Biomedical microelectromechanical systems (BioMEMS): Revolution in drug delivery and analytical techniques

Advancement in microelectromechanical system has facilitated the microfabrication of polymeric substrates and the development of the novel class of controlled drug delivery devices. These vehicles have specifically tailored three dimensional physical and chemical features which together, provide the capacity to target cell, stimulate unidirectional controlled release of therapeutics and augment permeation across the barriers. Apart from drug delivery devices microfabrication technology’s offer exciting prospects to generate biomimetic gastrointestinal tract models. BioMEMS are capable of analysing biochemical liquid sample like solution of metabolites, macromolecules, proteins, nucleic acid, cells and viruses. This review summarized multidisciplinary application of biomedical microelectromechanical systems in drug delivery and its potential in analytical procedures.


Introduction
Microelectromechanical systems (MEMS) is also called microfabricated devices, lab-on-chip, microsystems, micro-total analysis systems (micro TAS), which existed for more than 30 years, with several applications attaining commercial and/ or scientific success. Commercially, high-throughput, lowvolume-consumption technologies such as whole genome sequencing projects and drug discovery have created a need for these devices. Scientifically, the ability to design and control experiments at the micrometer scale has attracted the interest of biologists, who have started devising fundamental studies using this technology (Joel et al., 1999). MEMS techniques were originally developed in the microelectronics industry. Microelectronic process engineering is a discipline that developed due to the rapid growth of the integrated circuit (IC) industry. Traditionally, microelectromechanical systems (MEMS) have been used to produce functional devices on the micron scale, such as sensors, switches, filters, and gears, from silicon, the dominant material used throughout the IC industry. Microelectromechanical system (MEMS) techniques have enabled development of miniaturized diagnostic tools and high throughput screening assays for drug discovery and tissue engineering (Sant et al., 2011). Although in their embryonic, microfabrication technologies are being explored for drug delivery.
The review summarized detail account of how to prepare MEMS device, various strategies of it and application of MEMS in drug delivery and in analytical methods.

Strategies for fabricating patterned MEMS
There are four basic processes are used for the fabrication of MEMS. The first is photolithography or soft lithography, which transfers a pattern into a material. The second is thin film growth/deposition, in which thin films (usually on the order of micrometers in thickness) are grown or deposited onto a substrate. Etching, the third kind of process, creates features by selectively removing materials (either thin films or substrate) in defined patterns. The final kind of process is bonding, where two substrates (often structured and with thin films) are bonded together.
Photolithography process depicted in Fig. 1 is used to transfer a pattern envisioned by the designer into a material. A pattern, drawn with a computer assisted design (CAD) program (Fig. 1a), is transferred onto a mask (Fig. 1b). The mask is a glass plate that has on its surface a photo definable opaque material (usually chrome) in the desired pattern and is typically prepared by a mask vendor. If the features and tolerances in the pattern are relatively large (>20 lm), then one can use a simpler mask-making process (Duffy et al., 1998). After mask making, the pattern transfer begins when the substrate (Fig. 1c) is spin-coated with photoresist (Fig. 1d), a photosensitive organic polymer. The substrate and mask are brought into contact, and UV light is shown through the mask and onto the photoresist (Fig. 1e). Photoresist under the transparent portions of the mask will be exposed, causing it to become soluble in a developing solution. This is known as a positive photoresist (negative photoresist gives the inverse pattern). The wafer and mask are separated, and the exposed photoresist is removed in the developing solution (Fig. 1f). The photoresist can now be used as a protective mask to transfer the pattern into the underlying material via etching. When finished, the photoresist is removed.
Soft lithography is the collective name for a set of lithographic techniques Replica molding (REM), Microcontact printing (lCP), Micromolding in capillaries (MIMIC), Microtransfer molding (lTM), Solvent-assisted micromolding (SAMIM), and Nearfield conformal photolithography using an elastomeric phase shifting masks that has been developed as an alternative to photolithography and a replication technology for micro and nanofabrication (Xia et al., 1999). These techniques use a patterned elastomer (usually PDMS) as the mold, stamp, or mask to generate or transfer the pattern. Soft lithography offers immediate advantages over photolithography and other conventional microfabrication techniques for applications in which patterning of nonplanar substrates, unusual materials, or large area patterning is the major concerns. It is especially promising for microfabrication of relatively simple, single-layer structures for uses in cell culture, as sensors, as microanalytical systems, in MEMS, and in applied optics (Xia et al., 1999).
Microcontact printing (lCP) uses a soft polymeric stamp, usually made of PDMS, which has been formed by molding to a master made by conventional microfabrication. The stamp is ''inked'' with self-assembled monolayers (SAMs) of alkanethiols or alkylsilanes and placed on a gold-or silicon dioxide-coated surface, respectively. This transfers the molecules from the stamp to the substrate, where they form a self-assembled monolayer in the same pattern as the stamp. These patterned self-assembled monolayers can then be used as resists for etching or as passivation layers to prevent deposition. This method of pattern transfer is advantageous when working with non-cleanroom compatible materials or chemicals, or nonplanar substrates, although unresolved issues exist with multilevel pattern registration (Whitesides and Xia, 1998).

