Meeting Report

automated manner. As emerging tools Chips to Hits: and for , arrays have a number of advantages over conven- microfluidic technologies for tional methods of protein analysis using high-throughput analysis and 2D gel and mass spec- trometry, since they can be used to drug discovery detect with low concentrations in a high-throughput manner. September12-15, 2005, MA, USA In general, there are two types of pro- Ali Khademhosseini tein microarrays: microarrays and functional protein microarrays. Harvard-MIT Division of Health Sciences & Technology, Massachusetts Institute of Technology, Cambridge, Antibody microarrays are miniaturized MA 02139, USA; Department of Medicine, Brigham & Women’s Hospital, Harvard Medical School, Boston, enzyme-linked immunosorbent assays MA 02115, USA; Tel.: +1 617 253 3638; Fax: +1 617 258 8827; [email protected] that can be used to detect proteins with Expert Rev. Mol. Diagn. 5(6), 843–846 (2005) high sensitivity and selectivity. On the other hand, functional protein micro- arrays can be used to study protein inter- Methods of performing experiments One of the most successful applica- actions with other molecules. At Chips to cheaper, faster, easier, as well as with a tions of microarrays is for RNA analysis. Hits, Michael Snyder (Yale University, higher sensitivity and throughput, are This technology, which was pioneered USA) presented on the use of yeast pro- desirable for many scientific endeavors by Affimetrix, Inc., is now commonly tein microarrays to identify novel part- and commercial applications. In partic- used in many biologic and drug discov- ners within protein complexes, and to ular, improved technologies are benefi- ery studies to profile gene expression analyze the multifunctional nature of cial for the discovery of new drugs and within cells. DNA microarrays can be some proteins [1]. Using these assays, the analysis of biologic systems, where used to conduct large-scale quantitative novel functions could be found for even many different molecules must be ana- experiments that measure changes in well-studied proteins. Also, the use of lyzed in a combinatorial and concentra- gene expression for a particular tissue or protein arrays to understand phosphor- tion-dependant manner. A major type. The use of such technologies ylation mechanisms was demonstrated theme of the 2005 Chips to Hits confer- was a key aspect of the Chips to Hits by studying the binding of yeast protein ence recently held in Boston, Massa- conference, with many companies repre- kinases to large protein microarrays. chusetts, USA revolved around the senting their latest technologic advance- Using the data generated from pro- emergence of microarray and micro- ments and products. For example, Alan tein–protein, protein–DNA binding and fluidic technologies in addressing barriers Sachs (Merck Research Laboratories) phosphorylation experiments, network to high-throughput experimentation. presented on the use of DNA microar- maps could be generated that contain ray technology for enabling drug discov- much of the interaction and regulatory DNA & protein microarrays ery by using animal models and tumor pathways within a cell [2,3]. This ability Microarrays can be Authorused to perform samples to identify drug targets.Proofto construct regulato ry networks and to high-throughput experiments by localiz- Although gene arrays are good indica- analyze biologic systems may have great ing tests to small regions of a 2D sub- tors of RNA expression within a cell, implications in understanding the strate. By restricting the location of indi- they are not necessarily representative of underlying biologic mechanisms and vidual experiments, microarrays can the level of protein expression, due to identifying candidate drugs. dramatically miniaturize assays. Microar- complicated dynamics of translational Despite the remarkable potential of ray technologies have been widely used and post-translational modification proc- protein microarrays for understanding in academia and industry. It is estimated esses. Therefore, complementing gene biologic systems, significant technologi- that these technologies currently have a arrays with protein arrays is critical for cal challenges remain. Current research market of approximately 2 billion dollars producing a true picture of the state of thrusts in the area of microarrays involve with more than 350 companies selling the cell. It is envisioned that protein increasing the sensitivity of the assays, products. In addition, there are over arrays can be potentially used to perform minimizing false positives and increasing 150 academic institutes and approxi- protein–protein, protein–DNA, pro- the throughput of the assays. For exam- mately 10,000 researchers conducting tein–drug, or enzyme substrate screen- ple, since protein–protein interactions research in this field. ing assays in a sensitive, parallel and are complicated by the existence of many

