Recent progress in microfluidic devices for nucleic acid and antibody ...

Recent Progress in Microfluidic Devices for Nucleic Acid and Antibody Assays PONNAMBALAM RAVI SELVAGANAPATHY, EDWIN T. CARLEN, CARLOS H. MASTRANGELO, MEMBER, IEEE

AND

Invited Paper

Microfluidic devices are increasingly important in biomedical assays. In this paper, we review recent progress on these devices for chemical amplification, hybridization, separation, and detection of nucleic acids. Recent developments in microfluidic technologies for detection of protein binding events in immunoassay applications are also reviewed. Keywords—DNA, immunoassays, microelectromechnical systems (MEMS), microfluidic assays.

I. INTRODUCTION The use of miniaturization technologies in biomedical assays today is pervasive and steadily increasing. Miniaturization promises realization of assays with low reagent volumes and costs. Microelectromechnical systems (MEMS)-based microfluidic technologies permit scaling at the micrometer range; thus, they provide a path for implementing massively multiplexed, arrayed assays of small size that may be ultimately used in futuristic laboratory and point-of-care medical devices. In this paper, we summarize a few recent applications of MEMS microfluidic technology for two of the most important types of assays. Nucleic acid analysis is the basis for detection and identification of genetic pathogens and diseases, and protein binding is widely used for the detection of antigens and antibodies in immunoassays. II. NUCLEIC ACID ASSAYS The most pervasive examples of microfluidic technology are present in DNA assay chips. Several factors contribute Manuscript received March 4, 2003; revised March 10, 2003. P. R. Selvaganapathy is with the University of Michigan, Department of Electrical Engineering and Computer Science, Ann Arbor, MI 48109-2122 USA (e-mail: [email protected]). E. T. Carlen and C. H. Mastrangelo are with Corning-Intellisense, Wilmington, MA 01887 USA (e-mail: [email protected]; [email protected]). Digital Object Identifier 10.1109/JPROC.2003.813569

to this position. Among all the nucleic acids, DNA is fairly convenient to handle, whereas RNA is less so, owing to ribonuclease activity. Further, a large volume of widely accepted protocols for handling of nucleic acids already exists [1]. DNA is stable under laboratory conditions and interacts favorably with surfaces, unlike proteins that tend to denature. DNA is also inherently self-complimentary; hence, its identification and isolation can be performed using a single strand immobilized on the substrate to detect the complimentary strand in an analyte. The analysis of DNA samples requires one or several chemical processing steps, each step performing one of several “chemical toolkit” functions. These functions are digestion, chemical amplification, hybridization, separation, and detection. Specialized chips implementing these functions as well as integrated versions implementing sequences of these have been microfabricated with various technologies. A. Amplification Chips 1) Chemical Amplification: Biochemical amplification is a molecular recognition technique that chemically amplifies a small part of a large strand of DNA, exponentially (as large as 2 times), without simultaneous amplification of other genetic material present in equal or higher quantities in the solution. Amplification schemes generally exploit natural biochemical processes that regularly occur within the cells for replication and repair of DNA and expression of proteins. Enzymes (proteins which perform specific function in cells), like DNA polymerase, perform the task of replication of the DNA from a single strand of DNA present. This, along with the temperature-based denaturation and hybridization of double-stranded DNA, are used for an ingenious method of producing a large number of copies of a specific strand of DNA, as illustrated in Fig. 1. The strand to be amplified depends on the choice of primers, which are short synthetic chains of complementary nucleotides to the genetic sequence on either flanks of the

0018-9219/03$17.00 © 2003 IEEE

954

PROCEEDINGS OF THE IEEE, VOL. 91, NO. 6, JUNE 2003

Fig. 1. The PCR. The double-stranded fragment is first separated (denatured) into two strands by heating the sample to 92 C. The primers next attach to the 3 ends of DNA. Starting from the primer, the enzyme scans the single strands and reconstructs the complementary strand at 60 C–70 C, producing two double-stranded fragments. The cycle can then be repeated [2].

Fig. 2. [4].

Silicon microwell with integrated heater for PCR reaction

targeted section. The primers hybridize on either ends of the targeted section, and the polymerase enzyme constructs the rest of the chain between them, from the raw materials (single nucleotides), which are added with the primers. Once formed, these strands are denatured and a new strand is formed on the same template. Since the newly formed copy also forms another copy, the process grows exponentially. The cycle of denaturation and hybridization is done through temperature cycling, which lends itself for microfabrication. 2) PCR Microdevices: Early microfabricated polymerase chain reaction (PCR) devices were mostly disposable sample storage devices heated by external thermocyclers in which the reaction could occur quickly taking advantage of the rapid thermal diffusion in the small volume for heating and cooling [3]. These systems are capable of heating rates of 15 C/s and total cycle time around 30 min. The cycling was achieved at a rate four times faster and at much lower power than conventional tube-based thermocyclers. More recent developments have incorporated the heating systems into the chip, as shown in Fig. 2, using photolithographic construction techniques [4]–[7], thereby reducing the total cycling time to 15 min. Micromachined PCR devices have been fabricated with a variety of materials ranging from glass [8] and silicon-glass [9] to plastics [10]. Both thermal [12] and isothermal [7], [13] amplification techniques have been demonstrated. A major problem

Fig. 3. Schematic of a chip for flow through PCR. Three well-defined zones are kept at 95 C, 77 C, and 60 C by means of thermally controlled copper blocks. The sample is hydrostatically pumped through a single channel etched into the glass chip. The channel passing through the three temperature zones defines the thermocycling process [8].

associated with amplification microsystems is the large surface-to-volume ratio, where an increase in surface-to-volume ratio from 1.5 in PCR tubes to 20.4 in microfabricated systems [12] is typical. Various biocompatible surfaces have been investigated, including passivated silicon, fused silica, glass, and plastic with walls via coatings by chemical modification or physical adsorption. Passivation using oxide produces consistent and reproducible results [12], [14]. Several modifications have been introduced mainly in the methodology of the thermal cycling to produce efficient analysis within a shorter time. Instead of cycling the temperature in a reaction chamber with the PCR mixture static, a reaction format consisting of a microchannel passing through regions of microchip at three different temperatures repeatedly to perform denaturation, hybridization, and amplification was conceived by Kopp et al. [8]. In the design of the microchip, as shown in Fig. 3, the microchannel passed through the three regions 20 times giving a theoretical amplification of 2 . The total residence time of the reaction mixture in the channel was 10 min, a fivefold decrease in consumed time compared to commercial PCR system with 70% yield. Multiple simultaneous reactions can be carried out in the chip by introducing separate reagent streams in each loop. A modification of this scheme in capillary, with three temperature regions and pneumatic movement of a droplet of reaction mixture, was demonstrated by Erlich et al. [15] achieving 30 cycles in 23 min with good amplification efficiency. Instead of using artificial pneumatic movement, naturally developed Raleigh–Benard convection between two regions of differing temperature has also been used for performing PCR [16]. Shorter cycling times of 17 s/cycle have been achieved by the use of infrared radiation for heating and temperature control as opposed to the use of micromachined heaters by Landers et al. [17], [18]. Landers used infrared radiation (generated by a tungsten lamp), mediated heating, and compressed air cooling controlled by a feedback sensor to amplify DNA in capillaries and microchambers, the experimental setup of which is shown in Fig. 4. Recently, much smaller cycling times of 3 min for 30 cycles with heating and cooling rates

SELVAGANAPATHY et al.: RECENT PROGRESS IN MICROFLUIDIC DEVICES FOR NUCLEIC ACID AND ANTIBODY ASSAYS

955

Fig. 4. Setup of infrared heating of the PCR chamber.

of 36 C/s and 22 C/s have been obtained using grooves of 300- m depth for horizontal thermal isolation and a nonlinear feedback proportional integral control scheme [19]. Most microfabricated PCR devices require 2000 DNA copies/ L at volumes down to 1 L for amplification [14]. Amplification of a single DNA template molecule using microfabricated thin-film heaters and a fast cycle time of 30 s has recently been demonstrated by Mathies et al. [3]. They demonstrated quantized amplified product peak areas showing amplification from a single DNA template molecule, thus demonstrating the feasibility of microfabricated analysis devices for single cell studies. Amplification of DNA present inside cells, and its subsequent detection introduces a novel method of analysis for gene expression and has been demonstrated by Wilding [24]. White blood cells are isolated from the blood samples with the use of a microfabricated filter device. The PCR reaction mixture was added and thermocycling was done on-chip to amplify a 202-bp sequence of Exon-6 of the dystrophin gene directly from the genomic DNA inside the trapped white blood cells. This provides isolation of a specific cell type and amplification and analysis of genetic material within them without contamination of genetic material from other cell types also present in the sample. 3) Amplification Detection: Diode detectors have been used in microdevices for quantification of PCR products by electrochemiluminescence [21], [22] with detection limits of 40 fmol of DNA, but were substantially poorer than those achieved by conventional systems. Recently, devices integrating optical fibers directly with the PCR chambers have been reported. The design incorporates two optical fibers, one for excitation and one for collection of the emitted signal. The procedure used 5 mM of fluorescent dye for detection [23]. Another amplification-type technique for single nucleotide polymorphism (SNP) genotyping called pyrosequencing [61] has been used for mutation detection. This technique relies on the incorporation of nucleotides by DNA polymerase and the release of pyro-phosphate (PPi), which is converted to adenosine triphosphate ATP and subsequent light emission detected. The presence of a mutation will not allow the extension by polymerase and hence absence of light. A micromachined filter chamber device has been 956

Fig. 5. (a) Example of immobilized oligonucleotide probes showing hybridization of unknown to specific probe. (b) Probes are arranged as planar arrays. The hybridized regions can be detected by the fluorescence of the duplex.

developed for this purpose to detect mutations in a tumor suppressor gene [62]. B. Hybridization Chips 1) Hybridization Binding: The process of attraction and formation of loose hydrogen bonds between complimentary bases on two single-stranded DNA chains is known as hybridization. The specificity of the genetic material provides an extraordinary tool for detection of minute quantities of DNA in a solution with large amounts of nonspecific material. In hybridization arrays, several chains of single-stranded DNA are attached to a rigid substrate such as glass [25], silicon [26], or plastic [27], using a linker molecule as seen in Fig. 5. These immobilized probes can be either short-length synthetic oligonucleotides or longer-length cDNA fragments arranged in arrays [25], [27], [28]. A hybridization event is detected using fluorescent probes, which intercalate with the hybridized double-stranded DNA or by labeling them to sample-free DNA. Reading of these signals is achieved using confocal epi-fluorescence microscopy [29], [30] with cooled and intensified charge-coupled devices (CCDs). DNA microarray technology for large-scale genetic analysis can be fabricated in a massively parallel fashion [31]. Two of the main applications of DNA arrays that use the parallelism to its advantage are DNA sequence analysis and gene expression analysis [32], [33]. PROCEEDINGS OF THE IEEE, VOL. 91, NO. 6, JUNE 2003

Gene expression arrays [34] have subsets of gene transcripts (mRNA) as expressed in various cells and tissues corresponding to different proteins in wild type cells. The comparison of this comprehensive library to specific sets of transcripts expressed in cells under different conditions and stimuli provides valuable information about the mechanisms inhibiting or expressing different genes under a variety of stimuli such as drugs. These arrays have wide applicability in cancer diagnostics and pharmacogenomics. Sequence analysis arrays [35] typically have shorter length oligos corresponding to various disease and gene markers. The mutations of a single base in these short strands of DNA, known as SNPs, are extremely useful in finding the disease genes underlying complex diseases [36]. These arrays are also useful in the development of personalized drug delivery and early genetic risk assessments. 2) Microfabricated Arrays: Microarray systems vary between polynucleic acid immobilization, method of immobilization, nucleic acid formation, hybridization, and reading of hybridized arrays. DNA arrays of small oligos ( 25 bases) can be fabricated using photolithographic techniques based on the principle of photo-induced stepwise addition of bases to oligonucleotide chains [29]. Using these techniques as many as 10 probes/cm can be fabricated [37] with spot sizes around 10 m. This can cover all the combinatorial possibilities of a 10-mer sequence. Use of protecting photoresist layer [38] for definition of reaction sites increased the resolution producing pixel sizes of 8 m or even smaller, thus increasing the density by 50% [39] with efficiencies of 98%. A new maskless technique for photodefinition using digital micromirrors has been reported [40]. This method uses photogenerated acid (PGA) in solution to trigger deprotection of the 5 -OH group in conventional nucleotide phosphoramidite monomers (i.e., PGA-gated deprotection), as shown schematically in Fig. 6, with the rest of the reactions in the synthesis cycle similar to the routine synthesis of oligonucleotides. The complete synthesis process is accomplished using a conventional DNA synthesizer coupled to a 480 640 digital micromirror device (DMD). The resolution is poor with each micromirror corresponding to 30 30 m on the substrate; however, the fabrication time is considerably reduced. In addition, the oligo length increased as the patterns were generated digitally. Other methodologies have been used for fabrication of DNA arrays of longer chain lengths including syringe and pin-based contact mode spotting techniques [42] and piezoelectric ink jet nozzle based noncontact techniques [41] to form spots of presynthesized oligos, cDNAs, and gene fragments. 3) Hybridization Detection: The hybridization event is typically fluorescently detected. Labeled fluorescent tags are illuminated using a laser scanning system where the response is recorded using a CCD scanner, or confocal microscopy with a photomultiplier tube (PMT) reducing out-of-focus fluorescent light. The optical signal is increased if the oligos are immobilized in a three-dimensional (3-D) volume rather than a twodimensional plane. The 3-D volume of immobilized oligos using acrylamide gel pads as the suspension medium on glass

Fig. 6. Formation of isolated reaction sites and the microarray synthesis approach. (A) Droplet formation by filling and draining a PGA-P solution in a microarray reactor for the subsequent PGA-gated deprotection reaction. (B) Microscopic image of CH OH droplets formed on a microarray glass plate patterned by a nonwetting film. (C) Examples of digital light projection patterns generated on a desktop computer for selective PGA deprotection. Four masks are required for the A, C, G, or T residue per step growth in oligonucleotide array synthesis [40].