Substrate materials
Silicon is the most common material in microfabrication, owing to its role in the fabrication of integrated circuits and its excellent electrical properties, silicon also possesses outstanding mechanical properties, enabling the design of microelectromechanical structures (Petersen, 1982). For biological or medical microsystems, silicon may not be the material of choice (Joel et al., 1999). It is not optically transparent, preventing the use of transmission microscopy, and its cost can potentially be too large for disposable devices.
Glass is also used as substrate material in microfabrication although the range of micromachining processes for glass is less extensive than for silicon, glass provides some unique features, most notably optical transparency. Glass wafers are available in many different compositions and sizes. Two important examples are fused silica wafers and borosilicate wafers. Fused silica wafers are pure amorphous silicon dioxide (SiO 2 ). They can withstand high temperatures (T softening = 1580°C), are optically transparent down to short wavelengths, and have very low autofluorescence. Borosilicate wafers, of which the most common is Pyrex ª (Corning 7740), are much less expensive than fused silica (and can be less expensive than silicon). They can be easily bonded to silicon but cannot be exposed to the high temperatures needed for some thin-film depositions and have higher autofluorescence than fused silica (Joel et al., 1999).
Plastic is often the least expensive substrate material. The availability of mass production processes (e.g. injection molding, embossing) that can be extended to the microscale means that plastic devices can be extremely inexpensive to produce in volume. This allows for disposable devices, which minimizes issues of sterilization, clogging, and drift. For these reasons, a majority of commercial enterprises are using plastic microdevices, especially for disposable clinical applications. Most devices to date have been separation channels for capillary electrophoresis. In addition, the softness of plastics can mean poor dimensional tolerance and stability, and autofluorescence is often a problem (Joel et al., 1999).

Thin-film growth/deposition
Thin films are used for a variety of different purposes in microstructures masking materials, structural materials, sacrificial materials, and electrical devices, to name a few. They are formed by either chemical reaction driven processes or physical processes (Joel et al., 1999).
Chemical vapour deposition is a process that produces thin metal, ceramic, or compound films, through thermal oxidation in a gas chamber at an elevated temperature. Within the chamber the substrate interacts, at temperatures between 800 and 2000°C and pressures between millitorrs and torrs, with volatile precursors that react and decompose on the substrate a film of a metal (e.g. Al, Ta, Ti, Pt.), ceramic (e.g. Si 3 N 4 , B 2 O 3 , BN) or compound (Effenhauser et al., 1997). The gas mixture typically consists of reducing gas, like hydrogen (H 2 ), inert gases like nitrogen (N 2 ) or argon (Ar), and reactive gases such as metal halides and hydrocarbons. A typical chemical reaction sequence includes pyrolysis, reduction, oxidation, hydrolysis and co-reduction. For example, silicon nitride (Si 3 N 4 ) can be deposited by means of reaction of dichlorosilane gas (SiCl 2 H 2 ) with ammonia gas (NH 4 ) at temperatures between 700 and 800°C (Athanasios, 2011). The volatile byproducts can be blown away from the reaction chamber and neutralized before exposure to the environment. The chemical vapour deposition can also be plasma enhanced a method that functionalizes surfaces and is effective in depositing hydrophobic films on wafers.
Physical vapour deposition, such as sputtering or evaporation, is used to deposit thin films, layer by layer, onto substrates. This method employs mechanical or thermodynamic means for producing thin films and requires low-pressure vaporized environment to function (Athanasios, 2011).
Adsorption is a chemical deposition method that exploits hydrophilic head groups of self-assembled monolayers of, for example, hydrophobic trichlorosilanes (Cl 3 SiC n H 2n+1 ), or thiols (HSC n H 2n+1 ), which offer spontaneous binding on glass or metal surfaces (Schreiber, 2000).
Ion beam enhanced deposition influences the energy and charge states of the gas in the vapour phase and allows control over the energy state and crystallographic and stoichiometric form of the deposited films (Athanasios, 2011).
Deposition and patterning of biomolecules, most commonly proteins, are quite important in biological applications of microfabrication. Three predominant methods to accomplish this have been reported (Blawas and Reichert, 1998). The first method, protein adsorption, relies on physical adsorption of proteins in solution onto a substrate. The patterning is achieved either by dissolution of protein covered photoresist patterns or by constraining where the protein solution flows via microchannels (Blawas and Reichert, 1998;Delamarche et al., 1997;Folch and Toner, 1998). The co-cultures were made by patterning extracellular-matrix proteins in the former manner. The second method of biomolecular patterning is using photochemistry, where UV light shown through a patterned mask is used to activate or deactivate chemical species. The final method uses patterned self-assembled monolayers to selectively inhibit or allow protein attachment (Whitesides and Xia, 1998;Blawas and Reichert, 1998).

Etching
In microfabrication technology, patterning is the transfer of outlines of features (which define microchannels, microelectrodes, or other components) on the top of a substrate by means of ultraviolet illumination via a photomask. The photomask consists of a chromium layer where only part of it is transparent to the light. Photolithography is the method for producing structures by exposing photosensitive resists to ultraviolet light via successive photomasks and then removing the exposed areas of the photoresist in a development process. The photosensitive resist is a polymer, usually epoxy, which is sensitive to ultraviolet light. When the ultraviolet exposes specific areas, the photoresist polymerizes and resists to etchers. This replicates the patterns onto the photoresist film. The exposed areas of the resist film are being attacked by the developer and consequently removed. The unexposed areas remain resistant to the action of the etchant. The structure can be revealed after etching the exposed areas. Every wafer substrate may undergo many sequential etching cycles before completes (Athanasios, 2011).
Etching can be divided into wet (via liquid chemicals) or dry (via gas-phase chemistry) etching. Either method can lead to isotropic or anisotropic etching. Isotropic etching etches in all directions equally, leading to mask undercutting and a rounded etch profile (Fig. 2, left). Anisotropic etching is directional ( Fig. 2, middle, right) and is either chemically or physically (sputter etching) induced. In general, wet etching is more selective than dry etching, whereas anisotropic etches are more common with dry etching. Chemical etches are more selective than physical etches but amenable to fewer materials. The aim is to find a complementary set of materials and etchants, thus allowing selective pattern transfer (Joel et al., 1999).
Etching can be distinguished as two etching processes, wet etching and dry etching.
Wet chemical etching is widely used for producing microelectrodes and microfluidic channels on substrates. The wet etching requires acids, bases or mixtures to dissolve metals, silicon or glass, by immersing the substrate into the etching solution. For instance, gold can be etched using iodine solution. The etching can be isotropic or anisotropic. As an example, silicon AE1 0 0ae can be etched isotropically with HF-HNO 3 , which produces rectangular curved grooves by means of thermally grown SiO 2 mask. KOH can etch Si AE1 0 0ae or Si AE1 1 0ae anisotropically; the etchant should be selected carefully in order to have the etching rate of the masks considerably lower than that of the removable material. The final step in a wet etching process is to remove the resist by washing it away with an organic solvent (Athanasios, 2011).
Dry etching involves reactive ion etching with plasma, where the substrate is placed into a plasma chamber where a gas mixture is introduced and is ionized. The ionized gas mixture reacts with the surface of the substrate to be etched. As the ionized gas is highly energized, it removes the matter from the substrate. Xenon difluoride (XeF 2 ) is a dry vapour phase isotropic etcher for silicon. SF 6 in high-density plasma provides anisotropic high-aspect-ratio etching for silicon (Athanasios, 2011).

