Feature pubs.acs.org/ac
Solution Processed Micro- and Nano-Bioarrays for Multiplexed Biosensing This Feature article reports on solution dispensing methodologies which enable the realization of multiplexed arrays at the micro- and nanoscale for relevant biosensing applications such as drug screening or cellular chips. Giuseppe Arrabito† and Bruno Pignataro*,‡ †
Scuola Superiore di Catania, Via Valdisavoia 9, 95123, Catania, Italy Dipartimento di Chimica “S. Cannizzaro”, Università degli Studi di Palermo, V. le delle Scienze, Parco d’Orleans II, 90128, Palermo, Italy
‡
S Supporting Information *
printed feature size on the slide from micro- until nanoscale; multiplexing, i.e., the increase of the number and of the density of tested biological targets (biomolecules or cells), and finally biochip design, i.e., the careful development of smart platforms able to collect and compute real time biological information. As to the patterning resolution, the rapid realization and characterization of micro- until nanoscale architectures with a single molecule can be routinely achieved by nanolithography methods.3,4 Accordingly, nanoarray devices5 permit a dramatic decrease in the cost of the assay since, in comparison to microarrays, still a lower volume of reagents is required, response time is shorter, and higher sensitivity can be obtained up to the subfemtomolar level.6 Moreover, multiplexing still constitutes a challenge for modern biological patterning methods. In principle, the higher the biomolecular complexity of the array, the higher are the quantity and the quality (in term of density) of the attainable biological information from a single experiment. In biomolecular assays, multiplexing is strictly dependent on resolution. As to the complexity of the arrays and the number of features increase, reduction in size becomes more and more important, due to the fact that the area occupied by one array affects the amount of sample that can be used in a chip. In the following, we will consider examples of applications in which bioarray technology plays a fundamental role in important fields such as, for instance, biomolecular screening and cellular arrays. As to biomolecular assays, thanks to the continuous miniaturization efforts, they can be routinely performed in volumes of a few microliters in high-throughput microtiter plates (i.e., 1536-well microplates) executing more than 105 assays in a single screen, a great advantage with respect to perform the same reaction in conventional milliliter test tubes.7 However, the interest toward further assay miniaturization, driven by the increase of biological interactions and the necessity to save time, sample, as well as to increase sensitivity motivated the implementation of robotic liquid handling techniques able to dispense, in parallel, biological samples in the nanoliter scale. Currently employed robotic systems suffer from several issues such as high costs, complexity of the instrumental setup, and reliability of the
G. Arrabito
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icroarray technologies are nowadays relatively mature, their development starting with the onset of highdensity DNA arrays, then addressing with peptide and protein microarrays, and now prefiguring perspectives in the field of cellular arrays.1 Such high-throughput analytical tools are associated with smaller usage of sample volumes, decrease in use of reagents, rapid analyses, and increased sensitivity since, at the microscale, the chemical species have a shorter distance to diffuse than in conventional macroscale reaction vessels. Undoubtedly, the great advantage of these devices is their highly parallel nature and ability to interrogate hundreds to thousands of different sensing molecules in one experiment.2 The area of bioarray technology fabrication evolves along three different paths: patterning resolution, i.e., the decrease of the © 2012 American Chemical Society
Published: May 15, 2012 5450
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extracted data.8 Consequently, simplifications of the experimental setup, costs reduction of the handling devices, and further miniaturization are all highly desiderated. As to single-cells chips, one has to consider the analysis of cooperative patterning and geometrical effects on individual cell surfaces. The complex biochemical and morphological scenario of a cell determines a size requirement, i.e., different functional proteins should be patterned together below the size of a single cell. Since it is achieved, the response of the cell to such multiproteic subcellular dimensioned surfaces would be usable as a real-time detection of the biological function of the pattern9 into a single-cell. Finally, biochip design represents the last step in the development of a mature array technology, dealing with liquid automation and actuation via micro- and nanofluidic systems along with the optimization of the signal readout eventually via interfacing with micro- electronic components and finally signal extraction and processing. A review from Seidel and Niessner (ref 10) reports on the current state-of-the-art automation for analytical microarrays.