Fig. 7. Scanning electron microscope images of the front surface of a vertical microchannel microarray glass chip [45].

substrates has been reported [43]. The increase in signal has led to detection of rare transcripts in gene expression analysis with sensitivities 100 amol [44]. However, the use of gels introduces differential mobility based on the size difference between the samples, thus influencing hybridization kinetics and introducing analysis artifacts. An increase in the surface area for binding capture probes without the associated side effects of the gel can be achieved using small volumes of ordered porous substrates as seen in the SEM images in Fig. 7. Furthermore, the hybridization time can be substantially reduced if the analyte is allowed to flow through the array; therefore, the pores connect the upper and the lower surfaces of the chip [45]. Single-strand DNA (ssDNA) probes are immobilized on the sides of the vertical microchannels. Typically, each spot occupies a few hundred microchannels. This technique resulted in increased target capture and hybridization rates as well as a 60% reduction in spot size and an overall increase in spot density compared to experiments on flat glass substrates.


957

Fig. 9. (A) SEM image of cantilever device. (B) Deflection caused in the cantilever device due to hybridization. Reproduced from [63].

Fig. 8. Schematic diagram of the sandwich assay for electronic detection of nucleic acids. Reproduced from [51].

The signal-to-noise ratio (SNR) for single nucleotide discrimination is quite low if the reaction is based only on DNA hybridization. SNP determination is enhanced by target amplification using enzymes, such as polymerase [46] or ligase in addition to hybridization. Arrayed primer extension (APEX) is an alternative technique to array-based DNA sequence analysis by hybridization. The SNR of this technology is typically around 40:1, enabling the identification of heterozygous mutations with high confidence levels [48]. The method is based on a two-dimensional array of oligonucleotides, immobilized via the 5 terminal amino group on a treated glass substrate. Patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promotes sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a color change code of the primer sites. Several alternative methods have been investigated for detection of hybridization in microfabricated devices. Electron transfer measurements in double stranded (hybridized) DNA indicate that it is a conductor [49], [50]. Oligo chains are spotted on microelectrodes and electrical signals are used for hybridization detection using a separate electroactive molecule which complexes with specifically hybridized DNA [51] as shown in Fig. 8. Single base-pair mutations have also been detected using electroactive ferrocene labeled probes using alternating current voltammetry monitored at first harmonic [52]. Permittivity measurements and their variation in DNA solutions have also shown to indicate the event of hybridization. Higher losses and permittivity were observed in ssDNA than in dsDNA. This is thought to be a part of increased conductivity of the ssDNA and the subsequent increased electrical polarization [53]. Electrochemical sensors using DNA probes attached to an electrically active surface to detect the current and resistance changes caused by hybridization of target DNA have been investigated [54]. Hybridization detection can be made more 958

sensitive by coupling a redox active dsDNA intercalator with a catalytic mediator to provide chemical amplification of the signal [55]. A DNA array detection method is reported in which the binding of oligonucleotides functionalized with gold nanoparticles leads to conductivity changes associated with target-probe binding events [56]. The binding events localize gold nanoparticles in an electrode gap. These nanoparticles bridge the gap to facilitate silver deposition resulting in readily measurable conductivity changes. An unusual salt concentration dependent hybridization behavior associated with these nanoparticle probes was exploited to achieve selectivity without a thermal-stringency wash. Using this method, target DNA at concentrations as low as 500 fmol have been detected with a point mutation selectivity factor of 100 000:1. Mass transport of analyte to the immobilized recognition elements on the surface is the rate-limiting step in the analysis. The diffusion coefficient for DNA molecules is small compared with that of small molecules and decreases with the target length, so capture and hybridization is slow unless significant convective currents are introduced to enhance mass transport [58]. A number of modifications have been carried out to enhance the assay throughput. An electrical field has been used as an independent control parameter to enhance hybridization and improve stringency of nucleic acid interaction [26]. Application of positive fields to electrodes underneath individual spots allows rapid movement of target DNA to the site and, hence, hybridization occurs in seconds. Further application of negative signal forces the nonhybridized DNA away from the spots, enhancing accuracy. Detection limits can be improved with the use of an enzyme label from a bioelectrocatalytic reaction [59]. Attomole detection levels have been achieved for electrochemical hybridization detection of human cytomegalivirus DNA using horseradish peroxide as an indirect electrochemical label. Detection limits of 0.6 amol/mL were obtained [60]. 4) Beam, Gap, and Bead Microdevices: Cantileverbased techniques have been investigated to transform specific biochemical recognition such as hybridization into nanomechanical motion which can then be measured using in situ optical beam deflection techniques [63]–[65]. The cantilevers are 500- m long and 100- m wide and less than 1- m thick, as seen in Fig. 9(a). Each cantilever is coated on one side with a specific biomolecule. When immersed in the analyte solution, molecules of an injected substance dock on to the layer of receptor molecules. The docked molecules introduce surface stresses due to molecular forces, which PROCEEDINGS OF THE IEEE, VOL. 91, NO. 6, JUNE 2003

Fig. 11. DNA fragments are separated into bands due to their different drift velocities. The calibration ladder serves as a reference.

Fig. 10. Schematic diagram of the sandwich assay for electronic detection of hybridization in nucleic acids. Reproduced from [51].

deflect the cantilever as shown schematically in Fig. 9(b). Deflections of up to 10 nm and molecular forces of up to 300 pN were observed for hybridization of 16-mer probes [63]. Parallelization into integrated devices has been demonstrated with arrays of more than 1000 cantilevers [66]. Detection of single-base mismatches were also observed due to their reduced hybridization ability, which translated into reduced deflection. The signals were highly reproducible, with 1-nm accuracy [63]. While very sensitive, the cantilever technique suffers from bimetallic temperature-related effects as well as nonspecific binding effects. Another label-free technique for hybridization detection uses nanogap junctions [67]. Nanogap junctions consist of two polysilicon electrodes separated by a small gap (50 nm), shown in Fig. 10, such that the electrodes electrical double layers of ions overlap as a result of hybridization. Disruption of the ionic double layer reduces the net ion densities considerably; thus increasing the sensitivity of the dielectric measurements. Differences between the coiled configurations of ssDNA and the straight configuration of dsDNA, due to hybridization are exploited in hybridization detection by changes in dielectric properties. The hybridization of 10 M/L of target DNA to immobilized ssDNA on the electrode faces resulted in a 70% capacitance change of 60 pF [67]. The use of mobile solid support paramagnetic beads has been demonstrated for dynamic DNA hybridization (DDH) for gene expression analysis [68]. DDH offers several advantages. The hybridization process is dynamic because both the type and amount of paramagnetic beads, DNA target, and probe can be changed as needed, by pumping them into the microfabricated device. The hybridization reaction is fast and the reaction efficiency is high, since it is conducted in a small confined volume (2 nL), with continuous pumping of fresh probe solution. Multiple DNA samples can be introduced in different channels of the microdevice, and all samples can be analyzed at once. The concentration of probes from solution onto beads enhances sensitivity. C. Separation Microdevices 1) Separation of DNA: The determination of size of the DNA chain, which has been previously processed, is the principle method of DNA analysis. Size fragmentation analysis is used for DNA sequence determination using the

Sanger reaction scheme. Typically, the sample consists of DNA fragments of varying length mixed together. Electrophoresis (which is the most popular separation technique for DNA) separates these fragments based on their size by detecting their arrival time at the end of an obstacle course through which they pass. The obstacle course is a gel, which provides differential (proportional to logarithm of fragment size [69]) resistance based on size, charge, and mass. DNA fragments in solution are negatively charged; hence, an electric field is used to move the DNA through the gel medium as shown in Fig. 11. The resolution of separation is enhanced by separation voltage, but degraded by thermal convection caused by Joule heating that tends to cause band broadening. Separation resolution is also affected by the width of the injection plug, which can be precisely controlled in microfabricated systems. The separated fragments are detected using laser-induced fluorescence [70] at a fixed point toward the end of the separation to get the time and sequence of their arrival. This result is compared against the separation of a standard DNA ladder (sample with several DNA strands of uniformly increasing lengths) for sizing, as shown in Fig. 11. Alternatively, separation can be performed in four different lanes, with the DNA sample in each of the lanes undergoing Sanger reaction prior to separation, to provide sequence information. The Sanger reaction is an amplification scheme similar to PCR but with random termination of the complementary strand formation due to a small concentration of dideoxy nucleotides (ddNTPs). These special nucleotides terminate the polymerase duplication when captured. The reaction thus generates complementary strand fragments terminated at all possible positions of the matching dideoxide. When these fragments are separated in four separate lanes corresponding to the ddNTPs employed in the Sanger reaction, the relative positions of the fragments indicate the location in the sequence of a specific base; hence the sequence can be read out directly. 2) Gel Electrophoresis Microdevices: Manz et al. [71], [72] critically evaluated the benefits of microfabrication in separation systems. Joule heating and sample plug injection parameters were described. Micromachined devices are considerably faster compared to slab gel techniques, requiring from 1 min for short oligonucleotides [73] to less than 20 min for 500 base length DNA [74]. Simultaneous parallel high-throughput DNA sequencing using a 96-channel array format, as shown in Fig. 12, has been demonstrated with average read length of 430 bases [75]. Various analyses including nucleic acid sizing of short oligonucleotides [73] to


959

Fig. 12. (A) Overall layout of the 96-lane DNA sequencing microchannel plate (MCP). (B) Vertical cut-away of the MCP. The concentric PMMA rings formed two electrically isolated buffer moats that lie above the drilled cathode and waste ports. (C) Expanded view of the injector. (D) Expanded view of channel bend design to reduce dipersion. Reproduced from [75].

restriction fragments [76]–[78] and genotyping of specific genes for hemochromatosis [79], muscular dystrophy [80], and for short tandem repeats [88] have been performed in micromachined devices. A critical evaluation of the benefits of microfabrication of separation systems, including the increased dissipation of heat generated by Joule heating, and the ability to inject very small and well-defined sample plugs is described by Manz . Micromachined devices lead to considerably less analysis time than slab gel techniques, taking less than a minute for short oligonucleotides to less than 20 min for DNA sequencing with read lengths over 500 bases . Simultaneous parallel high-throughput DNA sequencing using a 96-channel array format, as shown in , has been demonstrated with average read length of 430 bases . Various analyses including nucleic acid sizing of short oligonucleotides to restriction fragments and genotyping of specific genes for hemochromatosis , muscular dystrophy , and for short tandem repeats have been performed in micromachined devices. A DNA sequencing microchip was demonstrated by Mathies et al. in 1995 [82] and subsequently improved to provide a high-speed high-throughput four-color sequencing [74]. One of the key design features of any microfabricated separation is the sample injection scheme. A smaller injection plug leads to faster separation with shorter channel length. These devices consist of two intersecting microchannels with 960

fluid reservoirs at each end. One of the reservoirs of the shorter channel is filled with the DNA sample, and the electric field is applied to move the DNA toward the other reservoir. In the process, DNA fills in the intersecting region between the two channels, which defines the injection plug. In the cross section injection scheme [89], [90], DNA fragments in the intersection are forced into the longer channel filled with the separation media while the rest of the DNA is forced back into the reservoirs, thus injecting a small but definable plug of sample for separation. Other injection schemes include the double T where the sample inlet and outlet arms of the injection channel are offset to allow geometrical definition of 50–500-pL sample plugs [91]. Narrower injection plugs can be formed by stacking at the boundary of a cross-linked gel [92]. In another scheme, a microfabricated porous membrane is used for preconcentration to increase the separation sensitivity [93]. The injection channel was fabricated to include a porous silicate membrane that is semipermeable and allows ionic current to pass through but not DNA. Two orders of magnitude increase in DNA concentration at the injection point is achieved. Several polymeric matrices have been used for separation purposes including cross-linked polyacrylamide [94] and noncross-linked gels such as linear acrylamide and hydroxyetylcellulose [83], [95]. Unlike cross-linked gels, the noncross-linked materials can be flushed out of the microchannels, and the chips can be reused. However, all these gels require filling and polymerization of the matrix after the fabrication of the chips in a serial manner leading to increased cost. 3) Artificial Gel and Matrix-Free Microdevices: Artificial gel systems have been fabricated using electron beam lithography with arrays of pillars 100 nm in size and 150–200 nm in separation [96]. These devices are largely used for coarse separations. Separation of 7.2- and 43-kbp phage DNA with twofold velocity differences has been reported. More recently, nanoimprint fabrication was used to fabricate artificial gel arrays on plastic substrates [97]. However, poor dc electrophoresis separations and requirements of pulsed fields were the main drawbacks in these array systems. One effort toward a matrix-free approach to DNA separations utilizes entropic traps. The separation column consists of lithographically defined deep (1.5–3 m) and shallow (75–100 nm) regions with a column width of 30 m, as shown in Fig. 13 [98]. When an electric field is applied across the length of the channel, the DNA molecules move toward the anode but are trapped when the depth of the shallow region is much smaller than DNA’s radius of gyration. The probability of escape from this trap is proportional to the width of the shallow region slit covered by the DNA molecule; hence, larger molecules have higher probability of escape. The mobility difference created due to channel design and DNA size lead to separation without the use of a matrix or pulsed electric field. Fragments in the range of 5–160 kilobase pairs were separated successfully in 15-mm-length channels. PROCEEDINGS OF THE IEEE, VOL. 91, NO. 6, JUNE 2003

Fig. 13. Nanofluidic separation device with entropic traps. (A) Cross sectional view. (B) Top view. (C) Experimental setup. Reproduced from [98].