Bonding
In many processes, there will be a desire to bond two substrates (possibly with thin films) together to form a hermetic seal. A common example is the bonding of a glass capping wafer to a structured silicon wafer to form an optically accessible sealed system. Many technologies have been developed to bond different materials together, either with or without intermediary layers (Schmidt, 1998).
Anodic bonding can fuse silicon or glass plates. The two opposing plates must come in contact with each other, in heat, with a high voltage applied across a conductive layer (200 nm Figure 2 Overview of (left) isotropic and (right) anisotropic etching, anisotropic etching by (middle) dry etching or by (right) wet anisotropic etching. silicon nitride (Si 3 N 4 ) or 120 nm Ni/Cr) developed intermediately, that causes diffusion of ions, which eventually fuses the two plates electrostatically. Anodic bonding conditions vary between 200 and 1500 V, at 200-450°C (Athanasios, 2011).
Thermal bonding can fuse glass or polymer plates. Thermal bonding is performed on coated with methylsilses-quioxane, or polysiloxane, surfaces at temperatures 150-210°C. Annealing for some hours in low vacuum produces bonds that withstand hundreds of N/cm 2 . Rapid glass bonding requires hot pressure at 570°C for 10 min under 4.7 N/mm 2 (Athanasios, 2011).
Photopolymer adhesives can bond any type of rigid substrates. This method is more tolerant for uneven surfaces since it provides a compressible cushioning layer that seals the chip. The adhesive layer can be benzocyclobutene (C 8 H 8 ) or UVcurable resins of micrometer thickness. It is possible to heat and separate the plates apart and rebond (Athanasios, 2011).
Other bonding techniques abound. To join two metal layers together, one can use eutectic or thermocompression bonding (Schmidt, 1998). Substrates can be bonded with adhesives, whereas plastics can be bonded by heating them to above their glass transition temperature and then compressing them (Martynova et al., 1997). PDMS can be reversibly hermetically bonded to glass or to itself by simple contact (Effenhauser et al., 1997) and can be irreversibly bonded to itself by oxidizing two pieces and placing them together (Duffy et al., 1998).

Application of MEMS in drug delivery
Biomedical microelectromechanical systems (BioMEMS) based drug delivery devices have become commercially feasible over the past several years due to converging technologies and regulatory accommodation. MEMS technology has been applied to the successful development of a variety of health care related products. Although research on microfabricated devices for biomedical applications, particularly in diagnostics, has rapidly expanded in recent years, relatively few researchers have concentrated on therapeutic applications of microfabrication technology, such as drug delivery. Combination products constructed using MEMS technology offer revolutionary opportunities to address unmet medical needs related to dosing. These products have the potential to completely control drug release, meeting requirements for on-demand pulsatile or adjustable continuous administration for extended periods, programmable dosing, sequential dose delivery and diagnostic feedback dispensing. MEMS technologies are significantly developed in recent years, providing a multidisciplinary foundation for developing integrated therapeutic systems. If small-scale biosensor and drug reservoir units are combined and implanted, a wireless integrated system can regulate drug release, receive sensor feedback, and transmit updates. For example, an ''artificial pancreas'' implementation of an integrated therapeutic system would improve diabetes management. The tools of microfabrication technology, information science, and systems biology are being combined to design increasingly sophisticated drug delivery systems that promise to significantly improve medical care. Several review articles are available regarding trends in microfabricated systems for drug delivery, with a few examples outlined here Hilt and Peppas, 2005;LaVan et al., 2003;Staples et al., 2006;Tao and Desai, 2003).

Programable
Microfabrication technology has been used to fabricate programmable devices as a new class of controlled release systems for drug delivery. These devices are particularly intriguing due to their small size, potential for integration with microelectronics and their ability to store and release chemicals on demand (Santini et al., 2000). The first experimental demonstration of a microchip with potential application in drug delivery was described by Santini et al. The ultimate goal of this approach is to develop a microfabricated device devoid of moving parts, but with the ability to store and release multiple chemical substances (Santini et al., 2000(Santini et al., , 1999. Drugs stored within reservoirs were sealed either with active or passive coatings. For example, in the original work the reservoirs were sealed with a thin layer of gold and released upon application of an applied electric potential that dissolved the coating. Subsequent version also included fully degradable polymeric systems with plastic seals.

Particulate systems
As their scale decreases, microfabricated devices can be delivered by ingestion (<1 mm), intra-tissue injection (<200 lm), inhalation (<100 lm) or released into circulation (<10 lm) (LaVan et al., 2003). Microfabrication methods, because of their ability to control microarchitecture and feature size, have been used successfully to develop novel nano/microparticles for applications in drug delivery. Silicon particles have been used as multistage drug delivery systems (Tasciotti et al., 2008) and for intravenous delivery (Martin et al., 2005). Several methods, including soft lithography, particle replication in non-wetting templates (PRINT), hydrogel templating, imprint lithography, and in situ photo polymerization in microfluidic channels have been developed to prepare homogeneous polymeric particles. Microstructures with complex geometries can influence anisotropic interactions with biomolecules and cells (Sant et al., 2011).