The success of a multiplexed microarray technology is not only connected to the fabrication methodologies but also to the development of suitable substrate surfaces for biomolecule immobilization and to the detection methods. As far as substrate surfaces are concerned, soft supports (i.e., polystryrene, nitrocellulose membrane), conventionally used for biochemical analysis (immunoblot), are typically not compatible with microarrays. This is due to the scarce biomolecular density, the high spot spreading, and the low signal/ noise ratio. Many groups make use of glass slides due to the low fluorescence background and the possibility to get high proteinbinding capacity by chemical functionalization via aminosilane, polylysine, or aldehydic groups.14 Also, conducting assays on solid substrates requires biomolecular attachment to its surface. In this sense, several chemical reactions are reported for peptide immobilization on surfaces. Peptide arrays are typically fabricated through in situ peptides synthesis, are built up in a linear fashion, and can be selectively immobilized by the first amino acid. Alternatively, peptides can be immobilized using bioorthogonal thiazolidine ring formation via a glyoxylyl group reaction with 1,2-amino thiols, 1,3 dipolar cycloaddition of terminal alkynes with azides, and Diels−Alder product formation of benzoquinone with cyclopentadiene and native chemical ligation. On the other side, protein immobilization requires more careful consideration,15 since it poses several consequences on the quality of the analysis influencing the inherent biological activity of the protein immobilized at the solid surface, the accessibility to the ligands present in solution and affecting reproducibility, selectivity, and device performance. The immobilization process should maintain protein activity and allow the correct orientation and accessibility to biomolecules for the correct analyte recognition at the solid− liquid interface. In particular, proteins can be immobilized by using covalent or noncovalent immobilization. Covalent attachment can be executed by employing random nonspecific cross-linking approaches via chemically activated surfaces (e.g., aldehyde, epoxy) or specific biomolecular affinity tag interactions (e.g., streptavidin−biotin, his-tag−nickel-chelates). The first ones can lead to poor preservation of protein biological activity, while the second ones allow proteins to maintain a correct orientation. In noncovalent interactions, hydrophobic (nitrocellulose, polystryrene) or positively charged (polylysine) surfaces are typically employed. These substrates are currently used in ELISA or Western blotting. In addition, by employing polyacrylamide gel pads and agarose thin films, it is possible to generate 3D proteic gel matrixes featured with higher capacity in protein immobilization in comparison to a 2D surface. Importantly, when conducting a multiplexed analysis on a multiprotein sample, different molecules often need to be analyzed at the same time. Problems can arise when proteins bind nonspecifically at surfaces. This can be significantly reduced by employing polymers like polyethyleneglycol because of its resistance toward protein adsorption in aqueous media. Finally, carbon nanotubes are also currently being developed as nanoscale building blocks for analytical devices because of their excellent mechanical, electrical, and electrochemical properties. They can be easily derivatized with different functional groups for covalent attachment of biomolecules in order to generate high-efficiency biosensing platforms.16 As far as detection methods are concerned, high-quality assays are required to translate specific multiplex biomolecular
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METHODS FOR MULTIPLEXING There exist several methods to produce patterns of biological materials on surfaces. However few of these are capable of multiplexing 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-down directly or indirectly may generate from micro- to nanoscale structures, bottom-up typically exploits intermolecular interactions to realize self-assembled or self-organized ordered structures. Admittedly, bottom-up self-assembly methods are also gradually gaining importance in biofabrication since they result in low-cost, solution based approaches allowing for programmed molecular architectures on 1-D, 2-D, and 3-D scales.11 However, supramolecular organization attainable from “bottom-up” approaches is often difficult to extend from nanoto mesoscopic length scales or does not permit an accurate placement of different desiderated structures on addressable regions of a surface. Top-down methods are currently the mostemployed in the field of biomolecular array fabrication due to the possibility to automate and precisely control the patterning process and the scaling on a large area. However, by considering multiplexing abilities, conventional top-down fabrication techniques like photolithography, electron beam lithography, and microcontact printing can pattern at nanometer resolution on large areas, but the number of different materials printable in parallel is limited, realized via indirect patterning approaches and requires clean rooms and expensive instrumentation. Multiprotein patterning by such techniques has been recently reviewed by Ganesan et al.12 Notably, photolithography affected by the employment of photoresist developers and heat treatments might affect the activity of delicate biomolecules. Great concerns reduce multiplexing abilities by microcontact printing due to contamination from the stamp material and the unprecise stamp/substrate alignment.13 These limitations motivated the development of new, original unconventional micro- and nano- top-down fabrication methods with high flexibility and ambient conditions operation, as well as lower costs as droplet microdispensing methods (namely, pin printing, inkjet printing, electrohydrodynamic, and pyroelectrodynamic printing) and nanotip printing techniques (serial dip pen nanopatterning, 1D and 2D Dip Pen Nanopatterning, and hollow tip dispensing). 5451