Another ingenious method of separation uses asymmetric Brownian ratchets [99]. These devices use anisotropic potential fields to trap small particles and molecules such as DNA fragments, as shown in Fig. 14. The Brownian ratchet system consists of a series of microfabricated interdigitated asymmetric electrodes. When a potential is applied to the electrodes, DNA fragments are trapped in a potential well near the positive electrodes. When the electric field is removed and the DNA fragments diffuse, with the smaller fragment moving the farthest. When the electric field is applied again, the smaller fragments, which are now sufficiently farther away, are trapped in the adjacent potential well. Repetition of this on–off process was used to transport DNA fragments 25, 50, and 100 bp in length at a concentration of 0.1 pmol/ L at varying speeds using potentials of 0.1 V applied between electrodes separated by 0.1 m and electrode pairs separated by 1 m. It has been estimated that the detection of SNPs requiring separation of a 24-mer DNA from a 25-mer DNA fragment could be accomplished in this type of device in 12 000 cycles of potential for a total separation time of 5.4 s on a 1.25-cm chip without the use of gels or polymer solutions [100], [101]. 4) Dynamic Electric Field Schemes: Effective separation can be achieved using short separation lengths using field inversion electrophoresis [102]. It involves application of an asymmetric electric field periodically switched at an 180 angle. The forward cycle at higher amplitude leads to movement of the entire plug of DNA fragments with very little separation. The reverse cycle with a smaller amplitude leads to greater separation with the smaller band traveling back faster than larger DNA fragments leading to band inversion. The frequency and the amplitude of the pulses can be tailored

Fig. 14. Brownian ratchet device is shown in schematic. Modulating the electric potential at the electrodes generates a ratchet-like potential energy surface for charged molecules such as DNA. Cycling the ratchet between an on state and an off state generates transport. Reproduced from [100].

to make a particular DNA size fragment have zero mobility after a complete cycle. It has been shown that a frequency between 10 and 20 Hz is suitable for separation of short DNA fragments. Single-stranded DNA fragments of 20, 40, and 60 bases have been separated using pulsed (10 Hz) fields (167 V/cm) with a 6-mm separation length. The possibility of achieving high resolution without an increase in separation length is attractive in identification and analysis of single-strand conformation polymorphism analysis. Cyclic electrophoresis has been used to reduce the size of microfabricated chips using closed-loop separation channels by connecting the end to the mouth [103]. In the first stage of each cycle, analysis is performed by the conventional procedure of constant-field capillary electrophoresis (analysis stage). The DNA fragment length-resolution of an analysis stage will eventually become inadequate due to reptation (end first elongation and migration of DNA in high electric fields), thereby decreasing the DNA length dependence of mobility. Thus, in the second stage of each cycle, the resolution of unanalyzed molecules is enhanced (enhancement stage) by reverse migration. The cycle repeats, thereby analyzing the entire DNA profile in several analysis stages that are separated by enhancement stages [104]. Separation between peaks can be increased indefinitely, with lower voltages and smaller channel lengths. Resolution will be proportionately increased, if diffusion-induced peak broadening does not occur. Microfabricated cyclic electrophoresis devices have been fabricated and demonstrated for the Hae X174 DNA ladder [105]. The separation enhancement stage was controlled at different parts of the ladder by adjusting the synchronization time between two stages [103].


961

Fig. 15. (a) Current blockage due to passage of 500 bp of dsDNA through nanopore. TEM image of the nanopore (b) before and (c) after ion beam sculpting. Reproduced from [110].

Low voltages can be used to generate high electric fields if the dc voltage travels along with the separated DNA fragments. This is achieved using arrays of microfabricated electrodes embedded along the separation channel. A small dc voltage applied across the pair of electrodes, enclosing the DNA bands, is sequentially moved downstream. DNA fragments of 1000 bases with 100-base resolution were separated with voltages of four to five times lower than required under normal conditions [106]. 5) Nanopores for Fragmentation Analysis: An interesting separation technique uses ion channels formed by -hemolysin, a protein whose ion channel is 5 nm long and 1.5 nm across at the narrowest point [107]. The channel diameter is sufficiently small to permit the passage of only one DNA molecular chain at a time. The molecule size can be determined by measurement of passage time signaled by a distinct change in the nanopore electrical conductivity. An open channel in 1M KCl solution conducts 120 pA when 120 mV is applied across it. An electric field applied across the pore forces ssDNA to be transported through the pore, which impedes the flow of current to 14 pA. This approach has been used to read blocks of nucleotides [108] and to identify oligomers using pores with covalently attached oligonucleotides [109]. Solid-state nanopores have been fabricated using Ar ion beam sculpting on a 60-nm hole in a silicon nitride membrane to form pores of 1.8 to 5 nm, as seen in Fig. 15, [110]. Ion currents of 1.66 pA were observed through these 5-nm pores in 1M KCl with a voltage of 120 mV across the pores. A reduction in current by 88% was observed with a 500-bp ssDNA passing through the pore, as shown in Fig. 15. Similarly, pore sizes from 100 nm to 1 m with pore lengths of 1 to 10 m were constructed in a polymeric substrate. A change in current represents the measure of the DNA fragment size crossing the pore, used to detect individual molecules of -phage DNA [111]. This device was able to distinguish between colloids whose diameters differ by 10%. 6) Detection of Separated Species: The most common detection technique for microfabricated separation systems 962

Fig. 16. Separation of the HaeIII digest of X174 RF DNA fragments in the integrated CE device. (A) Layout of the capillary electrophoresis chip with integrated detector. (B) Electropherogram using on-chip fluorescence detection. Reproduced from [114].

is laser-induced fluorescence (LIF) [112], [113], which can detect low concentrations of analyte. Single DNA molecule detection has been achieved on microfabricated separation chips using this technique [114]. However, fluorescence detection requires large and expensive supporting optical systems that can be simplified, at reduced sensitivity, with the use of blue LED excitation sources and on-chip fluorescence photodiode detector, as demonstrated by Webster et al. [114]. The layout of the separation chip with integrated detector is shown in Fig. 16. Separations carried out using SYBR-green-labeled dsDNA and HEC as the sieving matrix, the result of which is shown in Fig. 16, indicate an effective resolution of 30 000 theoretical plates on a 0.9-cm-long microchannel with a minimum detectable signal from 75 fg samples. Microfabricated devices have been developed to size and sort microscopic objects based on measurement of fluorescent properties [119]. With the development of high affinity intercalating DNA stains, it has become possible to directly measure the length of single molecules by quantifying fluorescence. The amount of intercalated dye is proportional to the length of the molecule, so measuring the total fluorescent intensity from a single molecule gives a direct measurement of its length. Resolution of 5% at 20 000 bp is posPROCEEDINGS OF THE IEEE, VOL. 91, NO. 6, JUNE 2003

III. PROTEIN IMMUNOASSAYS Protein function depends on its structure and shape. Like DNA, proteins can be digested, separated, bound to a surface or functional group, and tagged with markers. Protein function is selective, with specificity determined by finely tuned interactions between ligands and receptors. An extremely important application of these specific interactions is the identification of antibodies and antigens (analytes). Immunoassays are analytical experiments in which highly specific antibodies or antibody-related binding molecules are used for identification and quantification of target molecules. Antibody-based immunoassays are the most commonly used type of ligand-binding assays for the identification of a large variety of analytes, such as proteins, peptides, and low molecular weight molecules [120]. Immunoassays have been used in hospitals, laboratories, and industry for more than 40 years, providing scientists methods for detecting the prescence and amount of an antibody or antigen present in a sample. In the life sciences, immunoassays are used to study biological systems by tracking different proteins, hormones, and antibodies. In industry, immunoassays are used to detect contaminants in food and water, and in quality control to monitor specific molecules used during product processing [121]. A. Antibody Systems Fig. 17. (a) Salmonella PCR product sizing using indirect electrochemical detection of DNA. (b) Direct electrochemical detection of calf thymus DNA using microfabricated electrodes. Reproduced from [115] and [118].

sible. Linear measurements of DNA chain lengths up to 200 kbp have been observed with analysis of 3000 molecules in 10 min. This method in principle allows the measurement of extremely long DNA molecules because the signal increases with the length of the molecule. Electrochemical (EC) detection offers considerable advantages due to its sensitivity, selectivity, and ease of microfabrication and integration.Electrophoresis chips with integrated end column detectors have been used for indirect electrochemical detection of DNA [115], as shown in Fig. 17(a), with detection limits of 1.8 amol of DNA. Decoupling the separation and detection fields has been done with a palladium decoupler (using palladium’s capability to absorb and diffuse hydrogen and not allow gas evolution at the decoupler) to obtain noise levels of less than 15 pA. EC detectors have been incorporated into the separation column in an inline fashion using a novel decoupling scheme (allowing floating of the detection system on the separation field), which reduces dispersion of separated bands and eliminates gas evolution, was demonstrated with detection limits of 14.5 amol [117]. Such an EC detector was also used for direct detection of 50- g/ml calf thymus DNA tagged with electroactive ferrocene at the 5 end with noise levels of less than 1 pA, as shown in Fig. 17(b) [118].

1) Antibodies: Antibodies are proteins generated by animals in response to the invasion of a foreign molecule (antigen) into the body. Antibodies are found in blood and tissue fluids and will bind to an available antigen. Antibodies are immune-system-related proteins called immunoglobulins consisting of 110–130 amino acids in highly ordered sequences. Human antibodies are divided into five major classes, IgM, IgG, IgA, IgD, and IgE, based on their constant region structure and immune function. Mice have the same general isotypes as humans; however, IgG isotypes are divided into different subclasses. Each antibody consists of four polypeptides, two heavy chains, and two light chains forming a Y-shaped molecule. An amino acid sequence at the ends of the Y structure, or the antigen binding site, varies among different antibodies. The variable region includes the ends of the light and heavy chains. This variable region results in the antibody specificity for the binding antigen. The antigen binding sites are formed and domains. The locations of comby the opposing plement and Fc receptor binding sites within the heavy chain constant regions are approximations. The S-S regions refer to intrachain and interchain disulfide bonds. The N and C regions refer to amino and carboxy termini of the polypeptide chains, respectively. Despite the overall similarity, antibody molecules are divided into a smaller number of distinct classes and subclasses, based on minor differences in size, charge, solubility different and behavior as antigens. There are as many as antibody molecules in every individual, each with unique amino acid sequences in their antigen combining sites. This extreme diversity of structure accounts for the extraordinary


963

Fig. 18.

Schematic diagram of an immunoglobulin (IgG) molecule [125].

specificity of antibodies for antigens because each amino acid may produce a difference in antigen binding. 2) Antibody–Antigen Binding: Because antibodies are developed based on the specific 3-D structure of an antigen, or analyte, they are highly specific and will bind only to that structure. An antigen can be defined as any substance that may be specifically bound by an antibody molecule [122]. Each Fab fragment, the two upper fragments in Fig. 18, of the antibody contains one antigen-binding site formed by three light chain hypervariable loops and three heavy chain – fold, hypervariable loops held in position by the shown in Fig. 18. According to the traditional lock-and-key model, each antibody type has a unique hypervariable region that is specially shaped for its epitope. The antibody–antigen complex is not held together with covalent bonding; however, the interaction is very strong. Antigen–antibody interactions are similar to enzymatic reactions in that they are reversible. Upon engagement, the “key-and-lock” is held in place by electrostatic forces due to ionic bonds, hydrogen bonds, hydrophobic interactions, and van der Waals forces shown in Fig. 19. Since these individual interactions are weak, the antigen–antibody interaction requires a large number of these bonds and requires the antigen and antibody to be in close proximity. Typically, the noncovalent interactions operate on the scale of 1 [123]. 3) Antibody Affinity, Avidity, and Cross Reactivity: The strength of the total noncovalent interactions between a single antigen-binding site on an antibody, and a single epitope is the affinity of the antibody for that epitope. Antibodies with low affinity bind their antigens weakly and disassociate easily. Antibodies with high affinity bind their antigens stronger and do not disassociate easily. The association of antibody (Ab) binding its antigen (Ag) epitope can be expressed as follows: Ag 964

Ab

AgAb

Fig. 19. [125].