Oral drug delivery by BioMEMS
BioMEMS is the emerging approach in the field of oral drug delivery systems. They have significant potential to overcome some of the barriers of oral drug delivery through fabrication of asymmetrical devices with precise control over size and shape. Apart from drug delivery devices, microfabrication approaches can also enhance the field of oral drug delivery by designing biomimetic in vitro GI tract model systems that can aid in better prediction of drug absorption in vivo. Thus MEMS technique is used in the development of oral drug delivery system and in vitro cell culture models that can be used to evaluate the drug delivery efficacy (Sant et al., 2011).
Microfabrication also offers great opportunities to enhance the oral delivery of pharmaceuticals by allowing for precise control over shape, size, and geometry of delivery devices. Microfabricated devices can also increase drug loading capacities and provide better control over drug release. One approach for inducing greater levels of absorption and stability at the intestinal epithelium is the use of a multi-layered patch system. Patches are designed with layers of thin, flexible membranes: an impermeable backing, a drug reservoir, a rate-controlling membrane, and an adhesive. When the patch is applied, the drug begins to flow through the skin into the bloodstream at a rate regulated by the membrane, preprogrammed to keep the drug at an effective level. These properties are ideal for oral dosage forms intended for delivery to the intestinal mucosa. Microfabricated patch systems designed for oral drug delivery are capable of three main functions: (i) bioadhesive properties for retention of the dosage form, (ii) release drug in a controlled fashion, and (iii) provide unidirectional release toward the intestinal epithelium (Tao and Desai, 2005a,b,c).

Micropatch
The size of orally delivered particles has a great impact on their transit through the GI tract. Larger particles can get trapped in the mucus layer protecting the epithelium, resulting a relatively short residence time. Certain types of smaller Micro-and Nano-scale particles are known to permeate the epithelium (Tao and Desai, 2005a,b,c), but the uptake is largely restricted to Peyer's patches, which take up a small fraction of the brush border and lead to lymphoid tissue. This pathway carries the risk of toxic accumulation and poor bio-distribution.
Microfabricated patch systems are alternative to standard particulate delivery systems, such as microspheres. They are designed small enough to travel in between intestinal villi, maximizing the large absorptive surface area of the intestinal folds, but wide enough to prevent cell uptake. In contrast to particulate systems, micropatches are designed flat and thin to maximize contact area with the intestinal lining. At the same time, this flat design minimizes the side areas exposed to the constant flow of liquids through the intestine. The devices can be microfabricated to incorporate single or multiple drug reservoirs which can be loaded with any number of drugs/biomolecules of interest. These reservoirs, unlike multidirectional release from a spherical delivery system, allow for unidirectional release of the drug. Furthermore, regions of the device can be surface modified in order to incorporate cell targeting mechanisms which localize the vehicle at a specific site of action. Modification of microspheres is performed uniformly over the entire surface area, which increases instability and may induce rolling when exposed to flow (Sant et al., 2011). However, selective surface modification on only the reservoir side allows micropatches to stably anchor in an orientation which permits the released drug to follow the shortest diffusional pathway toward the intestinal epithelium. Fabrication processes for creating oral micropatches have been developed based on standard MEMS fabrication techniques including photolithography, etching, and thin film deposition, as well as soft lithography (Tao and Desai, 2005a,b,c).
Standard materials such as porous silicon and silicon oxide have been successfully used for microfabrication based drug delivery systems. Although silicon and glass are the materials of choice for electronic and mechanical devices, it is not clear if these materials are necessarily appropriate for all applications in biology and medicine (Quake and Scherer, 2000). Polymers allow for shorter fabrication times and potential large scale fabrication of complex drug delivery vehicles. In one such demonstration, poly(methyl methacrylate) (PMMA) microdevices were fabricated using an off-wafer process. Microdevices were also fabricated from SU-8, a chemically amplified; epoxy based negative photoresist typically used for producing ultra-thick resist layers. The use of SU-8 as a device material eliminates the need of a secondary patterning material and the dry etch procedure. Instead, multi-level processing can be used to create features in multiple layers. Repeated, aligned photolithography was used to define the backing, reservoir, and supplementary feature layers (Ainslie et al., , 2005. Asymmetrical microparticles were also fabricated from biodegradable polymers poly(DL-lactide-co-glycolide) (PLGA) and gelatin using soft lithographic techniques. In this manner, several batches of asymmetrical microparticles may be generated from a single master. The height of the resulting devices is determined by the height of the features in the PDMS master and the concentration of polymer in solution. Lateral resolution is determined by the features of the PDMS master and the solvents used. Guan et al. were able to combine methods of dip-coating, microcontact hot-printing and soft lithography to produce microdevices containing single-reservoir and multiple-reservoir systems as well as sustained release microcapsule depots in PLGA ( Fig. 3A-C).
Micropatch systems offer some advantages in drug loading over conventional solid dosage forms. They can contain reservoirs which can be loaded by microinjection with Pico to Nano-liters of a polymeric solution. Water quickly evaporates from these reservoirs leaving behind the drug contained in a timed-release polymer plug. Intimate contact between the micropatch and intestinal epithelium would provide a short diffusion distance, potentially negating the need for excipients to aid in dissolution. Using a specific type of polymer reservoir would predetermine the time and rate of release of drug from the reservoir; for example, a hydrogel that swells in response to a specific pH, solvent or temperature or a polymer with a known dissolution rate. Different polymers with various dissolution rates could then be used in separate reservoirs to obtain controlled release of several compounds. By capitalizing on surface-liquid interactions, it is also possible to utilize discontinuous de-wetting as a method for bulk filling of reservoirs (Guan et al., 2007).
Micropatch reservoirs can also be filled using photolithography. For example, after microdevice development, a photosensitive hydrogel precursor solution, consisting of a crosslinker, photoinitiator, poly(ethylene glycol) dimethacrylate (PEGDMA) and drug, can be spun into the empty reservoirs. Using a mask aligner, only the hydrogel within the reservoir is exposed to UV light and subsequently developed in water and isopropanol. Using repeated application and crosslinking, microdevice reservoirs can be loaded with multi-layered hydrogels for sequential delivery of therapeutics (Ainslie et al., , 2005. In the case of soft lithographic methods for fabrication of particles, drugs may be added to the prepolymer solution and incorporated into the device during the transfer process (Tao and Desai, 2005a,b,c).

