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					        Potential Applications of Polymer Microchips in Pharmaceuticals

Abstract
        Some of the most recent development in polymer based microchips have been reviewed.
Different methods used in fabricating polymer microchips are introduced. The importance and
applications of the polymer microchip in pharmaceutical and other areas are discussed. Finally,
the potential problems and development areas in polymer microchip technology are also
addressed.

Introduction
       Microprocessors have reshaped our economy, spawned vast fortunes, and changed the
way we live. Gene chips could be even bigger.
                      - David Stipp, Fortune, March 31, 1997

          Pharmaceutical discovery and development is a notoriously lengthy and costly process.
It is common practice to take over 13 years from time of a compound is synthesized to the time a
drug is marketed. The normal cost for bringing a drug into the market costs 300 millions
dollars. The length time and high cost come from the fact that many experiments have to be done
before a drug can win the approval of the regulating agency, such as the Food and Drug
Administration (FDA) of the US government. All of these experiments take time and cost
money. Due to competitive pressure, pharmaceutical companies are actively seeking
opportunities to shorten the time scale and reduce the cost in all aspects of drug discovery and
development. While advances in computation, structure chemistry, and molecular modeling are
facilitating rational design activities, empirical screening continues to play a crucial role in lead
identification. Because the ability to test large number of compounds quickly and efficiently can
provide a competitive advantage, high throughput screening (HTS) has become a key tool in
many companies. Whereas the numbers of compounds to be tested can be reduced by pre-
selection on the basis of diversity analysis or other criteria, there is a tendency to reduce the risk
of missing unexpected activity by designing screening programs that can test all of the available
compounds. Therefore, high sample throughput has become a key objective. For example, at a
weekly throughput of 2,500 compounds, it would take almost two years to finish screening a
collection of 250,000 compounds. A collection of 250,000 compounds is common in many
small-to-medium sized pharmaceutical companies. Apparently, the throughput needs to be
higher. In the last ten years, the throughput has increased from 10,000 per year per target in the
middle to later 1980s to 10,000 compounds per month per target (over 10 fold increase) in early
1990s to the 10,000 compounds per-week per target in the middle 1990s. Currently, we are
working on a throughput of over 10,000 compounds per day per target.
          Whereas increased throughput can be achieved to some extent by deploying additional
human resources, this is not usually an attractive option. Screening systems and associated
infrastructures are required that can sustain testing rates of many thousands of compounds per run
without the need for additional manpower. To achieve the necessary productivity, effective
integration of compound supply, assay operation and data management is essential.
          Micro-total chemical analysis (-TAS) system offers a novel way of achieving fast
separations with high resolution in a miniaturized setup including sample preparation such as
sample concentration, labeling, and digestion.
          Review [13]
 Review [14]: China
          Critical issues




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        Since the cost of fabrication microchips is prohibitive, people have worked very hard to
search for alternative solutions. The possible alternatives are polymer-based materials. These
polymer (plastic or rubber) chips offer several advantages: a) low cost, single-use devices, b)
multi-layer devices for complex fluidics, c) low native EOF-DNA separation without surface
coating, greater variety of surface modifications, flexible control of EOF for complex fluidics,
low temperature bonding of cover to chip base

Fabrication
        Previously, various polymer materials have been used in capillary electrophoresis.
Fluoropolymers, such as PTFE and poly(fluoroethylene-propylene), polyfluorocarbon,
polyethylene, poly(vinylchloride), polypropylene, and poly(butylene terephthalate) have all be
used for this purposes.
        Depending on the materials selected, several different methods have been used to
fabricate various polymer microchips. Eckstrom et al. [18] and Soane et al. [19] described the
use of various polymers, including fluoropolymers and silicone rubber, for producing
microfabricated fluid handling and electrophoretic devices. Effenhauser et al. prepared silicone
devices by casting protocol [6]. Kaltenbach et al. described the production of microseparation
channels in plastic substrates via laser ablation [20].