Schematic diagram of antibody–antigen binding forces

where is the association constant, is the disassociation constant, and AgAb represents the antibody to antigen binding. A measure of the affinity is defined as . Since the affinity is the association equilibrium constant, it can be calculated from the concentrations of the bound and unbound antibodies and antigens AgAb Ab Ag Affinity constants for antibodies are typically 10 to 10 M . The equilibrium constant for the dissociation is defined is a quantitative indicator of the stability as . of the AgAb interaction; very stable complexes have low Another term used to describe antigen–antibody interaction is avidity, which is the strength of multiple interactions PROCEEDINGS OF THE IEEE, VOL. 91, NO. 6, JUNE 2003

between antigen and antibody with multiple binding sites. All antibodies have at least two antigen binding sites represented as their (Fab) . Some antibodies, IgM and IgA, exist in secreted form as a multiantibody complex. Thus, pentameric IgM has ten potential binding sites and dimeric IgA has four potential binding sites, whereas IgG only has two binding sites. The sum of all the binding sites equals the avidity. Antibodies are antigen specific, but sometimes they are also cross reactive. This means that they recognize other antigens that look similar to the one for which they are specific. Cross reactivity occurs when two different antigens share the same or similar epitopes [125]. 4) Antibody Types: Most antigens possess multiple epitopes inducing proliferation and differentiation. Once purified from the blood, monoclonal and polyclonal antibodies are ideal assay reagents to detect and monitor specific target molecules with limited interferences from other substances. Monoclonal antibodies are produced by the daughters of a single B-cell; hence, they are completely homogeneous with respect to the type of immunoglobulin and binding to the antigenic epitope. Polyclonal antibodies are heterogeneous in that the polyclonal antisera will always be a mixture of various antibodies [124]. There are two main disadvantages to polyclonal antibodies. First, they will always possess multiple specificity, even after purification, and can never provide the monospecificity of monoclonal antibodies. The extraction process inevitably leads to varying antibody composition in terms of specificity [126]. A major improvement of ligand-binding assays has realized with the application monoclonal antibodies (mAbs). Ideally, monoclonal antibodies provide a solution to the problems faced with the use of polyclonal antibodies. The advantages of using monoclonal antibodies are that 1) each hybrid cell line produces one unique antibody; 2) there is potentially an unlimited antibody supply; 3) potentially all specificities (i.e., all combinations of antibody and corresponding complimentary antigen epitopes) can be obtained; 4) monoclonal antibodies can be genetically engineered; and (5) the technique is very general with respect to using antigens and the desired properties of the antibody [124]. B. Antibody Assays Conventional immunoassays require several time consuming steps such as reagent addition and washing before a signal is measured. Conventional immunoassays include: 1) radio immunoassays (RIAs); 2) enzyme immunoassays (EIAs); 3) fluorescent immunoassays (FIAs); and 4) enzyme-linked immunosorbent assays (ELISAs). The ELISA method is used most often for environmental field analysis because it can be optimized for speed, sensitivity, and selectivity, contains no radioactive materials, and is simpler to use than other immunoassay methods. Table 1 is a list of measurement sensitivities of common immunoassays. 1) Binding Strategies: Immunoassay binding strategies can be divided into three categories: direct, competitive, and sandwich assays [126]. Direct assays are performed by incubating the antigens with excess amounts of antibody. The

Table 1 Sensitivity of Conventional Immunoassays

The sensitivity depends on the binding affinity, epitope density, and distribution [125].

Fig. 20. Simplified protocol for conventional immunoassay. (a) Direct. (b) Competitive. (c) Sandwich [127].

sensitivity is directly proportional to the amount of antibody present. Fig. 20(a) shows the procedure for producing a direct assay. The antibody is bound to the substrate and a fixed amount of labeled antigen (black dot in Fig. 20) mixed with varying amounts of unlabeled antigen (gray dot in Fig. 20) or test sample are introduced to the sample. The amount of bound antibody–antigen is measured. Competitive assays used both labeled and unlabeled antigens, which compete for a limited number of antibody-binding sites. The signal intensity of the bound phase is inversely proportional to the concentration of the unlabeled. Fig. 20(b) shows the procedure for producing a competitive assay, where the antigen is bound to the substrate followed by adding a fixed amount of labeled antibody mixed with varying amounts of soluble unlabeled antigen or test sample. An alternative to the labeled analytes is the sandwich assays which uses two antibodies simultaneously bound to the same antigen, as shown in Fig. 20(c). The antibody is first bound to the substrate followed by adding varying amounts of labeled antigen are added to sample. Secondary


965

Fig. 21. Schematic of immunoRCA assay. Top left: Reporter conjugated to an oligonucleotide binds to the test analyte bound to the substrate. Top right: DNA circle hybridizes to a complementary sequence in the oligonucleotide. Bottom left: Excess reagents are washed away and DNA tag is amplified by RCA. Bottom right: Amplified product labeled with fluoro-labeled oligonucleotides [131].

antigen specific for nonoverlapping epitopes of the antibody is added. The amount of bound secondary antibody is measured. Sensitivity is related to the amount of primary antibodies present, with the response proportional to the antigen concentration. A limitation of this assay is that the required number antigens is large in order to have two binding sites for the secondary antibodies. The advantage is that the sample labeling step is eliminated, which is variable from sample to sample. In addition, sandwich assays have led to higher specificities [128] and dramatic improvements in performance and throughput [129]. 2) Labeling and Detection: Immunoassays use a large variety of labels including radioisotopes, fluorophores, and enzymes (color change) and magnetic and metal beads. Detection of antibody–antigen binding uses radioactivity, fluorescence, chemiluminescence or enhanced chemiluminescence (ECL), electrochemical, label-free surface plasmon resonance, and more recently a technique for signal enhancement of bound proteins using rolling circle DNA synthesis with fluorescent probes, immunoRCA [131]. The immunoassay sensitivity can be increased considerably through the use of chemical amplification techniques commonly used for nucleic acids. Advances in signal enhancement for analyte detection include chemiluminescent substrates for ELISA, immunoblotting [132], and signal amplification methods such as tyramide deposition [133]. However, for small sample sizes, an adaptation to the rolling circle amplification (RCA) reporter system [134] for the detection of protein antigen, immunoRCA, uses an olignucleotide covalently bound to an antigen as shown in Fig. 21. In the presence of circular DNA, amplification results in a long DNA molecule with hundreds of copies of the sequence that remain attached to the Ab, which can be detected in many different ways. Ultrasensitive detection of prostate-specific Ag (PAS) was performed on microspots. The immunoRCA was configured in an indirect sandwich format assay. This complex was detected with a polyclonal rabbit anti-mouse IgG 966

Fig. 22.

Number of publications in DNA and protein microarrays.

Fig. 23. Schematic diagram of preparation analysis and detection of antibody–antigen binding assays [129].

Ab conjugated to an oligonucleotide containing a sequence for priming an RCA reaction. Quantification of the fluorescence indicated a linear signal over two logs of PSA data down to 0.1 pg/ml. C. Microfluidic Immmunoassays The same microfluidic technologies discussed for nucleic acids analysis are beginning to be applied to protein assays. Micromachined devices in this area presently lack the sophistication of those used in DNA chips. They are categorized in two main areas of binding microarrays and bead or particle assays. Antibody arrays are an embryonic but rapidly growing technology. Fig. 22 shows the number of publications of nucleic acid and proteins arrays in the past decade [135]. D. Immunoassay Microarrays Antibody arrays is an embryonic and rapidly growing technology. Such arrays are used in the diagnosis of disease, PROCEEDINGS OF THE IEEE, VOL. 91, NO. 6, JUNE 2003

Fig. 24.

Planar waveguide immunoassay [129].

e.g., leukemia and cancer research and drug discovery [135], [136], [120]. Immunoassay microarrays are fabricated by high-density printing of arrays of antibody microspots on a solid substrate as shown in Fig. 23. The array thus consists of subnanoliter amounts of capture antibodies immobilized in rows and columns on a solid substrate. To detect the binding event, samples containing the analytes are incubated onto the array substrate. Unbound molecules are next washed away [see Fig. 23(b)], and labeled detection antibodies are allowed to bind to the captured analytes on the individual microspots. After washing away unbound detection antibodies, signals on each microspot are detected with a scanner [see Fig. 23(c)]. Signal intensities correlate with amount of captured analytes present in the sample [129], [130]. Printing of the protein spots onto substrates has been performed using DNA-based systems [137], [138]; however, these methods are problematic due to sample drying. Alternative techniques include humidified spotting chambers and glycerol rich protein solutions [139]. Microstamping methods have been reported which use a hydrogel stamper inked with the aqueous protein [140] or more recently a silicon microfabricated stamp with an stamp area of 350 350 m and 100- m pitch [141]. More recently, a technique using microstamper printing combined with microfluidics has been used to fabricate protein arrays on substrates for multiplexed immunoassays [142]. A micropatterned polydimethylsiloxane (PDMS) stamp is used to directly transfer adsorbed proteins to a glass substrate. Scanning probe microscopy (SPM) showed a high level of homogeneity with low nonspecific binding of nontargeted compounds. Microspot sizes around 5 m with 15- m pitch were achieved. Other printing methods include inkjet printing and electrospray [128]. An important aspect of immunosensors is the capability to simultaneously detect multiple analytes in a single sample for rapid quantitative analysis in clinical fluids [143], [144]. More recently, a fluorescence-based immunosensor has been reported for simultaneous analysis of samples [145]. A patterned array of antibodies, immobilized on the surface of the planar optical waveguide, are used to capture the desired an-

alytes present in the samples. Upon excitation of the fluorescent label, a CCD camera is used to detect the pattern of the AgAb complexes on the sensor surface. The reported detection limit for the assays described was 1 ng/mL. The simultaneous detection of 24 cytokines with detection limits comparable with physiological protein concentrations using a ELISA-based protein array system have been reported [146]. This method has the advantage of high specificity, characteristic of ELISA, high-sensitivity detection using ECL, and throughput using a microspot format. A multianalyte microarray immunoassay which uses mass sensing by direct near-infrared (NIR) fluorescence using an NIR label and image analysis has been reported [147]. Single and multianalyte sandwich assays of IgG subclasses have the sensitivity and specificity comparable to ELISA methods using 1/100 the capture antibody. Most microarray assays require the high sensitivity of fluorescence- or chemiluminescent-based detection schemes. The sensitivity of measurements has been improved by combining fluorescent labels with planar optical waveguide technology [129]. The capture antibodies are immobilized on the planar waveguide in a microarray format. A preincubated mix of sample and fluorescently labeled detection antibodies is added to the waveguide array. No washing steps are required of the unbound antibodies. Laser light is coupled into the planar waveguide, generating an evanescent field extending a few hundred nanometers into the solution, thus exciting the surface-bound fluorophores. Fig. 24 shows the planar waveguide microdevice. The speed of a dilute assay in planar microarray devices is generally determined by diffusion. Since the analyte is dissolved in the sample volume; the time required for a low concentration of sample to accumulate at the microarray surface can be significant. The diffusion time was reduced through the use of a flow-through cell arrangement. An array to analyze multiple samples simultaneously was developed using a sandwich assay format, with the antibodies immobilized in a patterned array on the surface of a planar waveguide [148]. Analysis on various mixtures of analytes resulted in the system’s ability to detect viral, bacterial, and protein analytes using a 14-min


967

Fig. 25. Schematic of micro flow cell for multianalyte sandwich immunoassay [150].

Fig. 26. Microfluidic bead immunoassay (A) chip cross section and (B) bead stopping area [154].

assay with sensitivities comparable to ELISA. The limits of detection for staphylococcal enterotoxin B (SEB) was reported 10 ng/mL. More recently, a fluorescence-based array which implements four different assay formats, i.e. direct, competitive, displacement and sandwich, on a planar waveguide has been reported [149]. The direct assay had the lowest detection limit of 1 ng/mL for a cyanine 5-diaminopentane-trinitrophenyl (Cy5-DAP-TNP) conjugate assay. The competitive and distance assays resulted in a detection limits of 20 ng/mL and 10 ng/mL, respectively, for a 2,4,6-trinitrotoluene (TNT) antigen assay. The sandwich assay displayed a detection limit for the 2,4,6- trinitrophenyl (TNP), a derivative of TNT, of 5 ng/mL. A microfluidic flow cell coupled sandwich immunoassay which uses check valves for sequential sample and reagent injection has been demonstrated [150]. Fig. 25 shows the schematic. A fluorescent sandwich immunoassay based on -fetoprotein (AFP), commonly used as tumor markers for hepatomas and germ cell tumors, was used to test the system. Anti-AFP monoclonal antibody was immobilized chemically on the silicon dioxide substrate. A 30-min reaction time was achieved. The speed of a dilute assay in planar microarray devices is generally determined by diffusion. Since the analyte is dissolved in the sample volume, the time required for a low concentration of sample to accumulate at the microarray surface can be significant. The diffusion time was reduced through the use of a flow-through cell arrangement.