Bioadhesion
As an alternate way to overcome short gut residence times and poor mucosal contact, increasing interest has been placed on bioadhesive systems that can delay the transit and prolong their residence at a specific site of delivery, thus enhancing the drug absorption process Serra et al., 2009). These mucoadhesive devices can protect the drug during the absorption process in addition to protecting it on its route to the delivery site. This in turn increases the drug concentration gradient due to intense contact. The general concepts and mechanisms of mucoadhesion have been reviewed in detail (Serra et al., 2009). In general, bioadhesion can be achieved through chemical or physical approaches.
In chemical bioadhesion approach the use of adhesion promoters such as linear or tethered polymer chains to promote bioadhesion during oral drug delivery. Mucoadhesion depends largely on the structure of the synthetic polymers used in controlled release applications and has been reviewed by Serra et al. Another strategy utilizing immobilization of lectin as targeting molecules for enhanced adhesion and specificity to intestinal epithelial models has been previously described (Lehr, 2000;Naisbett and Woodley, 1990;Wood et al., 2006). Post-fabrication chemical modification may be performed to immobilize bioactive targeting molecules to the surface of microdevices (Ainslie et al., 2005).
In vitro studies were performed using the Caco-2 cell line to measure the cytoadhesive properties of lectin-conjugated microdevices. Tomato lectin modified microdevices and microspheres were also studied in order to compare the effect of a tailored microdevice shape versus a traditional spherical shape on adhesion stability. It was found that the percent of microdevices remained consistently bound (68% of total applied) over consecutive washes while microspheres significantly decreased to approximately 17% (Tao and Desai, 2005a,b,c). Although microspheres have a larger surface area, or more importantly a larger lectin modified surface area, than the micropatch systems, microspheres appeared to be less stable when subjected to consecutive removal. The stability may be in part associated with the small fraction of the surface area which is directly in contact and anchoring to the cell monolayer at any given time. This suggests that the larger modified contact area of the flat micropatch device may provide a more stable interface. Additionally, studies have shown that these micropatch systems promote stable adhesion in the presence of mucin (Tao and Desai, 2005a,b,c), as well as under shear flow conditions (Ainslie et al., 2005).
By using physical approach for bioadhesion microdevice bodies can be designed to contain precisely shaped microneedles and microposts. These features may allow for the particle to more firmly adhere to the mucosa, potentially increasing drug permeability (Sant et al., 2011).
Microneedle systems were originally developed as an approach to enhance the poor permeability of the skin by creating microscale conduits for transport across the stratum corneum for transdermal drug delivery (Henry et al., 1998). The microfabrication of microneedles those are long and robust enough to penetrate this layer of skin, but short enough to avoid stimulating nerves, has the potential to make transdermal delivery of drugs more effective (Henry et al., 1998;McAllister et al., 2000), microneedle platforms have been fabricated in silicon, and also transferred into biodegradable carboxymethylcellulose, amylopectin, poly(lactic acid), poly(glycolic acid), and PLGA (Lee et al., 2008;Park et al., 2005). Tapered, needle-like structures have also been scaled down into the submicron range to provide adhesion and drug delivery in wet environments for potential applications in surgical, wound, and internal bandage systems (Mahdavi et al., 2008). Microneedles, in combination with infusion methods such as pressurized reservoirs and electrically controlled systems, have also been utilized for drug delivery. (Martanto et al., 2006;Roxhed et al., 2008). Furthermore, by modifying needle dimension and design to incorporate multiple channels and ports, optimized micro hypodermic needles and microprobes have also been developed for cellular, local tissue, or systemic delivery (McAllister et al., 2000).
The microneedle/micropost design principles can also be applied to oral drug delivery to increase the retention time of the microfabricated devices in the GIT. Using microfabrication techniques, oral microdevices can be designed to contain precisely shaped microposts/microneedles. These features may penetrate the mucus layer leading to anchorage of the particles/microdevices. For example, Guan et al. used a similar approach to fabricate a bilayered system of a poly(EGMAco-EGDMA) and crosslinked chitosan microparticles with self-folding arms. It is expected that by penetrating into the mucus layer, the arms may anchor microparticles, providing increased resistance to surface erosion of the mucus layer (Guan et al., 2007). This mechanism may also provide a means to ''grab'' the intestinal villi, also potentially leading to a longer retention time of the device ( Fig. 3D and E). In addition, the presence of microposts on oral microdevices may shed mucosa to increase the uptake of compounds into the blood vessels of the submucosa. Combined with the current chemically driven targeting mechanism, these microposts may provide a mechanically driven controlled release feature (Tao and Desai, 2005a,b,c).
Another physical method to enhance bioadhesion is the use of a particularly promising class of gecko-inspired or Nano structure based adhesives. Under Nano-adhesive conditions, as the number of adhesive elements per surface area increases, the surface area to volume ratio increases and van der Waals  Guan et al., 2007).
adhesion is predicted to increase (Spolenak et al., 2005). The microvilli present on the surface of the mucosal epithelia dramatically increase its surface area. Therefore, by creating a nanostructured microdevice to target the microvilli coated intestinal epithelium, it may be possible to generate strong bioadhesive forces due to geometric features alone. A standard vapour ''liquid-solid'' method for synthesizing silicon nanowires on flat wafer surfaces has been used in order to achieve growth of size-specific nanowires on microdevice surfaces (Fischer et al., 2009). Nanowire-coated devices were found to adhere to Caco-2 cells at a frequency of five times greater than non-coated devices under static conditions, and when tested under flow conditions, a median survival shear (the shear at which 50% of the devices detach) of 9.15 dyn/cm 2 was reported. Additionally, devices both chemically (tomato lectin) and physically (nanowire) modified for bioadhesion adhered as well or slightly better than unmodified devices under static conditions. However, under flow conditions and in the presence of mucin, these dually modified devices were found to be disadvantageous in terms of adhesion, with a median survival shear of 3.60 dyn/cm 2 . As lectins bind to both cells and mucus, adding a mucin layer introduces competition between these elements for binding to the lectin-modified nanowires, which may explain reduced adhesion. Therefore, geometrybased adhesion may offer distinct advantages over mucoadhesive chemistry in terms of mucosal tissue adhesion.

