome Project and of theHapMap Project and thanks to the continuous progresses inthe -omics sciences, it is now becoming possible to beneficiatefrom an impressively large amount of data that can permit oneto know the interactions and connections inside biologicalpathways both qualitatively and quantitatively. Since more andmore complex biological questions are then starting to arisederiving from this new exciting scenario, innovative andefficient methods that can address such issues of complexityfor investigating at the single “bio-entity” level (biomolecular,cellular etc.) are becoming necessary. Although biologicalmicroarrays fabricated by techniques as pin- and inkjet printingare already able to collect multiplexed information (untilthousands of different spots from a single sample), furtherefforts to increase information density via the increase ofmultiplexing (i.e., spot density and diversity in the same array)are needed to answer these recent questions of highercomplexity. A 10 000-fold increase in areal density, achievablewith the use of nanoscale patterns dispensing methods such asnano pen techniques would allow a biomolecular assay toscreen for a correspondingly large number of targets and a fixednumber of targets to be screened with a correspondinglysmaller sample volume and in a shorter amounts of time. In thissense, drug discovery efforts will be extremely facilitated by amolecular level knowledge of bioactive molecular scaffolds,structure−activity relationships, multitarget agents, and syner-gistic drug combinations against selected target or multipletargets, in order to fully apply a system-biology driven approachto drug discovery. Importantly, the ability to produce patternsby a certain complexity and with multiple materials at

Table 1. Main Features of the Solution Methods for Bio-Array Fabrication Reported in This Reviewa

techniques spot sizetime of

fabricationno. of spots printed in

parallelthroughput

(printed area)sensitivity

(fluorescence signal) automation main applications

pin printing 100−1000 μm >1 h 1−100 >1 cm2 until sub-nM yes

-drug screening-protein profiling-immunology-genomics-proteomics-metabolomics

inkjet printing 10−100 μm someminutes 1−100 >1 cm2 until sub-nM yes

-drug screening-protein profiling-immunology-genomics-proteomics-metabolomics

e-jet printing 0.1−10 μm minutes serial <1 cm2 until sub-nM yes -genomics

dip-pennanopatterning <0.1−10 μm <1 h 1−10 >1 cm2 by 2D pen

arrays until sub-pM no

-immunology-toxicology-proteomics-single-cell chips

photolithography <0.1−10 μm minutes serial >1 cm2 until sub-nM no-genomics-proteomics

microstamping 1−100 μm minutes serial >1 cm2 no info no-genomics-proteomics

aThe techniques in the main text are here compared to more conventional and expensive methods like photolitography and microstamping (seethe Supporting Information). In terms of multiplexed biosensing, the first typically have higher flexibility, ambient condition operations, andlower fabrication costs (refer to a chip of hundreds of spots) since they do not need clean rooms equipments and expensive instrumentalmaintenance.



subcellular levels will enable for addressing fundamental studies,at the single-cell level, including cellular migration, focal adhesion,cellular polarization and proliferation, neuronal development, andstem cell differentiation.Finally, as to diagnostic devices for the clinical practice,

reducing fabrication costs is a fundamental requisite. In thisrespect, inkjet printing results in a simple and accessiblefabrication method due to its simplicity and friendly operativeconditions. Second, detection assays need to be short,information-rich, and possibly validated by the medicalcommunity. Efforts have to be spent to integrate this high-quantity of biological information with the medicinal practice.

■ ASSOCIATED CONTENT*S Supporting InformationAdditional information as noted in text. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Dipartimento di Chimica “S. Cannizzaro”, Universita diPalermo, V. le delle Scienze, Parco D’Orleans II; Ed.17−90128Palermo, Italy. Phone: +39 091 645 98 46. Fax: +39 091 590015. E-mail: [email protected].

NotesThe authors declare no competing financial interest.

Biography

Giuseppe Arrabito, born in Ragusa in 1984, is currently a Ph.D.student in Nanoscience at Scuola Superiore di Catania. He received aBachelor’s Degree in Chemistry in 2006 (with Diploma from ScuolaSuperiore in 2007) and a Master’s Degree in Biomolecular Chemistryin 2008 (with Diploma from Scuola Superiore in 2009). His mainresearch interests are in the field of micro- and nano-biological arraysrealized by solution dispensing methodology for drug screening andcell-biology applications. Bruno Pignataro, born in Bologna in 1972, isProfessor of Physical Chemistry at the University of Palermo. Hereceived a degree in chemistry in 1995 and a Ph.D. in materials sciencein 2000 at the University of Catania. His research interests focus onordered molecular materials at solid surfaces including theirapplication in electronics (transistors, photovoltaic cells, biosensors,and drug screening devices).

■ ACKNOWLEDGMENTSWe are grateful to Superlab (Consorzio Catania Ricerche) forhis hospitality and Antonino Scandurra and GiuseppeFrancesco Indelli for useful discussions. Italian MiUR (PRIN2008 Program) and the University of Palermo are acknowl-edged for funding.

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Solution Processed Micro- and Nano-Bioarrays for Multiplexed Biosensing