1) Flow-Through Packed Bead Cell: Fig. 26 shows an example of a micromachined flow-through cell with a dam obstruction that immobilizes immunosorptive beads forming a bed. This bed-bead immunoassay microsystem significantly improved enabled assay speed from 2 days to 35 min [154]. The system has inlet holes and a reaction area with a holding area for the microbeads for detection. Microbeads precoated with antibody were injected into the system. The sample was then circulated through the flow cell. Next, the sample was washed away, and biotinylated antibody, buffer, and streptavidin-peroxidase were injected into the system. After a final washing operation, the substrate solutions were injected into the system and the resulting enzyme was monitored with a thermal lens microscope. A calibration curve of a peptide hormone concentration yielded a 1-ng/mL sensitivity. 2) Magnetic Beads: Magnetic beads can be used to provide an electronic sensing mechanism for binding. Magnetically labeled assays have low background noise and can be used to manipulate biological systems. An electrochemical immunoassay-based biochemical detection system, which uses a magnetic bio-bead approach, has been reported [155]. A filterless bioseparator using magnetic beads carrying the immobilized antibodies in the form of dendrimers for selective biorecognition is introduced. The separator can hold selected beads in a fluid flow up to 1 mm/s when a 30-mA dc current is applied to an electromagnet. The system uses a bead-based sandwich enzyme immunoassay. The enzyme label is alkaline phosphatase, the enzyme substrate is p-aminophenyl phosphate (PAPP), and the enzyme product is p-aminophenol (PAP). The PAP is electrochemically detected by oxidation. An immunoassay, which uses an array of Hall sensors, fabricated using standard CMOS technology, has been used for this purpose [156]. The Hall sensor chip was coated with a protein-coated gold surface. A simple protocol for this assay is shown in Fig. 27. The test liquid was added [see Fig. 27(B)]. Next, magnetic beads coated with an appropriate antibody against the target antigen were added [see Fig. 27(C)]. The sample was magnetically washed [see Fig. 27(D)]. An external magnetic field normal to the sensor surface was then applied. The induced field generated by the bound beads was measured as a resistance change with the fabricated sensor. A rare-earth magnet was placed above the sensor surface for 60 s before the measurement. The change in resistance corresponds to the number of

E. Microfluidic Bead and Particle Assays A prevailing trend in the development of bioanalytical assays is the display of biochemical reagents on synthetic microbeads [151]–[153]. The incorporation of fluorescent tags into proteins as well as bead-based surface chemistries has resulted in new types of immunoassay systems. Bead-based immunoassay systems use microspheres as a substrate for capturing biomolecules as opposed to planar substrates [129]. Bead assays contain an immunosorptive area much larger than that of planar arrays, and since beads are exposed to the entire sample, collection takes place from the entire volume. Beads are mobile in the sample increasing the rate at which diffusion-limited binding takes place on their surface, (i.e. diffusion lengths equal to their inter-bead separation). Beads are easily manipulated, collected from the sample, and immobilized for readout purposes. 968


Fig. 27.

Fig. 28.

Simplified magnetically labeled bead assay [156].

Portion of sensor array with 3-m magnetic beads [156].

bound beads. Fig. 28 shows the Hall sensor array used for assay detection. Scanning all 256 elements required 2 min. The average SNR was about 13 dB. This device has single magnetic bead sensitivity immobilized for readout purposes. 3) Microparticles: An important consideration in multianalyte based bead particles is the identification of the bead. Each bead has a specific type of antibody or antigen bound to it. Colored beads are commonly used for this purpose. For assays with a large number of analytes, beads can be fabricated with identification markers such as bar codes or combinations of colors. A new approach to perform parallel multianalyte immunoassay, which uses metallic microparticles bearing an engraved bit-type signature for bimolecular coding, has been reported [157]. Micromachined beads consisting of rectangular gold-coated magnetic particles 100 m wide and 20 m thick with a set of etched holes for identification markers were used for a multiplexed immunoassay as shown in Fig. 29. Antibody binding is detected via chemiluminescence, with detection limit of 0.01 g/mL as shown in Fig. 30. F. Capillary Electrophoresis Assay Most conventional immunoassay assays use immunosorbent techniques, in which reagents are adsorbed onto surfaces, involving many fluid handling steps. The unique fluid delivery capabilities of the microelectrophoresis systems provide an important alternative method of automating immunoassays eliminating conventional robotics used in

clinical laboratories today [158]. The application of capillary electrophoresis to immunoassays for the rapid and efficient separation and quantification in immunoassays has been reported [158]–[160]. Since antibody–antigen complexes possess different mobilities among the various constituents, electrophoresis appears to be a natural mechanism to generate rapid immunoassays with immediate on-line quantiation. Nearly all reported devices use LIF detection of fluorescent labeled conjugates [161]. A microscale capillary electrophoresis device has been used to separate the reaction products of a homogeneous reaction using monoclonal mouse IgG in mouse ascites fluid with a direct assay. A linear calibration curve of up to at least 135 g/mL with 3% for the mouse antibovine serum albumin IgG assay was shown. A theophylline assay resulted in a detection limit of 1.25 ng/mL in diluted serum [158]. More recently, a multiplexed microfluidic system capable of performing six immunoassays simultaneously using on-chip mixing, reaction, and affinity capillary electrophoresis has been reported [162]. Fig. 31 shows the schematic layout of the microfluidic system. An LIF detection scheme was used in combination with a single-point galvo-scanner to step a mirror from channel to channel, collecting multiple points for each site. Detection limits of 30 pM for fluoroscein (FL) were obtained, with both qualitative and quantitative immunoassays being conducted. The separations were completed in less than 20 s, and it is speculated that a calibrated data set can be completed for all six assays in less than 1 min. IV. CONCLUSION The clinical and life science need for DNA and protein information far outweighs the ability of conventional assays. Conventional assays tend to be slow and expensive. Assay processing time and cost can be significantly reduced through the miniaturization of such systems. Micromachined devices continue to evolve, and their full impact has yet to be realized. This paper discusses recent progress of microfluidic devices specific to nucleic acid and antibody assays. The progress in the last few years in nucleic assays has led to dramatic improvements in realizing microscale devices for


969

Fig. 29. Process leading to parallel multianalyte immunoassays using a sandwich assay format. Antibodies were tagged with particles coded for a specific location of the substrate [157].

GLOSSARY 3

5

Antibody (Ab)

Fig. 30. Assay calibration curve for IgA, IgG, and IgM immunoglobulins using chemiluminescent detection. Measurement range 0.001–10 and 0.01 g/mL detection limits [157].

Antigen (Ag)

Antimouse IgG

Adenine triphosphate (ATP)

Base pair (bp)

Fig. 31. Schematic layout of system. Access ports and detection zone are labeled for the estradiol (E ) assay. A is the optical alignment channel, B is the buffer, S is E , S is Ab, SW is sample waste, and BW is buffer waste [162].

Complementary DNA (cDNA) Cytokine

genomic based assays. Rapid technological advancements in applying microdevices to genomics and proteomics, specifically for antibody assays, has led to an increase in commercial applications of microarray and bead assays. 970

The nucleic acid strand has a free hydroxyl (or phosphate) on a 3 carbon (carbon atoms in the sugar ring are numbered from 1 to 5 ). The nucleic acid strand has a free hydroxyl (or phosphate) on a 5 carbon (carbon atoms in the sugar ring are numbered from 1 to 5 ). Multifunctional glycoproteins produced by the immune system of vertebrates and are essential for the prevention and resolution of infection by microorganisms. Also called immunoglobulins. Any substance or material that is specifically recognized by antibody or a T cell receptor. A monoclonal antibody that reacts with high affinity to all mouse IgG subclasses via the Fc region. A riboncleoside triphosphate precursor of RNA. Also called adenoside 5’-triphosphate. Other precursors include GTP, CTP, UTP, dATP, dGTP, dCTP, and TTP. The common nucleic acid bases adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U) pair selectively with each other in nucleic acids. The A–T (or T–A) and C–G (or G–C) base pairs form the "rungs" of the DNA double helix, holding the two complementary strands together. Base pairing can also occur within ssDNA or RNA molecules. A complementary DNA copy of an mRNA molecule synthesized in vitro by reverse transcription. Low molecular weight regulatory proteins or glycoproteins secreted by white blood cells and various other cells in the body in response to a number of stimuli.


Dideoxynucleotide triphosphate (ddNTP) Deoxyribonucleic acid (DNA)

DNA Polymerase

Dystrophin gene

Epitope

Exon-6

Gel

Hybridization

Monoclonal antibodies

mRNA

Oligonucleotide

Polymerase chain reaction (PCR) Phage

Polyclonal antibodies

These special nucleotides (ddATP, ddCTP, ddGTP, and ddTTP) terminate the polymerase duplication when captured. A high molecular weight molecule composed of two chains coiled into a double helix, DNA is an almost universal component of living matter. It is located on the chromosomes in the cell nucleus, and consists of nucleoproteins. The set of DNA molecules form the chromosomes, which contain all the information of the genetic code. An enzyme that catalyzes the synthesis of DNA from the 5 to the 3 using a template DNA. Carries instructions for a muscle protein and is flawed in Duchenne and Becker muscular dystrophies (DMD and BMD). Immunologically active regions of an antigen that bind to antigen-specific membrane receptors on lymphocytes or secreted antibodies. Block of nucleotide sequence within a eukaryotic gene which is not removed from the initial RNA transcript. The semisolid compound used to form a collection of fine pores (or gel matrix) through which macromolecules are forced to migrate during gel electrophoresis. A technique that uses the ability of complementary sequences in ssDNA or RNA to pair with each other forming a double helix. Can take place between two cDNA or two RNA sequences. Antibodies derived from a single lineage of lymphocytes. They all have perfectly identical characteristics, in particular the same specificity for the antigen. A molecule that is a type of copy of the DNA chain. It synthesizes cellular proteins based on the genetic information encoded in the DNA. A short length of single stranded polynucleotide chain usually < 30 residues in length. A method that produces several million copies of a single fragment of DNA in several amplification cycles. Bacteriophages are the largest virus group responsible for destroying or modifying bacteria. A mixture of different antibody types specific for antigens an individual is exposed.

Primer

Probe

Ribonucleic acid (RNA)

Single nucleotide polymorphism (SNP)

Single strand DNA (ssDNA) Transcription

A small sequence of a single-stranded DNA or RNA bases (oligonucleotide) which attaches to the template DNA or RNA, and it initiates a complementary strand. Any molecule that specifically binds to a nucleic acid sequence or to a protein that is being sought, and which can be labeled such that the target can be detected. A polymer of monoribonucleotides found in cells and some viruses. Both prokaryotic and eukaryotic cells contain mRNA, tRNA, and rRNA, all involved in the accurate conversion of genetic information (in the form of DNA) into protein. Variations of single base pairs scattered throughout the human genome that serve as measures of the genetic diversity in humans. About 1 million SNPs are estimated to be present in the human genome, and SNPs are useful markers for gene mapping studies. A single chain of deoxyribonucleotides which can be either linear, with free 5 and 3 ends, or circular. Term used to describe the synthesis of RNA from a DNA template.

REFERENCES [1] F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, Eds., Short Protocols in Molecular Biology. New York: Wiley, 2002. [2] L. Stryer, Biochemistry, 3rd ed. New York: Freeman, 1988. [3] P. Wilding, M. A. Shoffner, and L. J. Kricka, “PCR in a silicon microstructure,” Clin. Chem., vol. 40, pp. 1815–1818, 1994. [4] M. A. Northrup, M. T. Ching, R. M. White, and R. T. Watson, “DNA amplification with a microfabricated reaction chamber,” in Proc. 1993 Int. Conf. Solid State Sensors and Actuators, 1994, pp. 924–926. [5] A. T. Wooley, D. Hadley, P. Landre, A. Mello, R. A. Matheis, and M. A. Northrup, “Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device,” Anal. Chem., vol. 68, pp. 4081–4086, 1996. [6] M. A. Burns, C. H. Mastrangelo, T. S. Sammarco, F. P. Man, J. R. Webster, B. N. Johnson, B. Forrestor, D. Jones, Y. Fields, A. R. Kaiser, and D. T. Burker, “Microfabricated structures for integrated DNA analysis,” Proc. Nat. Acad. Sci., vol. 93, pp. 5556–5561, 1998. [7] M. A. Burns, B. N. Johnson, S. N. Brahmasandra, K. Handique, J. R. Webster, M. Krishnan, T. S. Sammarco, P. F. Man, D. Jones, D. Heldsinger, C. H. Mastrangelo, and D. T. Burke, “An integrated nanoliter DNA analysis,” Science, vol. 282, pp. 484–487, 1998. [8] M. U. Kopp, A. J. Mello, and A. Manz, “Chemical amplification: Continuous flow PCR on a chip,” Science, vol. 280, pp. 1046–1048, 1998. [9] P. Belgrader, J. K. Smith, V. W. Weedn, and M. A. Northrup, “Rapid PCR for identity testing using a battery powered miniature thermal cycler,” J. Forensic Sci., vol. 43, pp. 315–319, 1998. [10] T. B. Taylor, P. M. StJohn, and M. Albin, “Microgenetic analysis system,” in Proc. MicroTAS, J. D. Harrison and A. Van den Berg, Eds, 1998, pp. 261–266. [11] H. Yu, P. Sethu, T. Chan, N. Kroutchinina, J. Blackwell, C. H. Mastrangelo, and P. Grodzinski, “A miniaturized and integrated plastic thermal chemical reactor for genetic analysis,” in Proc. MicroTAS, 2000, pp. 545–548.