Microfabricated in vitro models
Development of physiologically relevant three-dimensional (3D) in vitro models is another area where microfabrication can advance the field of oral drug delivery. The drug development process is a long and expensive process with only one out of ten drug candidates in clinical trial reaching final FDA approval stage. The number of new molecular entities that are approved by the FDA is also declining with 53 approved in 1996 and only 19 approved in 2010 (Mullard, 2011). The main reason for this low success rate is poor prediction of drug efficacy and toxicity in preclinical testing. The current drug testing paradigm is based on 2D cell monolayers and in vivo animal models before clinical trials in humans. Although 2D cell monolayer-based assays are routinely used for drug efficacy and toxicity testing, such systems often fail to recapitulate microenvironmental context and in vivo biological complexity. Such systems are static and do not mimic the exchange of metabolites between the tissues, physiological shear stress, fluid flow dynamics as experienced by cells in vivo (Park and Shuler, 2003). In vivo animal models allow testing drug distribution, efficacy and toxicity under physiological conditions; however, there are differences in animal and human physiology making extrapolation of animal data to human difficult. Animal studies are also expensive, time consuming and ethically controversial. Better in vitro model systems are necessary to accurately predict drug efficacy and toxicity. Microfabrication approaches have been proposed recently to develop physiologically relevant, in vitro 3D tissue models to reduce or replace animal studies. These include various strategies such as 'organ-on-a-chip' (Moraes et al., 2011), 'body-on-a-chip' (Esch et al., 2011), 'lung-on-a-chip' (Huh et al., 2010) and 'perfused multi-well liver tissue' (Domansky et al., 2010).
Conventional approaches to study drug absorption across the intestinal mucosa are classified into in vivo, in situ and in vitro models and have been reviewed elsewhere. The most accepted and widely used in vitro absorption model consists of Caco-2 cells seeded on a polycarbonate membrane in a transwell device (Peppas and Carr, 2009). When cultured as a monolayer, Caco-2 cells differentiate to form brush border microvilli on the upper side of the monolayer and contain both tight junctions and brush border associated enzymes. In this model, test compounds are added on the apical side of the Caco-2 cell monolayer, and compounds penetrating the cell are monitored at the basolateral side of the monolayer. Although used successfully to model oral drug absorption (Yazdanian et al., 1998), this model still has limitations. For example, low permeabilities have been observed in vitro compared to in vivo data for the drugs that are transported through paracellular transport route. Similarly, compounds with low solubility/dissolution may have less absorption than that predicted by the Caco-2 system since oral absorption may be limited by low solubility. In addition, Caco-2 monolayers are planar in geometry, and do not accurately represent the brush border topography. Also, the use of a static monoculture neglects the influence of mucus-secreting goblet cells, and peristalsis on drug absorption. Furthermore, these monolayers cannot be used to predict the bioavailability of compounds susceptible to hepatic first pass clearance.
Microfabricated devices can potentially better model the GI surface topography and can also be used to better control the mechanics of cell-cell and cell-substrate interactions. Current efforts in this field can be divided into three approaches: (i) engineered intestinal tissues; (ii) microfluidic-based approaches and (iii) microscale cell culture analogs (lCCA).