10.1586/14737159.5.6.843 © 2005 Future Drugs Ltd ISSN 1473-7159 843 Khademhosseini post-translational modifications, such as the identification of cues that induce identifying drug target interactions and phosphorylation and glycosylation, pro- desired cell responses. Also, the materials for evaluating phenotypic changes result- tein arrays must be fabricated that can that yield desired responses could be used ing from the expression of specific pro- sense such interactions by using post- as templates for tissue scaf- teins in the cells. translationally modified proteins or spe- folds. Such an approach is a radical Other types of cell microarrays have cific . Also, technological bar- change from traditional methods of also been developed in which cells are riers exist for protein arrays that must be developing new biomaterials, where pol- patterned on surfaces comprised of adhe- optimized to further facilitate their wide- ymers have been individually developed sive or nonadhesive regions. As adhesive spread use for biologic studies. Current and tested for their effect on cells. In cells are seeded on these substrates, they research ranges from enhancing the func- addition, the effect of natural ECM mol- adhere to the patterned regions. Alterna- tionality of proteins by using spacer mol- ecules on cell fate can be evaluated in a tively, microwells have been used, in ecules, to minimizing nonspecific protein high-throughput manner. For example, which cells, including nonadhesive cells, adsorption through surface modification. combinatorial matrices of various natural are plated within the microwells and For example, Jason Armstrong (Bio- ECM proteins were tested for their abil- remain physically separated from each Layer, Austria), presented the use of bio- ity to maintain the function of differenti- other. Cell arrays have been used to study mimetic surfaces to improve various ated hepatocytes and to induce hepatic cell behavior in a clonal manner [14,15] or assays in the range of 6- to 50-fold. differentiation from murine ES cells [9]. to study the effects of drugs on cells in a These technologies may provide addi- Other emerging microarray technolo- high-throughput manner [16,17]. Current tional functionality and sensitivity to gies include patterned polysaccharide limitations with cell array technologies today’s microarray technology. and bead arrays [10]. Polysaccharide arrays include lack of suitable technologies to can be used to study the interaction of for many different chemicals as well Other polymeric microarrays chemical libraries or drugs with polysac- as maintaining proper cellular Microarrays fabricated from polymers or charides. Such interactions will be partic- in culture. Methods of integrating cell extracellular matrix (ECM) proteins can ularly important in finding new drugs arrays within microfluidic channels, gen- also be used to test extracellular condi- that interact with the cell surface polysac- erating patterned co-cultures and multi- tions on cellular behavior or to test charide molecules, and are potentially phenotype cell arrays are emerging fea- molecular libraries. Such libraries are use- important tools for understanding the tures which may solve these challenges. ful for and have interaction of sugars with other mole- The cell array technology requires further already yielded candidates that have been cules in the emerging filed of . development, but has the potential to shown to induce osteogenesis [4] and car- lend itself to a broad range of functional diomyogenesis [5] from embryonic stem Cell microarrays ultrahigh-throughput cell-based assays. (ES) cells as well as the dedifferentiation Cellular micropatterning can be used to of committed cells [6]. Recently, research- control cell shape and subsequent behav- ers in Robert Langer and Sangeeta Bha- ior (i.e., migration, proliferation, differ- Microfluidics is a potentially powerful tia’s groups (Massachusetts Institute of entiation and apoptosis) [11,12]. Cell method of performing high-throughput Technology, USA) have used robotic arrays are also a potentially powerful tool experiments that encompass a series of spotters capable of dispensing and for performing high-throughput bio- techniques used for controlling the flow immobilizing nanoliters of material to logic studies. There have been a number and reaction of minute amounts of liq- fabricate microarrays, where cell–matrix of studies that have demonstrated the uids or gases. Microfluidic systems have a interactions can be optimized in a high- power of cell-based assays. For example, number of benefits, which include throughput manner. For example,Author syn- Sabatini and colleagues have Proof demon- reduced waste, improved