971

[12] M. A. Shoffner, J. Cheng, G. E. Hvichia, L. J. Krika, and P. Wilding, “Chip PCR: Surface passivation of silicon glass microchips for PCR,” Nucleic Acids Res., vol. 22, pp. 375–379, 1998. [13] T. Tang, G. Ocvirk, and D. J. Harrison, “Isotermal DNA reactions and assays in microfabricated capillary electrophoresis systems,” in Proc. Transducers, 1997, pp. 523–527. [14] C. J. Cheng, M. A. Shoffner, G. E. Hvichia, L. J. Krikca, and P. Wilding, “Chip PCR: Investigation of different PCR amplified systems in microfabricated silicon-glass chips,” Nucleic Acids Res., vol. 24, pp. 380–385, 1996. [15] J. Chiou, P. Matsudaria, A. Sonin, and D. Erlich, “A closed-cycle capillary polymerase chain reaction machine,” Anal. Chem., vol. 73, pp. 2018–2021, 2001. [16] M. Krishnan, V. Ugaz, and M. A. Burns, “PCR in a Rayleigh Benard convection cell,” Science, vol. 298, pp. 793–794, 2002. [17] B. Giordano, J. Ferrance, A. F. R. Höhmer, S. Swedberg, and J. P. Landers, “PCR in polymeric microchips: DNA amplification in less than 240 seconds,” Anal. Biochem., vol. 291, pp. 124–132, 2001. [18] A. F. R. Huhmer and J. P. Landers, “Noncontact infrared heat-mediated thermocycling in capillaries for effective polymerase chain reaction (PCR) in nanoliter volumes,” Anal. Chem., vol. 72, pp. 5507–5512, 2000. [19] D. S. Yoon, Y.-S. Lee, Y. Lee, H. J. Cho, S. W. Sung, K. W. Oh, J. Cha, and G. Lim, “Precise temperature control and rapid thermal cycling in a micromachined DNA polymerase chain reaction chip,” J. Micromech. Microeng., vol. 12, pp. 813–823, 2002. [20] E. T. Lagally, I. Medintz, and R. A. Matheis, “Single molecule DNA amplification and analysis in an integrated microfluidic device,” Anal. Chem., vol. 73, pp. 565–570, 2001. [21] Y. T. Hsue, R. L. Smith, and M. A. Northrup, “A microfabricated electrochemiluminescence cell for the detection of amplified DNA,” in Proc. IEEE Int. Conf. Solid-State Sensors and Actuators, 1995, pp. 768–771. [22] Y. T. Hsue, S. D. Collins, and R. L. Smith, “DNA quantification with an electrochemiliminescence microcell,” in Proc. IEEE Int. Conf. Solid-State Sensors and Actuators, 1997, pp. 175–178. [23] C. G. J. Schabmueller, J. R. Pollard, A. G. R. Evans, J. S. Wilkinson, G. Ensell, and A. Brunnschweiler, “Integrated diode detector and optical fibers for in situ detection within micromachined polymerase chain reaction chips,” J. Micromech. Microeng., vol. 11, pp. 329–333, 2001. [24] P. Wilding, L. J. Krikca, J. Cheng, G. Hvichia, M. A. Shoffner, and P. Fortine, “Integrated cell isolation and polymerase chain reaction analysis using silicon microfilter chamber,” Anal. Biochem., vol. 257, pp. 95–100, 1998. [25] R. Lipshutz, D. Morris, M. Chee, E. Hubbell, M. Kozal, N. Shah, N. Shen, R. Yang, and S. Fodor, “Using oligonucleotide probe arrays to access genetic diversity,” Biotechniques, vol. 19, pp. 442–447, 1995. [26] R. G. Sosnowski, E. Tu, W. F. Butler, J. P. O’Connell, and M. J. Heller, “Rapid determination of single base pair mutations in DNA hybrids by direct electric filed control,” Proc. Nat. Acad. Sci., vol. 94, pp. 1119–1123, 1997. [27] R. S. Matson, J. Rampla, S. L. Pentoney, P. D. Anderson, and P. Coassin, “Biopolymer synthesis on polypropylene supports: Oligonucleotide arrays,” Anal. Biochem., vol. 224, pp. 110–116, 1995. [28] J. D. Hoheisel, “Oligomer-chip technology,” Trends Biotech., vol. 16, pp. 465–469, 1998. [29] S. P. A. Fodor, R. P. Rava, X. C. Huang, A. C. Pease, C. P. Holmes, and C. L. Adams, “Multiplexed biochemical assays with biological chips,” Nature, vol. 364, pp. 555–556, 1993. [30] G. Ramsay, “DNA chips: State of the art,” Nature Biotech., vol. 16, pp. 40–44, 1998. [31] M. J. Heller and A. Guttman, Eds., Integrated Microfabricated Biodevices. New York: Marcel Dekker, 2001. [32] M. Chee, R. Yang, E. Hubbell, A. Berno, X. C. Huang, D. Stern, J. Winkler, D. J. Lockhart, M. S. Morris, and S. P. A. Fodor, “Accessing genetic information with high density DNA arrays,” Science, vol. 274, pp. 610–614, 1996. [33] J. G. Hacia, L. C. Brody, M. S. Chee, S. P. A. Fodor, and F. C. Collins, “Detection of heterozygous mutations inBRCA1 using high density oligonucleotide arrays and two color fluorescence analysis,” Nat. Genet., vol. 14, pp. 441–447, 1996. [34] M. Schena, D. Shalon, R. Heller, A. Chai, P. Brown, and R. Davis, “Parallel human genome analysis: Microarray based expression monitoring of 1000 genes,” Proc. Nat. Acad. Sci., vol. 93, pp. 10 614–10 619, 1996.

972

[35] D. M. Grant and M. S. Phillips, Technologies for the Analysis of Single-Nucleotide Polymorphisms: An Overview, W. Kalow, U. A. Meyer, and R. Tyndale, Eds. New York: Marcel Dekker, 2001, vol. 113. [36] S. V. Tillib and A. D. Mirzabekov, “Advances in analysis of DNA sequence variation using oligonucleotide microchip technology,” Curr. Opin. Biotechnol., vol. 12, pp. 53–58, 2001. [37] R. C. Anderson, G. McGall, and R. J. Lipshutz, “Polynucleotide arrays for genetic sequence analysis,” in Topics in Current Chemistry, A. Manz and H. Becker, Eds. Heidelberg, Germany: Springer-Verlag, 1998. [38] G. McGall, J. Labadie, P. Brock, G. Wallraff, T. Nguyen, and W. Hinsberg, “Light directed synthesis of high density oligonucleotide arrays using semiconductor photoresists,” Proc. Nat. Acad. Sci., vol. 93, pp. 13 555–13 560, 1996. [39] J. E. Beecher, G. H. McGall, and M. J. Goldberg, “Chemically amplified photolithography for the fabrication of high density oligonucleotide arrays,” Polymer Mater. Sci. Eng., vol. 76, pp. 597–598, 1997. [40] X. Gao, E. LeProust, H. Zhang, O. Srivannavit, E. Gulari, P. Yu, C. Nishiguchi, Q. Xiang, and X. Zhou, “A flexible light-directed DNA chip synthesis gated by deprotection using solution photogenerated acids,” Nucleic Acids Res., vol. 29, pp. 4744–4750, 2001. [41] M. Schena, R. A. Heller, T. P. Theriault, K. Konrad, E. Lachenmeier, and R. W. Davis, “Microarrays: Biotechnology’s discovery platform for functional genomics,” Trends Biotechnol., vol. 16, pp. 301–306, 1998. [42] D. Shalon, S. J. Smith, and P. O. Brown, “A DNA microarray system for analyzing complex DNA samples using two color fluorescent probe hybridization,” Genome Res., vol. 6, pp. 639–645, 1996. [43] E. Timofeev, S. Kochetkova, A. Mirzabekov, and V. Florentiev, “Regioselective immobilization of short oligonucleotides to acrylic copolymer gels,” Nucleic Acids Res., vol. 24, pp. 3142–3148, 1996. [44] D. Lockhart, H. Dong, M. Byrne, M. Follettie, M. Gallo, M. Chee, M. Mittmann, C. Wang, M. Kobayashi, H. Horton, and E. Brown, “Expression monitoring by hybridization to high density oligonucleotides arrays,” Nature Biotechnol., vol. 14, pp. 167–168, 1996. [45] V. Benoit, A. Steel, M. Torres, Y. Yu, H. Yang, and J. Cooper, “Evaluation of three-dimensional microchannel glass biochips for multiplexed nucleic acid fluorescence hybridization assays,” Anal. Chem., vol. 73, pp. 2412–2420, 2001. [46] T. Pastinen, A. Krug, A. Metspalu, L. Peltonen, and A. C. Syvanen, “Minisequencing: A specific tool for DNA analysis and diagnostics on oligonucleotide arrays,” Genome Res., vol. 7, pp. 606–614, 1997. [47] K. Gunderson, X. Huang, M. Morris, R. Lipshutz, D. Lockhart, and M. Chee, “Mutation detection by ligation to complete n-mer DNA arrays,” Genome Res., vol. 8, pp. 1142–1153, 1998. [48] A. Krug, N. Tonisson, I. Georgiou, J. Shumaker, J. Tollett, and A. Metspalu, “Arrayed primer extension: A sloid phase four color DNA resequencing and mutation detection technology,” Genetic Testing, vol. 4, pp. 1–7, 2000. [49] T. J. Meade and J. F. Kayyem, “Electron transfer through DNA: Site-specific modification of duplex DNA with ruthenium donors and acceptors,” Angew. Chem. Int. Ed. Engl., vol. 34, pp. 354–358, 1995. [50] F. D. Lewis, T. Wu, Y. Zhang, R. L. Letsinger, S. R. Greenfield, and M. R. Wasielewski, “Distance-dependent electron transfer in DNA hairpins,” Science, vol. 277, pp. 673–676, 1997. [51] J. F. Kayyem, “DNA diagnostics using electronic detection,” Clin. Chem. Supplement, vol. 45, 1999. [52] J. Yu, Y. Wan, H. Yowanto, J. Li, C. Tao, M. D. James, C. L. Tan, G. F. Blackburn, and T. J. Meade, “Electronic detection of single-base mismatches in DNA with ferrocene-modified probes,” J. Amer. Chem. Soc., vol. 123, pp. 11 155–11 161, 2001. [53] A. G. Georgakilas, A. A. Konsta, K. S. Haveles, and E. G. Sideris, “Dielectric study of the double helix to single coil transition of DNA,” IEEE Trans. Dielect. Elect. Insulation, vol. 5, pp. 26–32, Feb. 1998. [54] E. Palecek, “Past, present and future of nucleic acids electrochemistry,” Talanta, vol. 56, pp. 809–819, 2002. [55] E. M. Boon, D. M. Ceres, T. G. Drummond, M. G. Hill, and J. K. Barton, “Mutation detection by electrocatalysis at DNA modified electrodes,” Nature Biotechnol., vol. 18, pp. 1096–1100, 2000. [56] S. J. Park, T. A. Taton, and C. A. Mirkin, “Array based electrical detection of DNA with nanoparticle probes,” Science, vol. 295, pp. 349–350, 2002.