         Casting
         Polydimethylsiloxane (PDMS) is one of the most common materials used to make
polymer microchips. PDMS comes in liquid form and can be cured at room temperature. The
most commonly used PDMS reagents are Sylgard® 184 (Dow Corning, Midlan, MI), which
contains two components. When they are mixed at 1:10 ratio, it may be allowed to settle for one
hour to remove the air bubble. Then, the device can be cured within three hours at 75 °C or 24
hours at room temperature. In general, the fabrication of PDMS devices is similar to the process
of glass chip fabrication. Fig 1a is the general scheme of the fabrication steps [2]. In this case,
the micromachining fabricated glass mold is used to fabricate PDMS device directly. Whitesides
and his group developed a new procedure that allowed them to rapidly prototype masks and to
obtain the PDMS chip device within 24 hours [9].
         One of the beauties of the PDMS device is that the PDMS chips can be hermetically
sealed to a flat glass, such as a microscope plate or to a silicone slab. Thus, PDMS device offers
a significant advantage over glass chips in that it can be peeled off and cleaned right away in case
the capillaries are clogged. Another advantage is that this process can be accomplished at low
temperature (as compared to over 600 °C for making the glass chips) for the whole process,
making it feasible to interface with other electronic device [8].

         Injection Molding
         Microfabricated electrophoretic separation devices can also be produced by an injection
molding process [1]. Injection molding affords the possibility of producing hundreds of
thousands of essentially identical microfabricated plastic separation devices from a single master
in a cost-effective manner. These separation devices have demonstrated high-resolution
separations of several biological analytes with total run times of only a few minutes.
         The strategy for production of injection molded chips are illustrated in Fig 1b, which
shows a silicon master is made by micro-fabrication process first. Then, a nickel electroform
mother was formed based the silicon master. Multiple daughter forms can then be generated from
the mother form. Usually, more than 10,000 injection-molded chips can be made from a single
nickel daughter form. The surface profiles of the silicon master, the electroform mother, and the
injection molded chips are shown in Fig 2. It is clear to see that the profile of the injection
molded plastic chip is not perfect in the surface area. This is a place that polymer chemists can



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help to improve. Scanning electron micrographs (SEM) of channels in the injection-molded chips
indicated that the shape of the channels is quite smooth.

         Etching and photoablation
         Deep X-ray etching was used to generate capillaries (width equals 50 m; depth equals
50 m) on polymethylmethacrylate (PMMA) [14]. UV excimer laser photoablation can be used
to micro-machine polymer substrates not only to drill microchannel structures but also to change
the surface physical properties of the substrates [15]. Witt et al. used an excimer laser to ablate
microstructures into polyimide to create micro-channels with "built-in" alignment feature [16].
The micro-alignment features provide a micro-fluidic interface that integrates into the HPCE
format.

Evaluation

         System Set-up
         The experimental setup for the evaluation of the microchips is shown in Fig. 3. This is
for a simple design with only one separation channel and a cross sample injection channel. The
four electrodes are connected to the header connector for easy operation.
         Whereas there are several detection methods, including electrical chemical [3, 31], MS
[4, 32], and UV [5], the most common method of detection is laser induced fluorescence (LIF)
detection. The detection system can be done using an inverted microscope. The basic optical
pass is shown in Fig. 4a. An alternative design of the detection scheme is to introduce the light
source from the side of the chip and collect the fluorescence from the top using a CCD (charge
coupled device) camera (Fig 4b [2] frame 3).

         Ohm's Law
         In addition to the physical profiling of the channels, it is crucial to evaluate the
relationship between the current and voltage (Ohm's law). Based on the fundamental theory of
capillary electrophoresis (CE), the separation efficiency is proportional to the voltage. Thus,
higher voltage is desirable. In conventional CE experiments, the capillary is usually 25-70 cm
long and the electric field strength is at only a few hundred volts per centimeter due to the
limitation of the power supply (30 K at the upper limit). With microchip, the separation length is
usually only a few centimeters. It is possible to apply very high electric field strength onto the
chip to achieve fast separation with high efficiency. But, too high electric field will result in an
electric breakdown. A comparison study to compare the Ohm's law from the acrylic polymer
chip and the fused silica capillary indicated that the response curve matched very well below
6000 volts (approximately 1500 V/cm).