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of the most widely used methods to fabricate fundamental tools for genomics18 and protein function determination.19 The uniformity in spot formation depends on several parameters as sample viscosity and pin and substrate surface properties. Spot dimensions depend on the printing speed, on the solution surface tension, and on the solution wettability of the substrate surface.20 Two pin printing typologies can be distinguished: solid pin printing and split pin printing. In solid pin printing, the pin is loaded 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 several time-consuming sample reloads needed to realize a single microarray. 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 and Diamond21 employed split pin printing to realize a chemical compound microarray consisting of nanoliter droplets of glycerol. By means of the addition of successive reagents to each spot via aerosol deposition, they were able to profile the kinetics of protease screening reactions in single droplets (Figure 1A) reaching subnanomolar detection limits for human plasmin cleavage of substrates like (CBZ-FR)2-R110. Nielsen et al.22 employed split pin printing to fabricate multiplexed antibodies arrays, having sensitivities comparable to standard ELISA methods, and being able to monitor the activation, uptake, and signaling of ErbB receptor tyrosine kinases. They integrated these arrays with 96-well microtiter plates in order to identify and analyze small molecule inhibitors of signal transduction processes with high speed and precision (Figure 1B). These array could quantify purified antigens over a 1 000-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 which glyoxylyl peptides were immobilized by site-specific α-oxo semicarbazone ligation and proteins by physisorption. By the employment of an immunofluorescence assay, the microarray permitted to achieve sensitive and specific serodetection of multiple antibodies directed against pathogens as different as hepatitis C virus, hepatitis B virus, human immunodeficiency virus, Epstein−Barr virus, and syphilis. Such peptide−protein microarrays showed good specificities and sensitivities (0.01 mg/mL) for antibody detection. More recently, Lin et al.24 developed a reliable multiplexed peptide microarray-based immunoassay (peptide covered the primary sequences of αS1-casein, αS2-casein, β-casein, κ-casein, β-lactoglobulin) fabricated by pin printing for large-scale epitope mapping of food allergens in milk. The epitopes identified via fluorescence detection by the peptide microarray were consistent with those identified by conventional analytical methods. By employing replicate arrays of an immunolabeled serum pool, reproducibility was evaluated, while specificity and sensitivity were determined by employing serial dilution of the serum pool and a peptide inhibition assay. Notwithstanding pin printing is the most known array fabrication methodology, it is a tedious and time-consuming process. Indeed, after several prints, pin tends to degrade. When a pin is used to print multiple solutions, it must be washed and cleaned to avoid cross contamination. Indeed, because of the impact at contact, the pin structure deformation, and the clogging from contaminants, pin-based arraying is prone to suffer from slide-to-slide inconsistency. 20
interaction phenomena into observable and quantitative parameters. Typically, this is achieved through measurements of radioactivity, photon absorption, or photon emission. Albeit successfully applied in high-throughput screening, radioactive methods have become much less popular because of hazards in handling radioactive materials and the possibility of nonradioactive alternatives. Photon emission is by far the dominant assay methodology due to its ability to deliver speed, accuracy, and sensitivity necessary for successful assays. This is achieved primarily through fluorescence and chemiluminescence. While with fluorescence the energy needed for producing excited states is gained through light absorption, with chemiluminescence energy results from exothermic chemical reactions. This difference 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 the background is virtually absent since no photons need to be introduced in the samples leading to higher sensitivities. However, to date there is a scarce number of chemiluminescence assays along with a limited development for multiplexing. On the other side, thanks to the high number of different fluorophores, multiplexing via fluorescence can be much more easily achieved allowing multibiomolecular process assays on a microarray scale. Also, fluorescence methods always require labeling strategies which can pose synthetic challenges and multiple label issues and may exhibit interference. For this reason, label-free detection techniques (for example, surface plasmon resonance (SPR), atomic force microscopy (AFM), nanowires, ellipsometry) are starting to acquire more and more importance since they eliminate the need for secondary reactants. For a review on this topic you may refer to Ray et al.17 Finally, colorimetric detection constitutes a label-free method whose use is increasing, especially in clinical applications. They are quite