sensitivity/pre- thetic biomaterial arrays have been fabri- strated the parallel transfection of hun- cision, reduced cost, reduced energy con- cated to test the interaction of stem cells dreds of genes in a microarray format [13]. sumption, miniaturized experiments and with various extracellular signals [7]. In In this technique, a printed array of full- an increase in the speed of the reactions this approach, thousands of polymeric length open reading frames of the genes by reducing times. With typical materials were synthesized and their in expression vectors, along with lipid channel dimensions of 5–100 µm, effect on differentiation of human ES transfection reagents, were used to trans- devices comprised of complex networks cells [7] or mesenchymal stem cells [8] was fect cells. The phenotypic effects of vari- of fluidic microchannels and intercon- evaluated. These interactions have led to ous genes can then be analyzed. One nects can be generated around the size of unexpected and novel cell–material inter- potential use of the cell microarrays may postage stamps. actions. Although the molecular mecha- be to test the repression or silencing of Many companies have been developing nisms associated with the biologic genes in a sequence-specific manner microfluidic technology for various high- responses have yet to be clarified, such using small, single-stranded antisense oli- throughput applications such as immu- technology may be widely applicable in gonucleotides, or small interfering RNA. noassays, diagnostic devices, single mole- cell–microenvironment studies and in Cell array systems can be utilized for cule DNA and protein detection as well

844 Expert Rev. Mol. Diagn. 5(6), (2005) Chips to Hits as cell separation. Research is also being equipment. In order to stream of sample is layered over the performed in many academic laborato- alleviate these needs, soft lithographic immobilized capture molecules, and ries on novel applications of microfluidic approaches have been developed. In soft binding events produce changes in the technology. For example, researchers , polymers are cured on a sili- diffractive light signal. Using total inter- from the University of Chicago, USA, con master, which is microfabricated nal reflectance diffraction, a pattern is and other laboratories have demonstrated once and can be repeatedly used to gen- generated in which the diffraction of light the use of two-phase droplet systems to erate molded replicas. Polydimethyl- can be detected and quantified. Research generate droplets within microfluidic siloxane (PDMS), an elastomeric and in this technology is under way in aca- channels that could be used as microreac- transparent material, has been exten- demic and commercial sectors, and has tors [18,19]. These nanoliter plugs can be sively used for this fabrication approach. significant potential to improve today’s used for high-throughput screening of However, there are potential disadvan- detection methods. Alternatively, micro- compounds and can be used to perform tages associated with PDMS, which cantilevers can be used within micro- multiple chemical reactions. Microfluidic include its hydrophobicity and inability devices for the detection of biologic mole- drops can also be used for studying crys- to resist protein adhesion. In order to cules. Microcantilever biosensors are tal growth and nucleation, which could address these problems, PDMS surfaces label-free, real-time and silicon-based be used for many chemical screening or have been coated with nonbiofouling tools in which the free end of the canti- protein purification assays. molecules such as PEGs [20] and polysac- lever moves up or down upon a biorecog- One of the limitations of microfluidic charides [21]. Alternatively, many micro- nition event. Microcantilevers can be technologies has been the micro- to fluidic devices have used the physisorp- conjugated with proteins or DNA, and macroscale interface. In most applica- tion of serum proteins, such as albumin, can be fabricated in a highly parallel and tions, the number of inlets is limited by to minimize surface biofouling of func- miniaturized fashion, making them useful physical geometries of the tubing and tionalized microchips. Alternatives to for high-throughput analysis. syringe . Research is currently PDMS include molding of polymethyl- underway in many aspects of interfacing methacrylates-based [22] or fluoropoly- Commercialization microfluidic chips to their surroundings. mer-based [23] materials. These alterna- The commercialization of microarray A number of approaches have been devel- tive materials and other emerging and microfluidic technologies is an oped in order to eliminate external techniques are advantageous with ongoing process as demonstrated by the pumps. In one scheme, microchannels respect to