         Electro-osmotic flow
         The electro-osmotic flow (EOF) is another parameter that needs to be evaluated. Simply,
EOF is the bulk movement of the liquid in the micro-channel under electrical field. For the
injection molded plastic chips, the EOF was significantly lower than that in fused silica capillary
[1]. But, in the PDMS chips, EOF comparable to that in the fused silica capillary was obtained
[2]. In addition, the change of EOF as a function of buffer pH was also similar to that in fused
silica capillary [2] (Frame 5).

Applications
        Microchip offers lower reagent consumption, faster separations, and integrated sample
handling steps, such as on-chip mixing, dilution and labeling, and the possibility of running



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multiple analyses in parallel by microfabrication of a multi-channel device. As expected,
polymer microchip devices could lead to a variety of applications, such as DNA analysis (sizing,
sorting and sequencing), combinatorial library screening and rapid immunoassay testing.

         DNA Analysis
         Gene Chip:
         The Affimetrix (Santa Clara, CA) GeneChip probe array is packaged for single-use
convenience, each probe array contains from tens to hundreds of thousands of different
oligonucleotide probes, where their sequences, lengths and locations within the array are known.
For example, GeneChip HIV PRT probe arrays contain over 15,000 different probes. They are
designed to perform high-accuracy sequence analysis on protions of the HIV-1 virus [33]. Most
of the current GeneChips are made of glass, it is anticipated that polymer chips will soon replace
glass chips due to the significant potential of cost savings.
         DNA probes can also be attached to PDMS devices and then used as sensor for the
precise recognition and subsequent detection of a specific complimentary DNA targets for
diagnosis and genetic screening [3].
         DNA and Cell Sorting and Sequencing:
         Asymmetric flow cells etches on silicon chips show promise as size separation tool to
sort DNA [10]. This technique has unique advantage in that it does not involve any gels and can
be easily automated. Cell sorting can also be done using similar technique [7].
         Fig 5 shows the separation of DNA on an acrylic injection molded chip in less than three
minutes using a 4-cm separation length [1].

        DNA Sequencing:
        Due to the development of Human Genome Project in the past few years, DNA
sequencing is one of the most rapidly developed area [21]. By using 95-sample capillary array
electrophoresis, a throughput that is 50 to 100 times greater than that of conventional slab gels
can be achieved [22].

Protein Analysis
        Since microchip device facilitates the on-chip mixing and reaction, it is convenient to
derivative proteins with fluorescent reagents such as 2-toluidinonaphthalene-6-sulfonate (TNS).
The separation of human serum proteins, IgG, transferrin, -1antitrypsin, and albumin, can be
separated and detected by LIF after reacting with TNS within 50 seconds [12]. The separation of
BSA and IGG was accomplished in less than 2 min using the injection molded chip (Fig. 6).
        Several groups have reported the use of microchip based immunoassays for the analysis
of monoclonal mouse bovine serum albumin, cortisol, and serum theophylline [12].

Pharmaceuticals
         Since enzymes are the common targets for many drug development programs,
characterizing enzymes is very important in pharmaceutical industry. This involves the
determination of the equilibration constant (Km) and the maximum reaction rate (V max). Once
characterized, the enzyme can be used as target to evaluation various compounds for potential
drug candidate based on their inhibition to the enzyme. Microchip has been reported for this
application.
         It is expected that HTS will be the common means for pharmaceutical discoveries. We
have obtained the separation of reaction mixtures in 96 capillaries simultaneously within 8 min.
This means that it is possible to screen over 150,000 compound per 24-hour day. When similar
strategy is applied to microchip, it is expected that the throughout could be even higher. Another
significance is that the above screening throughput is achieved based on separation, which means



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the quality of the screening results will be much more reliable and much less false "hit". If no
separation is needed, microchips with over 10,000 wells on a single microchip is commercially
available and thousands of compounds can be assayed within a few minutes are possible in the
near future.