attractive because the associated experimental setups permit multiplex analysis at relatively low cost. The principle of detection is based on the formation of a soluble or insoluble colored product leading to measurable spot/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 give information about their automation capabilities. In order to give a broader picture of this field, in the Supporting Information we briefly discuss the latest advancements of photolithography, microcontact printing methods, electrodeposition, scanning electrochemical microscopy, and modified scanning atomic microscope tips for biological patterning in multiplexed format, as well. The presented examples of biomolecular patterning, self-assembled monolayer methodologies, and site specific immobilization are frequently employed as fundamental means to acquire accurate molecular-scale control of biomaterial deposition on surfaces. Together with a description of the fabrication process for each different technique, we discuss the multiplex analytical abilities of the array devices providing information about the number of different analytes investigated in parallel, the selectivity, and sensitivity of the bioassays executed in an array format.
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MULTIPLEXING VIA PIN PRINTING Pin printing is currently among the most diffused patterning technique in biological applications. In particular, the parallel printing with multiple heads to create DNA microarrays is one 5452
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Figure 1. Multiplexed biochemical assays by pin- and inkjet printing methods. (A) Profiling of protease activity with three fluorogenic substrates in microarrays fabricated by pin-printing. The blue fluorescence resulted from the cleavage of a thrombin substrate, and the red and green fluorescence resulted 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 compartmentalization of 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 is proportional 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. Reprinted with permission from ref 26. Copyright 2008 Elsevier. A mixed BAT/PEG thiol solution is first spotted on a gold-coated microscope slide. A streptavidin layer is added and then two biotinylated proteins (b-HRP and b-BSA) are deposited in a second step and then antibodies are introduced sequentially. 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 pixel distribution, and F stands for foreground pixel distribution. Reprinted from ref 34. Copyright 2010 American Chemical Society.
in the picoliter to the nanoliter range. It has the great advantage to allow precise and controlled parallel deposition of small volumes (typically from the picoliter to the nanoliter range) of multiple biological materials using independent jets (i.e., independent controlled microchannels) on almost any possible type of substrates (solid, gel, liquid surfaces) being a contactless tool. Moreover, the hardware integration with automated translation stages enables precise pattern placement and registration for preparing multilayered patterns with different biomaterials. In contrast to thermal inkjet based on resistive or laser heaters, piezoelectric systems employ piezoelectric actuators, such as lead zirconate titanate (PZT), to dispense fluids, thereby reducing possible damage to the biomaterial.26
Also, because of the required physical contact, it could be possible to damage either the substrate or the deposited proteins.
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INKJET PRINTING FOR MULTIPLE SOLUTIONS DISPENSING Inkjet printing is a soft and robust micropatterning technique due to its rapidity and low cost nature, not requiring the use of masks, stamps, or other costly and time-consuming conventional processing equipment.25 It permits the dispensing of fluid droplets by generating a pressure pulse within a confined liquid, causing its ejection from a micrometric orifice (nozzle), the drop volume being affected by the nozzle size and typically 5453
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platform in which multiple reactions are conducted by piezo deposited nanoliter droplets maintained on a glass slide for the in situ preparation of thousands of derivatives of phenylboronic acid and their successive accurate activity screening on the NS3/4A protease of the hepatitis C virus.33 Recently, we applied piezo-electric inkjet technology for drug screening applications.34 Picoliter drops containing a model substrate (D-glucose)/inhibitor (D-glucal) couple were serially inkjet printed on a target enzymatic monolayer (glucose oxidase) linked to a glutaraldehyde activated silicon oxide solid surface (Figure 1D). The solid supported enzymatic surface was rinsed in water to remove the physisorbed layer finally resulting in a compact protein monolayer (height 3.6 nm) covalently linked to the silicon oxide surface. It was possible to fabricate microarrays showing quality factors comparable to pin printing spotting along with high density, high throughput (10 spots/s), and a simple colorimetric detection that results in sensitivity sufficient for the discrimination between D-glucose (only substrate) 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 inkjet printed on indium tin oxide (ITO) electrodes. Then capture anti-IgG antibodies were coupled through peptide bond formation to acidic functional groups on the nanotubes. Immunoassay were conducted via electrochemiluminescence on luminophore [Ru(bpy)2PICH2]2+] and IgG coated silica nanoparticles. An excellent detection limit of 1.1 ± 0.1 pM of IgG was found.16 Such results are promising for future development of multiplexed assays.