[57] D. P. Little, T. J. Cornish, M. J. O’Donnell, A. Braun, R. J. Cotter, and H. Koster, “MALDI on a chip: Analysis of arrays of low-femtomole to subfemtomole quantities of synthetic oligonucleotides and DNA diagnostic products dispensed by a piezoelectric pipet,” Anal. Chem., vol. 69, pp. 4540–4546, 1997. [58] B. Tinland, A. Pluen, J. Strum, and G. Weill, “Persistence length of single stranded DNA,” Macromolecules, vol. 31, pp. 5763–5765, 1997. [59] D. J. Caruana and A. Heller, “Enzyme amplified amperometric detection of hybridization and of a single base pair mutation in an 18 base oligo on 7 m diameter microelectrode,” J. Amer. Chem. Soc., vol. 121, pp. 220–225, 1999. [60] F. Azek, C. Gossiord, M. Joannes, B. Limoges, and P. Brossier, “Hybridization assay at a disposable electrochemical biosensor for the attomole detection of amplified human cytomegalovirus DNA,” Anal. Biochem., vol. 284, pp. 107–113, 2000. [61] A. Ahmadian, B. Gharizadeh, A. Gustafsson, F. Sterky, P. Nyrn, M. Uhl, and J. Lundeberg, “SNP analysis by pyrosequencing,” Anal. Biochem., vol. 280, pp. 103–110, 2000. [62] A. Russom, A. Ahmadian, H. Andersson, W. Wijngaart, J. Lundeberg, M. Uhlen, G. Stemme, and P. Nilsson, “SNP analysis by allele specific pyrosequencing extension in a micromachined filter chamber device,” in Proc. Micro Total Analysis Systems, J. M. Ramsey, Ed., 2001, pp. 22–24. [63] J. Fritz, M. K. Baller, H. P. Lang, H. Rothuizen, P. Vettiger, E. Meyer, H. J. Guntherodt, C. Gerber, and J. K. Gimzewski, “Translating biomolecular recognition into nanomechanics,” Science, vol. 288, pp. 316–318, 2000. [64] G. H. Wu, H. F. Ji, K. Hansen, T. Thundat, R. Datar, R. Cote, M. F. Hagan, A. K. Chakraborty, and A. Majumdar, “Origin of nanomechanical cantilever motion generated from biomolecular interactions,” Proc. Nat. Acad. Sci., vol. 98, pp. 1560–1564, 2001. [65] R. Raiteri, M. Grattarola, and R. Berger, “Micromechanics sense biomolocules,” Mater. Today, pp. 22–29, Jan. 2002. [66] P. Vettiger, “The “millipede”—More than one thousand tips for future AFM data storage,” IBM J. Res. Develop., vol. 44, pp. 323–327, 2000. [67] J. S. Lee, S. Oh, Y. K. Choi, and L. Lee, “Label free dielectric detection of DNA hybridization with nanogap junctions,” in Proc. Micro Total Analysis Systems, Y. Baba, Ed., 2002, pp. 305–307. [68] Z. H. Fan, S. Mangru, R. Granzow, P. Heaney, W. Ho, Q. Dong, and R. Kumar, “Dynamic DNA hybridization on a chip using paramagnetic beads,” Anal. Chem., vol. 71, pp. 4851–4859, 1999. [69] P. D. Grossman and J. C. Colburn, Capillary Electrophoresis: Theory and Practice. New York: Academic, 1992. [70] M. D. O’Neill, “Sequencers benefit from solid state detectors,” Laser Focus World, vol. 10, pp. 135–142, 1995. [71] A. Manz, D. J. Harrison, E. M. Verpoorte, and H. M. Widmer, “Planar chips technology for miniaturization of separation systems: A developing perspective in chemical processing,” in Advances in Chromatography, P. R. Brown and E. Gruska, Eds. New York: Marcel Dekker, 1993, pp. 1–66. [72] Z. Ronai, C. Barta, M. Sasvari-Szekely, and A. Guttman, “DNA analysis on electrophoretic microchips: Effect of operational variables,” Electrophoresis, vol. 22, pp. 294–299, 2001. [73] C. S. Effenhauser, A. Paulus, A. Manz, and H. M. Widmer, “High speed separation of ‘anti-sense’ oligonucleotides on a micromachined capillary electrophoresis device,” Anal. Chem., vol. 66, pp. 2949–2953, 1994. [74] S. Liu, Y. Shi, W. Ja, and R. A. Mathies, “Optimization of highspeed DNA sequencing on microfabricated capillary electrophoresis channels,” Anal. Chem., vol. 71, pp. 566–573, 1999. [75] B. M. Pagel, C. A. Emrich, G. J. Wedemayer, J. R. Scherer, and R. A. Mathies, “High throughput DNA sequencing with a microfabricated 96 lane capillary array electrophoresis bioprocessor,” Proc. Nat. Acad. Sci., vol. 99, pp. 574–579, 2002. [76] A. T. Woolley and R. A. Mathies, “Ultra-high-speed DNA fragment separations using microfabricated capillary array,” Proc. Nat. Acad. Sci., vol. 91, pp. 11 348–11 352, 1994. [77] C. S. Effenhauser, G. J. M. Bruin, and A. Paulus, “Integrated chip-based capillary electrophoresis—A review,” Electrophoresis, vol. 18, pp. 2203–2213, 1997. [78] R. M. McCormick, R. J. Nelson, M. G. Alonso-Amigo, D. J. Benvegnu, and H. H. Hooper, “Microchannel electrophoretic separations of DNA in injection-molded plastic substrates,” Anal. Chem., vol. 69, pp. 2626–2630, 1997.

[79] A. T. Woolley, G. F. Sensabaugh, and R. A. Mathies, “High-speed DNA genotyping using microfabricated capillary array electrophoresis chips,” Anal. Chem., vol. 69, pp. 2181–2186, 1997. [80] J. Cheng, L. C. Waters, P. Fortina, G. Hvichia, S. C. Jacobson, J. M. Ramsey, L. J. Kricka, and P. Wilding, “Degenerate oligonucleotide primed-polymerase chain reaction and capillary electrophoretic analysis of human DNA on microchip-based devices,” Anal. Biochem., vol. 257, pp. 101–106, 1998. [81] D. Schmalzing, L. Koutny, A. Adourian, P. Belgrader, P. Matsudaria, and D. Erlich, “DNA typing in 30 seconds with a microfabricated device,” Proc. Nat. Acad. Sci., vol. 94, pp. 10 273–10 278, 1997. [82] A. T. Woolley and R. A. Mathies, “Ultra-high-speed DNA sequencing using capillary electrophoresis chips,” Anal. Chem., vol. 67, pp. 3676–3680, 1995. [83] J. M. Ramsey, S. C. Jacobson, and M. R. Knapp, “Microfabricated chemical measurement systems,” Nature Med., vol. 1, pp. 1093–1096, 1995. [84] A. Manz, N. Graber, and H. M. Widmer, “Miniaturized total chemical analysis systems: A novel concept for chemical sensing,” Sens. Actuators, vol. B1, pp. 244–248, 1990. [85] J. D. Harrison, K. Fluri, K. Seiler, Z. Fan, C. S. Effenhauser, and A. Manz, “Micromachining a miniaturized capillary electrophoresisbased chemical analysis system on a chip,” Science, vol. 261, pp. 895–897, 1993. [86] D. Schmalzing, L. Koutny, O. Salas-Solano, A. Adourian, P. Matsudaria, and D. Erlich, “Recent developments in DNA sequencing by capillary and microdevice electrophoresis,” Electrophoresis, vol. 20, pp. 3066–3077, 1999. [87] C. H. Mastrangelo, M. A. Burns, and D. T. Burke, “Microfabricated devices for genetic diagnostics,” Proc. IEEE, vol. 86, pp. 1769–1787, Aug. 1998. [88] V. Dolnk, S. Liu, and S. Jovanovich, “Capillary electrophoresis on microchip,” Electrophoresis, vol. 21, pp. 41–54, 2000. [89] Z. H. Fan and J. D. Harrison, “Micromachining of capillary electrophoresis injectors and separators on glass chips and evaluation of flow at capillary intersections,” Anal. Chem., vol. 66, pp. 177–184, 1994. [90] S. C. Jacobson, R. Hergenroder, L. B. Koutny, R. J. Warmack, and J. M. Ramsey, “Effects of injection schemes and column geometry on the performance of microchip electrophoresis devices,” Anal. Chem., vol. 66, pp. 1107–1113, 1994. [91] L. L. Shultz-Lockyear, C. L. Colyer, Z. H. Fan, K. I. Roy, and D. J. Harrison, “Effects of injector geometry and sample matrix on injection and sample loading in integrated capillary electrophoresis devices,” Electrophoresis, vol. 20, pp. 529–538, 1999. [92] M. A. Burns, C. H. Mastrangelo, T. S. Sammarco, P. F. Man, J. R. Webster, B. N. Johnson, B. Foerster, D. K. Jones, Y. Fields, A. R. Kaiser, and D. T. Burke, “Microfabricated structures for integrated DNA analysis,” Proc. Nat. Acad. Sci., vol. 93, pp. 5556–5561, 1996. [93] J. Khandurina, S. C. Jacobson, L. C. Waters, R. S. Foote, and J. M. Ramsey, “Microfabricated porous membrane structures for sample concentration and electrophoretic analysis,” Anal. Chem., vol. 71, pp. 1815–1819, 1999. [94] S. N. Brahmasandra, J. R. Webster, D. T. Burker, C. H. Mastrangelo, and M. A. Burns, “On-chip DNA band detection in microfabricated separation systems,” in Proc. SPIE, vol. 3515, 1998, pp. 242–251. [95] W. M. Sunada and H. W. Blanch, “Polymeric separation media for capillary electrophoresis of nucleic acids,” Electrophoresis, vol. 18, pp. 2243–2254, 1997. [96] S. W. Turner, A. M. Perez, A. Lopez, and H. G. Craighead, “Monolithic nanofluid sieving structures for DNA manipulation,” J. Vac. Sci. Technol. B, vol. 16, pp. 3821–3824, 1998. [97] V. Studer, P. Youinou, A. Pepin, A. Lebib, Y. Chen, and Y. Baba, “Nanoimprint fabrication of DNA electrophoresis chips,” in Proc. Micro Total Analysis Systems, J. M. Ramsey, Ed., Netherlands, 2001, pp. 201–202. [98] J. Han and H. G. Craighead, “Separation of long DNA molecules in a microfabricated entropic trap array,” Science, vol. 288, pp. 1026–1029, 2000. [99] R. W. Hammond, J. S. Bader, S. A. Henck, M. W. Deem, G. A. McDermott, J. M. Bustillo, and J. M. Rothberg, “Differential transport of DNA by a rectified brownian motion device,” Electrophoresis, vol. 21, pp. 74–80, 2000. [100] J. S. Bader, R. W. Hammond, S. A. Henck, M. W. Deem, G. A. McDermott, J. M. Bustillo, J. W. Simpson, G. T. Mulhern, and J. M. Rothberg, “DNA transport by a micromachined brownian ratchet device,” Proc. Nat. Acad. Sci., vol. 96, pp. 13 165–13 169, 1999.


973

[101] J. S. Bader, M. W. Deem, R. W. Hammond, S. A. Henck, J. W. Simpson, and J. M. Rothberg, “A brownian-ratchet DNA pump with applications to single-nucleotide polymorphism genotyping,” Appl. Phys. A, vol. 75, pp. 275–278, 2002. [102] M. Ueda, Y. Endo, H. Abe, H. Kuyama, H. Nakanishi, A. Arai, and Y. Baba, “Field inversion electrophoresis on a microchip device,” Electrophoresis, vol. 22, pp. 217–221, 2001. [103] A. Manz, L. Bousse, A. Chow, T. B. Metha, A. Kopf-Sill, and J. W. Parce, “Synchronized cyclic capillary electrophoresis using channels arranged in a triangle and low voltages,” Fresenius J. Anal. Chem., vol. 371, pp. 195–201, 2001. [104] G. A. Griess, A. Choi, A. Basu, J. W. Valvano, and P. Serwer, “Cyclic capillary electrophoresis,” Electrophoresis, vol. 23, pp. 2610–2617, 2002. [105] N. Burggraf, A. Manz, E. Verpoorte, C. S. Effenhauser, and H. M. Widmer, “A novel approach to ion separations in solution: Synchronized cyclic capillary electrophoresis,” Sens. Actuators B, vol. 20, pp. 103–110, 1994. [106] Y.-C. Lin, “Design of low voltage-driven capillary electrophoresis chips using moving electrical fields,” Sens. Actuators B, vol. 80, pp. 33–40, 2001. [107] L. Z. Song, M. R. Hobaugh, C. Shustak, S. Cheley, H. Bayley, and J. E. Gouaux, “Structure of staphylococcal alpha-hemalysin, a hepatameric transmembrane pore,” Science, vol. 274, pp. 1859–1866, 1996. [108] M. Akeson, D. W. Deamer, W. Vercoutere, R. Braslau, and H. Olsen, “Use of a nanoscale pore to read short segments within single polynucleotide molecules,” presented at the NATO Advanced Research Workshop Polymer Structure and Transport Confined Spaces, Bikal, Hungary, 1999. [109] S. Howorka, S. Cheley, and H. Bayley, “Sequence specific detection of individual DNA strands using engineered nanopores,” Nature Biotechnol., vol. 19, pp. 636–639, 2001. [110] J. Li, D. Stein, C. McMullan, D. Branton, M. J. Aziz, and M. A. Golovchenko, “Ion beam sculpting at nanometer length scales,” Nature, vol. 412, pp. 166–169, 2001. [111] O. A. Saleh and L. L. Sohn, “A quantitative nanoscale coulter counter,” in Proc. Micro Total Analysis Systems, J. M. Ramsey, Ed., 2001, pp. 54–56. [112] J. D. Harrison, K. Seiler, A. Manz, and Z. Fan, “Chemical analysis and electrophoresis systems integrated on glass and silicon chips,” in Proc. Int. Workshop Solid-State Sensors and Actuators, 1992, pp. 110–113. [113] T. Tang, G. Ocvirk, and D. J. Harrison, “Iso-thermal DNA reactions and assays in microfabricated capillary electrophoresis systems,” in Proc. IEEE Int. Conf. Solid-State Sensors and Actuators, 1997, pp. 523–526. [114] B. B. Haab and R. A. Mathies, “Single molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams,” Anal. Chem., vol. 71, pp. 5137–5145, 2001. [115] J. R. Webster, M. A. Burns, D. T. Burke, and C. H. Mastrangelo, “Monolithic capillary electrophoresis device with integrated fluorescence detector,” Anal. Chem., vol. 73, pp. 1622–1626, 2001. [116] A. T. Woolley, K. Q. Lao, A. N. Glazer, and R. A. Matheis, “Capillary electrophoresis chips with integrated electrochemical detection,” Anal. Chem., vol. 70, pp. 684–688, 1998. [117] P. Selvaganapathy, M. A. Burns, and D. T. Burke, “Inline electrochemical detection for capillary electrophoresis,” in IEEE MEMS Conf., 2001, pp. 451–454. [118] P. Selvaganapathy, “Components for an integrated microfabricated electroanalysis system,” Ph.D. dissertation, Univ. Michigan, Ann Arbor, 2002. [119] H. P. Chou, C. Spence, A. Scherer, and S. Quake, “A microfabricated device for sizing and sorting DNA molecules,” Proc. Nat. Acad. Sci., vol. 96, pp. 11–13, 1999. [120] R. P. Ekins, “Ligand assays: From electrophoresis to miniaturized microarrays,” Clin. Chem., vol. 44, no. 9, pp. 2015–2030, 1998. [121] M. Madou and J. Joseph, “Immunosensors with commercial potential,” Immunomethods, vol. 3, pp. 134–152, 1993. [122] B. Alberts, D. Bray, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, Essential Cell Biology: An Introduction to the Molecular Biology of the Cell. New York: Taylor & Francis, 1998. [123] A. P. Johnston and M. W. Turner, Immunochemistry Vols. 1 & 2. New York: Oxford Univ. Press, 1997. [124] P. Tijssen, “Practice and theory of enzyme immunoassays,” in Laboratory Techniques in Biochemistry and Molecular Biology, R. H. Burdon and P. H. van Knippenberg, Eds. Amsterdam, The Netherlands: Elsevier, 1985, vol. 15, pp. 39–150.