Conclusion
         Microchip based total chemical analysis system (-TAS) is gaining more than ever
popularity in biological, clinical and pharmaceutical applications. Various polymer materials can
be used as substitute for glass or silicon materials to reduce the cost of the total systems. Various
methods can be used to make different polymer microchips. These polymer-based microchips
have show performance characteristics comparable to chips made of glass or silica. For example,
under the conditions tested, the thermal dissipation in acrylic chips is comparable to cylindrical
fused silica capillaries. High-resolution separations of DNA, proteins and other compounds can
be attained in polymer microchips. The potential in HTS, due to the feasibility of parallel
analysis, of these microchips has attracted many pharmaceutical companies to invest large
amount of money in pursuing the advancement of this technology.


Future Development
         Despite the great potential, there are still several hurdles before the microchip technology
can be fully utilized in pharmaceutical industry.
         First, the surface characteristics will play an important role. One of the limitations of the
injection-molded chip is the relatively low EOF. This problem may be overcome by coating the
surface with proper layers. It is possible to generate EOF as high as the fused silica capillary.
         Second, the background fluorescence of plastics is typically higher than fused silica.
This problem may be avoided if other detection methods are chosen. However, this problem will
remain as long as LIF is the primary detector. Maybe material scientists can help us to solve this
problem by finding better materials.
         Third, the dielectric breakdown of plastics is generally lower than silica or glass. This
characteristic limits the maximum applicable electric potential.
         Protein/DNA adsorption onto chip walls is another problem we have to deal with when
polymer microchip is used for protein and DNA separations. Fortunately, there have been a lot of
experiences and knowledge accumulated in dealing with this problem in the CE field.
         Cover-plate sealing is another issue when polymer materials are used to make
microchips. The sealing should not affect the characteristics of the channels and should not block
the channels. Fortunately, there are many good sealant available for selection.

References
[1] a) ] R. M. McCormick, R. J. Nelson, M. G. Alonso-Amigo, D. J. Benvegnu, and H. H.
Hooper, Mcirochannel Electrophoretic Separations of DNA in Injection-Molded Plastic
Substrates, Anal. Chem. 1997, 69, 2626-2630. B) R. M. McCormick, R. J. Nelson, M. G. Alonso-
Amigo, D. J. Benvegnu, H. Y. Wang, T. D. Boone, H. H. Hooper, Mcirochannel Electrophoretic
Separations of DNA and Proteins in Injection-Molded Plastic Substrates, HPCE'97 poster,
Anaheim, Feb., 1997.
[2] G. Ocvirk, M. Munroe, T. Tang, R. Oleschuk, K. Westra, D. J. Harrison, Important
parameters for CE with PDMS devices, HPCE'99 poster.
[3] B. Davis, S. A. Brooks, and W. G. Kuhr, PDMS microfluidic chip for direct detection of DNA
with an integrated electrochemical detector, HPCE'99 poster.
[4] Q. F. Xue, F. Foret, Y. M. Dunayevskiy, P. M. Zavracky, N. E. Mcgruer, and B. L. Karger,
Anal. Chem. 1997, 69, 426-430.