Accordingly, for achieving spots with high resolution and welldefined morphology without the formation of undesirable satellites, the drop rheological properties have to be optimized by adding viscous, surfactant and biocompatible additives. In this sense we showed that additives like glycerol can increase fluid viscosity at a sufficient level in order to permit satellites free droplet formation along with the retaining of the biological activity.27 Several papers report on the realization of multiplexed arrays via piezodispensing nanoliter−picoliter droplets for realizing accurate biomolecular assays involving DNA hybridization on functionalized surfaces28 and for characterizing interactions involving delicate proteins. Antibodies have been one of the reagent of choice for the preparation of protein microarrays by inkjet printing. Delehanty and Ligler reported on the employment of noncontact printer for realizing an antibody microarray biosensor for multiplex detecting protein and bacterial analytes (cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii) both individually and in samples containing mixtures of them. Capture antibodies were biotynilated and immobilized as spots on the surface of an avidin-coated glass microscope slide. The assay were executed under flow conditions using fluorescent tracer antibodies which were able to bind to analytes recognized by spotted antibodies.29 The limit of detection was of few nanograms of analyte per milliter or 104 cfu of bacterial cells per milliliter. Microarray assays require minutes versus the hours need by conventional ELISA (times needed for incubation and multiple wash steps). More recently, Li et al. showed the fabrication of three-dimensional alginate hydrogel droplet microarrays to entrap antibody-coated microbeads within spots inkjet printed on glass. Mass transport during assays is greatly favored by 3D spot architecture and high gel porosity. The microarrays enabled multiplexed sandwich immunoassays to detect six proteins including cytokines (TNF-alpha, IL-8, MIP/CCL4) and breast biomarkers (CEA and HER2) in buffer solutions and 10% serum by employing fluorescence sandwich assays with sensitivity down to the pg/mL range.30 Hasenbank et al.26 showed multiprotein patterns by inkjet printing for setting up a biosensor assay (Figure 1C). The method involved two sequential patterning steps. At first, the deposition onto a gold-coated surface of a mixed thiol layer (BAT/PEG thiol solution) to provide oriented binding capabilities in a nonfouling background; second, the deposition of multiple biotinylated proteins (b-HRP and b-BSA) (Figure 1C). Antibodies specific to each of the two proteins were introduced in a development step in the SPR microscope in order to execute detection and highly specific binding of the antibodies to the immobilized proteins. In addition, Sukumaran et al. showed the advantage given by the combination of enzyme encapsulation techniques in alginate and microarray methods for an integrated screening platform for CYP450 featured with nanomolar sensitivity detection31 by fluorescence microscopy. On the other hand, Lee et. al prepared a miniaturized 3D cell-culture array (the data analysis toxicology assay chip or DataChip) for high-throughput toxicity screening of drug candidates and their cytochrome P450-generated metabolites getting results comparable to those of 96-well plate assays.32 Together with heterophase assays, piezo inkjet droplet formation enables droplet microarray fabrication in order to conduct enzymatic assays in liquid droplets. On the basis of this approach, Mugherli et al. set up a robust enzymatic microarray
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ELECTROHYDRODYNAMIC AND PYRO-ELECTROHYDRODYNAMIC PRINTING Advancements in technologies for high-resolution noncontact liquid dispensing are constituted by methods like electrohydrodynamic (E-jet)35 and pyroelectrohydrodynamic (pyro e-jet) printing.36 They enable submicrometer lateral resolution but have not been implemented for high-throughput and multiplexed applications yet. Recently, a low cost, automatic 1 h
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