Engineered intestinal tissues
The small intestinal epithelium consists of an epithelial monolayer of enterocytes, goblet cells, and Peyer's patches resting on a basement membrane. The absorptive surface area is enhanced through the topographical arrangement of this monolayer into finger-like projections (villi) and well-like invaginations (crypts). In addition to enhancing surface area, this spatial arrangement also dictates cell behavior. Wang et al. used simple microfabrication approaches to create biomimetic crypt-like microarchitecture on polymer substrates (Wang et al., 2009). Caco-2 cells seeded on such substrates showed higher metabolic activity and lower alkaline phosphatase activity compared to the flat substrates signifying influence of topography on cell phenotype. In a follow-up study, authors patterned type I collagen membrane using soft lithography to study the synergistic effect of crypt-like topography and extracellular matrix (ECM) proteins (fibronectin and laminin) on Caco-2 adhesion, proliferation, differentiation and tight junction formation (Wang et al., 2010). It was found that crypt-like topography had short term effects on cell phenotype whereas substrate chemistry (ECMprotein coating) had more prominent and long term effects on intestinal epithelial cell behaviors. Insight from such studies can be useful in developing biomimetic in vitro intestinal models for drug absorption.
Gunawan et al. created immobilized physiological protein gradients (laminin and collagen type I) similar to those found in small intestinal crypts using microfluidic gradient generator.
Results revealed region-specific expression of p27 (prodifferentiation marker) and proliferating cell nuclear antigen (PCNA; proliferative cells), markers that are linked to the cell cycle progression, when intestinal epithelial cells were cultured on immobilized counter-gradients of laminin and collagen I. Such studies are necessary to understand the role of various ECM proteins on the intestinal epithelial renewal along the crypt-villus axis (Gunawan et al., 2005).
Another emerging approach to engineer biomimetic GI tract models is the use of hydrogels. Hydrogels have attracted great attention for 3D cell cultures since they mimic the ECM and can be easily modified to generate tailored microenvironments (Slaughter et al., 2009;Peppas et al., 2000). Additionally, hydrogels are amenable to various micromolding and soft lithographic techniques for drug delivery (Guan et al., 2007) as well as tissue engineering applications (Aubin et al., 2010).
Recently, Sung et al. developed a biomimetic GI tract model using laser ablation combined with sacrificial molding in microscale collagen hydrogels mimicking actual density and the size of human intestinal villi (Fig. 4) (Sung et al., 2011). Caco-2 cells seeded onto the structure covered the whole structure in three weeks resembling finger-like intestinal villi covered with epithelial cells. Thus, microfabrication approaches can be used to recapitulate the in vivo microenvironment to construct physiologically realistic in vitro models of intestinal villi that can improve the predictability of drug absorption studies.

Microfluidic based approaches
The field of microfluidics is gaining popularity in drug discovery, development and personalized biomolecular diagnostics due to their ability to provide fluid flow in the physiological range. Moreover, microfluidics offers spatial and temporal fluidic control in a biomimetic environment enabling long-term cell culture and differentiation (Shaw et al., 2011). Thus, microfluidics is becoming an integral part of the cell-based assays to predict oral drug absorption.
For realistic prediction of oral drug bioavailability, an in vitro model should incorporate all major physiological obstacles to the drugs entry into systemic circulation. These include the transport properties of the epithelium, liver metabolism, and the vascular transport that links them. Transwell co-culture models have been designed to include apical caco-2 monolayers along with hepatocytes in the basolateral compartment (Lau et al., 2004). However, these models use large liquid to cell ratios and are devoid of circulation of medium. To overcome these limitations, perfused co-culture system can be designed using microfluidic-based approaches.
To generate a biomimetic microenvironment for drug absorption studies, a perfused co-culture system was designed using microfluidics that enhanced cytochrome P450 (CYP) 1A1/2 activity (Choi et al., 2008). In another study, Mahler et al. co-cultured mucous secreting HT29-MTX goblet-like cells with Caco-2 cells to mimic intestinal cell populations and HepG2/C3A cell line as liver cell populations. The presence of HT-29 cells resulted in Caco-2 cell layer covered with mucus when cultured in physiologically realistic ratios (Mahler et al., 2009a,b).
Microfluidics has been used to design a bioreactor system with physiologically meaningful flow conditions to study various epithelial cell transport processes (Jang et al., 2011). Fabricated a bilayer microfluidic system with integrated Trans Epithelial Electrical Resistance (TEER) measurement electrodes to evaluate kidney epithelial cells under physiologically relevant fluid flow conditions. The apical and basolateral fluidic chambers were connected via a transparent microporous membrane. The top chamber contained microfluidic channels to perfuse the apical surface of the cells whereas the bottom chamber acted as a reservoir for transport across the cell layer and provided support for the membrane. TEER electrodes were integrated into the device to monitor cell growth and evaluate cell-cell tight junction integrity in real time. Such bioreactors can be easily integrated with perfused co-culture systems that closely mimic GI epithelial barriers along with first pass metabolism described above (Ferrell et al., 2010). Kimura et al. have developed a microfluidic device embedded with a stirrer-based micropump to create on-chip perfusion, and an optical fiber connection for on-line fluorescence detection for drug screening and toxicity testing (Fig. 5) (Kimura et al., 2008). In another study, a microfluidic device containingmicro-hole arrays was fabricated to reduce the Caco-2 culture time (Yeon and Park, 2009). In vivo permeabilities in the human and rat intestine are highly correlated with those measured by the microfluidic device. However, the limitation of the device is that tight cell junctions are not formed since single cells are trapped in each micro-hole for a short period. Consequently, this system cannot be applicable for drugs transported through tight junctions; however, it can still be used for drugs that are transported passively or actively with the aid of transport proteins (Yeon and Park, 2009).

Microscale cell culture analogs (lCCA)
A drug's absorption, distribution, metabolism, and excretion (ADME) is a result of interaction between various cells, tissues and organs that are interconnected by vasculature. Physiologically based pharmacokinetic (PBPK) mathematical models that describe an organism as a set of interconnected tissue or organ compartments based on vasculature have been designed to calculate the time-dependent distribution of a drug in various tissues (Brown et al., 1997). An important advancement in the field of drug screening is to integrate multiple miniaturized organ model systems into a single device to recapitulate the potential interaction between different organs in determining the drug's ADME. The concept of microscale cell culture analog (lCCA) is a physical representation of PBPK model where different cell types are cultured in small chambers interconnected by fluidic channels (Khamsi, 2005). Such systems offer versatile in vitro models to study drug's biotransformation, and interaction between different tissues in determining drug's response (both efficacy and toxicity). Fabrication and applications of macroscopic and microscopic CCAs have been discussed in detail in recent reviews (Park and Shuler, 2003;Esch et al., 2011).
Most of the previous work with lCCAs was done to mimic intravenous administration of a compound as the drug was added directly into the circulating culture medium . More recently, GI tract lCCAs have been developed that include digestion, a mucus layer, and physiologically realistic cell populations to determine oral bioavailability of drugs ( Fig. 6) (Mahler et al., 2009a,b). The GI tract lCCA was used together with a systemic lCCA to demonstrate absorption, distribution, metabolism and toxicity of a widely used analgesic and antipyretic drug, acetaminophen. The authors showed that acetaminophen was absorbed and metabolized by GI cells, then circulated to the liver cell compartment. Liver cells were capable of metabolizing the drug into reactive metabolite resulting in a dose dependent toxicity to the liver cells (Mahler et al., 2009a,b).
Combination of microfabrication with microfluidics has allowed precise control over microscale structures. In addition, the ability to pattern physiologically relevant cell types, as well as to manipulate geometry of the substrate in 3D and flow patterns/hydrodynamic shear stress in the physiological range upon the cells takes us one step closer to creating wholebody-on-a-chip for efficient screening of drug efficacy and toxicity. Reduction in the amount of sample, spatiotemporal fluidic control, easy fabrication and reduced cost makes it more attractive for high throughput drug screening and can further reduce the cost of drug development if integrated earlier in the drug development process. Potentially, such systems can be used as an alternative to animal models in drug screening.