their mechanical and protein emergence of many start-up companies. sealed under vacuum have been formed biofouling properties. Throughout the conference, potential in which the exposure of the liquid to the Finally, the widespread use of micro- routes and challenges to commercializa- vacuum can be used to draw the fluidic technologies and microarrays for tion were discussed. Richard Fisler into the channels for subsequent analysis. high-throughput applications is limited (Beachhead Consulting) discussed the Alternatively, hydrophilic surface modifi- by the detection methods. Research in market for such technologies and noted cation of microchannels using plasma detection methods has focused on that, as technologies have treatment, dextran or poly(ethelyne gly- increasing the sensitivity, portability, cost shown, promising technologies often col) (PEG) have been presented as meth- and size of these devices. Various modes take longer to deliver market success ods of spontaneously delivering fluids of detection are currently in use. These than initially anticipated. Entrepre- into the channels. Finally, methods of range from - and absorb- neurs need to realize that, even though using gravitational forces have been used ance-based optical methods, to other it takes a relatively small amount of to drive the fluids within the microchan- approaches such as surface plasmon reso- money to demonstrate the proof-of-con- nels. In one approach, forcesAuthor generated by nance (SPR) and diffractive Proof optics tech- cept for a product, it takes much more a rotating disk are used to drive the fluids nology. In many cases, fluorescence has money and time to commercialize a suc- through the microfluidic channels. The been used as the desired source of detec- cessful product. Also, for a new techno- is filled at the center of the disks and tion due to its high sensitivity and accu- logy to reach commercial success, it it is subsequently carried through the racy. However, fluorescent detectors are needs to be much more advantageous channels using microchannels. not easily integrated within portable over existing technologies. One example To make microfluidic technologies devices. SPR is an alternative method of is Affymetrix, who generated a new mar- cheaper and more mechanically robust, detection that provides potential advan- ket based on their GeneChip® technol- research is underway to improve micro- tages. SPR works by measuring the bind- ogy over a 12-year period. Despite the channel fabrication. Traditionally, ing of an analyte to a ligand or receptor barriers to enter the market, the future microchannels have been fabricated in on the surface. Since SPR measures the looks promising. For example, despite silicon or glass using cumbersome litho- changes in the mass on the surface in a the initial caution and hesitation in graphic methods. However, these tech- real-time and quantitative manner, it is marketing products niques require extensive technologic not necessary to label the analyte. In dif- or high-throughput microfluidic assays expertise and access to clean rooms and fractive optics technology, a flowing for drug discovery, certain applications

www.future-drugs.com 845 Khademhosseini of these technologies have become tools has become a bottleneck for patenting. diagnostic applications since they perform that are routinely used in basic research This increase in the number of patent experiments with higher sensitivity while and drug discovery. applications is a further indication that using less reagents. Current research Microarray and microfluidic techno- the future brings many opportunities in a revolves around increasing the sensitivity logies also face and pose unprecedented competitive market environment for and the speed of these technologies, while challenges to intellectual property law. As emerging companies. minimizing their size and cost. The inte- discussed by Kathleen Williams (Palmer gration of microfluidic and microarray & Dodge LLP), in the last decade, the Conclusions technologies may also lead to unmatched number of patent applications in Microarray and microfluidic technologies throughput for diagnostics and screening or lab-on-a-chip technol- have significant potential in increasing the applications. With respect to commercial- ogy has increased from approximately 300 throughput of existing assays for analyzing ization, although commercially successful to 4000 per year. This increase has been molecules (such as DNA, RNA, proteins products have been generated, many difficult to handle for agencies such as the and polysaccharides) as well as cells. These opportunities remain that rely on US Patent and Trademark Office, which technologies could also be used for many improved technologies and niche markets.

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