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[5] Z. Liang, N. Chiem, G. Ocvirk, T. Tang, K. Fluri, D. J. Harrison, Anal. Chem. 1996, 68,
1040-1048.
[6] C. S. Effenhauser, G. J. M. Bruin, A. Paulus, M. Ehrat, Anal. Chem. 1997, 69, 3451-3457.
[7] H. P. Chou, C. Spence, A. Fu, A. Scherer and S. Quake, Disposable micro devices for DNA
analysis and cell sorting, in International Workshop on Solid State Sensor and Actuator (Hilton
Head'97), pp11-13, South Carolina, June 8-11, 1998.
[8] P. F. Man, D. K. Jones and C. H. Mastrangelo, IEEE, 1997, 0-7803-3744-1/97, pp311-316.
[9] D. C. Duffy, J. C. McDonald, Schueller, O. J. A., G. W. Whitesides, Anal. Chem., 1998, 70,
4974-4984.
[10] a) S. Borman, C&EN 33-34, March 9, 1998. b) D. Ertas, W. D. Volkmuth, T. Duke, R. H.
Austin, Physical Review Letters, 1998, 80, 1548 and 1552.
[11]
[12] C. L. Colyer, T. Tang, N. Chiem, and D. J. Harrison, Electrophoresis, 1997, 18, 1733-1741.
[13] J. Major, Review
[14] S. A. Soper, S..M. Ford, Y. Xu, S. Qi, S. McWhorter, S. Lassiter, D. Patterson, and R. C.
Bruch, Proc. SPIE-int. Soc. Opt. Eng., 1999, 3602 (Advances in fluorescence snesing technology
IV), 392-402.
[15] J. S. Rossier, A. Shwarz, F. Reymond, R. Ferrigno, F. Bianchi, and H. H. Girault,
Electrophoresis, 1999, 20 (4-5), 727-731.

[16] K. Witt, P. Kaltenbach, L. Mittelstadt, R. Brennen, S. Swedberg, T. van de Goor, K. Robotti,
and S. Udiavar, Miniaturized planar columns in polymeric support media for liquid phase
analysis, HPCE'98, Orlando, Fl., Feb. 1998.

[18] B. Eckstrom, G. Jacobson, O. Ohman, H. Sjodin, World Patent WO91-16966, 1991.
[19] D. S. Soane and Z. M. Soane, US Patent 5,126,022, 1992.
[20] P. Kaltenbach, L. Mittelstadt, and S. Swedberg, US Patent 5,500,071, 1996.
[21] A. T. Woolley & R. A. Mathies, Anal. Chem., 1995, 67, 3676-3680.
[22] P. C. Simpson, D. Roach, A. T. Woolley, T. Thorsen, R. Johnston, G. F. Sensabaugh, and R.
A. Mathies, PNAS online (http://www.pnas.org/cgi/content/full/95/5/2256), 1998, 95, 2256-2261.

[31] P. F. Gavin and A. G. Ewing, J. Microcolumn Separations, 1998, 10, 357-364.
[32] F. Xiang, Y. Lin, J. Wen, D. M. Matson, and R. D. Smith, Anal. Chem. 1999, 71, 1485-
1490.
[33] T. Studt, Gene chip technologies transform biological research, R&D Magazine, Feb. 1998,
38-42. Also see http://www.affimetrix.com.




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                                                               Silicon Master



                                                     Nickel Electroform Mother



                                                    Nickel Eletroform Doughter



                                                            10,000's IM Chips


                           Fig 1 Procedures of making the injection mold plastic chips


Fig 2 Profilometer scans of silicon master, nickel electroform mother, and injection dolfed chip
([1, Mccormick]).
Fig. 3([1, Mccormick]).

Fig 4a ([1, Mccormick]).

Fig. 5 ([1, Mccormick]).
Fig 6 (1, Mccormick]).



Abstract .......................................................................................................................................................... 1
Introduction .................................................................................................................................................... 1
Fabrication ...................................................................................................................................................... 2
   Casting ........................................................................................................................................................ 2
   Injection Molding ....................................................................................................................................... 2
   Etching and photoablation .......................................................................................................................... 3
Evaluation ....................................................................................................................................................... 3
   System Set-up ............................................................................................................................................. 3
   Ohm's Law.................................................................................................................................................. 3
   Electro-osmotic flow .................................................................................................................................. 3
Applications.................................................................................................................................................... 3
   DNA Analysis ............................................................................................................................................ 4
   Protein Analysis.......................................................................................................................................... 4
   Pharmaceuticals .......................................................................................................................................... 4
Conclusion ...................................................................................................................................................... 5
Future Development ....................................................................................................................................... 5
References ...................................................................................................................................................... 5




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