974

[125] R. A. Goldsby, T. J. Kindt, and B. A. Osborne, Kuby Immunology, 4th ed. New York: W.H. Freeman, 2000. [126] T. Vo-Dinh, M. J. Sepaniak, G. D. Griffin, and J. P. Alarie, “Immunosensors: Principles and applications,” Immunomethods, vol. 3, pp. 85–92, 1993. [127] A. K. Abbas, A. H. Lichtman, and J. S. Pober, Cellular and Molecular Immunology. Philadelphia, PA: W. B. Sanders, 1991. [128] D. S. Wilson and S. Nock, “Function protein arrays,” Curr. Opin. Chem. Biol., vol. 6, pp. 81–85, 2001. [129] T. O. Joos, D. Stoll, and M. F. Templin, “Miniaturised multiplexed immunoassays,” Curr. Opin. Chem. Biol., vol. 6, pp. 76–80, 2001. [130] M. F. Templin, D. Stoll, M. Schrenk, P. C. Traub, C. F. Vohringer, and T. O. Joos, “Protein microarray technology,” Drug Discovery Today, vol. 7, no. 15, pp. 815–822, 2002. [131] B. Schweitzer, S. Wiltshire, J. Lambert, S. O’Malley, K. Kukanskis, Z. Zhu, S. F. Kingsmore, P. M. Lizardi, and D. C. Ward, “Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection,” Proc. Nat. Acad. Sci., vol. 97, no. 18, pp. 10 113–10 119, Aug. 29, 2000. [132] E. P. Diamandis and T. K. Christopoulos, Eds., Immunoassay. San Diego, CA: Academic, 1996. [133] R. P. van Gijlswijk, H. J. Zijlmans, J. Wiegant, M. N. Bobrow, T. J. Erickson, K. E. Adler, H. J. Tanke, and A. K. Raap, “Fluorochrome-labeled tyramides: Use in immunocytochemistry and fluorescence in-situ hybridization,” J. Histochem. Cytochem., vol. 45, pp. 375–382, 1997. [134] P. M. Lizardi, X. Huang, Z. Zhu, P. Bray-Ward, D. C. Thomas, and D. C. Ward, “Mutation detection and single-molecule counting using isothermal rolling-circle amplification,” Nature Genet., vol. 19, pp. 225–232, 1998. [135] S. P. Lal, R. I. Christopherson, and C. G. dos Remedios, “Antibody arrays: An embryonic but rapidly growing technology,” Drug Discovery Today, vol. 7, no. 18, pp. S143–S149, 2002. [136] T. Kodadek, “Protein microarrays: Prospects and problems,” Clin. Biol., vol. 8, pp. 105–115, 2001. [137] H. Zhu et al., “Global analysis of protein activities using proteome chips,” Science, vol. 293, pp. 2101–2106, 2001. [138] B. B. Haab, M. J. Dunham, and P. O. Brown. (2001) Protein microarrays for highly parallel detection and quantiation of specific proteins and antibodies in complex solutions. Genome Biology [Online] Available: http://genomebiology.com/2001/2/2/research/0004 [139] G. MacBeth and S. L. Schreiber, “Printing proteins as microarrays for high-throughput function determination,” Science, vol. 289, pp. 1760–1763, 2000. [140] B. D. Martin, B. P. Garber, C. H. Patterson, and D. C. Turner, “Direct protein microarray fabrication using hydrogel ‘stamper’,” Langmuir, vol. 14, pp. 3971–3975, 1998. [141] M.-J. Lee, H. Huang, C.-K. Chou, Y.-C. Tsai, F.-G. Tseng, and C.-C. Chieng et al., “Diagnostic antigens and antibodies patterned by micro stamping system,” in Proc. Micro Total Analysis Systems, vol. 1, Y. Baba et al., Eds., 2002, pp. 461–463. [142] S. W. Howell, H. D. Inerowicz, L. Guirl, F. Regnier, and R. Reifenberger, “Immunosensing using fabricated protein microarrays,” in Nanotech, vol. 1, 2003, pp. 74–77. [143] R. M. Wadkins, J. P. Golden, L. M. Pritsiloas, and F. S. Ligler, “Detection of multiple toxic agents using a planar array immunosensor,” Biosens. Bioelectron., vol. 13, pp. 407–415, 1998. [144] U.S. Patent 5 677 196, 1997. [145] C. A. Rowe, S. B. Scruggs, M. J. Feldstein, J. P. Golden, and F. S. Ligler, “An array immunosensor for simultaneous detection of clinical analytes,” Anal. Chem., vol. 71, pp. 433–439, 1999. [146] R.-P. Huang, R. Huang, Y. Fan, and Y. Lin, “Simultaneous detection of multiple cytokines from conditioned media and patient’s sera by an antibody-based protein array system,” Anal. Biochem., vol. 294, pp. 55–62, 2001. [147] J. W. Silzel, B. Cercek, C. Dobson, T. Tsay, and R. J. Obremsko, “Mass-sensing, multianalyte microarray immunoassay with imaging detection,” Clin. Chem., vol. 44, no. 9, pp. 2036–2043, 1998. [148] C. A. Rowe, L. M. Tender, M. J. Feldstein, J. P. Golden, S. B. Scruggs, B. D. MacCraith, J. J. Cras, and F. S. Ligler, “Array biosensor for simultaneous identification of bacterial, viral, and protein analytes,” Anal. Chem., vol. 71, pp. 3846–3852, 1999. [149] K. E. Sapsford, P. T. Charles, C. H. Patterson Jr., and F. S. Ligler, “Demonstration of four immunoassay formats using the array biosensor,” Anal. Chem., vol. 74, pp. 1061–1068, 2002. [150] N. Honda, S. Shoji, M. Isomura, Y. Ashihara, K. Akahori, and H. Sato, “Fabrication of microflow cell for multianalyte sandwich immunoassay using Teflon micro check valves,” in Proc. Micro Total Analysis Systems, J. M. Ramsey and A. van den Berg, Eds., 2001, pp. 403–404.


[151] L. B. Bangs, “New developments in particle-based immunoassays: Introduction,” Pure Appl. Chem., vol. 68, pp. 1873–1879, 1996. [152] S. B. Marchenko, A. A. Zhdanov, I. A. Gritskova, and S. Y. Zaitsev, “Modeling of the interfacial absorption layer of polystyrene particles by the langmuir technique,” Polymer Sci. Ser. A, vol. 43, pp. 273–277, 2001. [153] H. Andersson, W. van der Wijngaart, and G. Stemme, “Micromachined filter-chamber array with passive valves for biochemical assays on beads,” Electrophoresis, vol. 22, pp. 249–257, 2001. [154] K. Sato, M. Tokeshi, T. Odake, H. Kimura, T. Ooi, M. Nakao, and T. Kitamori, “Integration of an immunosorbent assay system: Analysis of secretory human immunoglobulin A on polystyrene beads in a microchip,” Anal. Chem., vol. 72, pp. 1144–1147, 2000. [155] C. H. Ahn, T. Henderson, W. Heineman, and B. Halsall, “Development of a generic microfluidic system for electrochemical immunoassay-based remote bio/chemical sensors,” in Proc. TAS Workshop, 1998, pp. 225–230. [156] T. Aytur, P. R. Beatty, and B. Boser, “An immunoassay platform based on CMOS hall sensors,” in Proc. Solid-State Sensor, Actuator and Microsystems Workshop, 2002, pp. 126–129. [157] Z.-L. Zhi, Y. Morita, and E. Tamiya, “Microfabricated addressable particles for multianalyte immunoassay,” in Proc. IEEE 16th Annu. Int. Conf. MEMS, 2003, pp. 411–414. [158] N. Chiem and D. J. Harrison, “Microchip-based capillary electrophoresis for immunoassays: Analysis of monoclonal antibodies and theophylline,” Anal. Chem., vol. 69, pp. 373–378, 1997. [159] F.-T. A. Chen and S. L. Pentoney, “Characterization of digoxigenin-labeled B-phycoerythrin by capillary electrophoresis with laser-induced fluorescence application to homogeneous digoxin immunoassay,” J. Chromatography A, vol. 680, pp. 425–430, 1994. [160] L. B. Koutny, D. Schmalzing, T. A. Taylor, and M. Fuchs, “Microchip electrophoretic immunoassay for serum control,” Anal. Chem., vol. 68, pp. 18–22, 1996. [161] J. J. Bao, “Capillary electrophoresis immunoassays,” J. Chromatography B, vol. 699, pp. 463–480, 1997. [162] S. B. Cheng, C. D. Skinner, J. Taylor, W. E. Lee, M. Jolivet, G. Picelli, and D. J. Harrison, “Multichannel microchip system for rapid calibration and immunoassay,” in Proc. Micro Total Analysis Systems, A. van den Berg, Ed., 2000, pp. 533–536.

Ponnambalam Ravi Selvaganapathy was born in Madras, India, in 1977. He received the B.S. degree in chemical and electrochemical engineering from Central ElectroChemical Research Institute (CECRI), Karaikudi, India, in 1998. During his research at CECRI, he worked on cathodic disbondment of polymer coatings due to corrosion and on development finite element software for corrosion monitoring and detection. He received the M.S. and Ph.D. degrees in electrical engineering and computer science from the University of Michigan, Ann Arbor, in 2001 and 2002, respectively. He also received the M.S. degree in nuclear engineering (materials) from the University of Michigan in 2002. From 1998 through 2002, he was a Research Assistant at the Center for Integrated MicroSystems at the University of Michigan, working on a variety of projects related to MEMS and BioMEMS processes and device development including electrochemical detection in capillary electrophoresis systems, electroosmotic pumping systems, prevention of bubble generation in electrically active microfluidic systems, thermal phase change polymers, development of microfabrication techniques for porous polymers in microsystems, development of a contact-free technique for pH measurement using the Kelvin probe, and investigation of microplasmas as a fluorescence excitation source for DNA.

Edwin T. Carlen received the B.S. and M.S. degrees in electrical engineering from Oakland University, Rochester, MI, in 1989 and 1993, respectively, and the Ph.D. degree in electrical engineering from the University of Michigan, Ann Arbor, in 2001. His thesis work resulted in the realization of novel polymer-actuated microvalves. From 1990 to 1993, he was a Design Engineer in the Engine Controls Group, Automotive and Industrial Electronics Group, Motorola, Northbrook, IL. From 1993 to 1996, he was a Research Engineer in the Advanced Engineering Division, Ford Motor Co., Dearborn, MI. From 1996 through 2000, he was a Research Assistant at the Center for Integrated MicroSystems, University of Michigan, working on a variety of projects related to MEMS and BioMEMS process and device development. Since 2000, he has been working for Corning-Intellisense, Wilmington, MA, as a MEMS Development Engineer. His research focuses on the development of microfabrication and micromachining technologies for solid-state sensors and microactuators and novel material development for MEMS and BioMEMS applications. Dr. Carlen was a Coauthor of the 1999 Best Paper Award from the IEEE TRANSACTIONS ON SEMICONDUCTOR MANUFACTURING editorial staff.

Carlos H. Mastrangelo (Member, IEEE) was born in Buenos Aires, Argentina, in 1960. He received the B.S., M.S., and Ph.D. degrees in electrical engineering and computer science from the University of California, Berkeley, in 1985, 1988, and 1991, respectively. His graduate work concentrated on the applications of microbridges in microsensor technology. From 1991 through 1992, he worked at the Scientific Research Laboratory, Ford Motor Company, Dearborn, MI, developing microsensors for automotive applications. From 1993 to 2000, he was first an Assistant Professor, then an Associate Professor of electrical engineering and computer science at the Center for Integrated Microsystems, Univesity of Michigan, Ann Arbor. He is currently Vice President of Engineering at Corning-IntelliSense Corporation, Wilmington, MA. His research focuses on microsensor and microelectromechnical system applications and technology, microfluidic systems, and integration, design, and modeling of complex fabrication processes. Dr. Mastrangelo received the AT&T Ph.D. Fellowship Award in 1987. In 1991, he received the Sakrison Award for the best dissertation in the department. He received the 1991 Counsel of the Graduate Schools/University Micro-films Distinguished Dissertation Award for the best technical dissertation in the United States and Canada. He received a 1993 NSF Research Initiation Award and a 1994 NSF Young Investigator Award. He also received the 1999 Best paper award from the IEEE TRANSACTIONS ON SEMICONDUCTOR MANUFACTURING for his early contributions to semiconductor and MEMS process flow synthesis.


975