Microneedles intended for transdermal drug delivery
One alternative to oral delivery and intravenous injection is the administration of drugs across the skin. This approach seeks to avoid any degradation of the molecules in the gastrointestinal tract and first-pass effects of the liver associated with oral drug delivery as well as the pain of intravenous injection (Polla et al., 2000;Hadgraft and Guy, 1989;Henry et al., 1998). It also offers the possibility to continuously control the delivery rate over extended periods of time (Henry et al., 1998). However, conventional trans-dermal drug delivery is severely hindered by the outer 10-20 mm of skin, a barrier of dead tissue called the stratum corneum (McAllister et al., 2000). The development of microneedles for transdermal drug delivery came about as an approach to enhance the poor permeability of the skin by creating microscale conduits for transport across the stratum corneum (Henry et al., 1998). The development of microneedles that are long and robust enough to penetrate this layer of skin, but short enough to avoid stimulating nerves has the potential to make transdermal delivery of drugs more effective (McAllister et al., 2000).
By adapting microfabrication technology, three dimensional arrays of sharp-tipped microneedles can be made for transdermal drug delivery (McAllister et al., 2000;Polla et al., 2000;Henry et al., 1998). To fabricate microneedles, a deep reactive ion etching process is commonly used. In this process, a chromium masking material is deposited onto silicon wafers and patterned into dots that have a diameter approximately equal to that of the base of the desired needles. When placed in the reactive ion etcher, the wafers are exposed Figure 5 Representation graphic of the integrated microfluidic device, Caco-2 cells are cultured only on the semipermeable membrane in the AP side culture chamber. The stir-bar is driven by motor-controlled permanent magnets beneath the device. Figure adapted from Kimura et al., 2008).
to carefully controlled plasma of fluorine and oxygen, which causes a deep vertical, etch and slight lateral under etching. The regions on the wafer that are protected by chromium remain and eventually form the microneedles. Etching is allowed to proceed until the masks are undercut and fall off, leaving behind an array of silicon spikes (Henry et al., 1998). The aspect ratio of the microneedles can be adjusted by simply modifying the ratio of flow rates of SF 6 and O 2 . Hollow silicon needles can also be fabricated using deep reactive ion etching in an inductively coupled reactive ion etcher. The deep etch creates arrays of holes through the silicon wafer (the needle lumen) and the microneedles are formed by reactive ion etching around these holes (McAllister et al., 2000).
Arrays of solid silicon microneedles have been fabricated with individual needles measuring 150 lm in lengths, 80 lm in diameter at the base, with a radius of curvature less than 1 lm (Henry et al., 1998). Hollow needles have also been microfabricated with similar dimensions, but containing hollow bores anywhere from 5 to 70 mm in diameter, depending on (Davies, 1997). To test their durability, the solid needles were inserted into skin with gentle pushing, an approximate force of 10 N. All but a few percent of the microneedles remained intact. Even in these few needles, only the top 5-10 lm was damaged (McAllister et al., 2000;Henry et al., 1998). Additionally the array of microneedles could also be removed without additional damage and could also be reinserted into skin multiple times.
Quantification of transdermal transport of various molecules with and without inserted microneedle arrays was used to assess any increase in skin permeability leaving (Fig. 7). Insertion of the microneedles increased permeability only 1000-fold because the microneedles or the silicon plate may have blocked access to the microscopic holes (Henry et al., 1998). When the microneedles were removed after 10 s, permeability increased by 10,000 fold (Henry et al., 1998). Removal after 1 h increased skin permeability by 25,000-fold (Henry et al., 1998). Elevated permeability after microneedle insertion was found to remain at approximately the same level for as long as 5 h (Henry et al., 1998). Hollow microneedleswere also capable of insertion into the skin without any extensive damage to the microneedles or skin (McAllister et al., 2000). In addition, the improved design of these needles increased skin permeability further still (McAllister et al., 2000).
More recently, the original microneedle design has been further refined to provide better control over drug delivery. Silicon micro-hypodermic needles have been fabricated in combination with heat-controlled bubble pumps (McAllister et al., 2000). Hollow metal microneedles have been fabricated by defining molds in epoxy and filling them by electrodepositing metal (McAllister et al., 2000). Similarly, polysilicon microneedles have been fabricated with reusable molds (McAllister et al., 2000). Polysilicon microneedles are likely to be most cost effective and have the potential to produce single use disposable platforms (McAllister et al., 2000). These types of needles, combined with a pressurized reservoir to generate a drug delivery pump, have already been incorporated into a wear able drug infusion system to deliver insulin. Furthermore, by modifying needle dimension and design to incorporate multiple channels and ports, optimized micro-hypodermic needles and micro-probes can be developed for cellular, local tissue, or systemic delivery (McAllister et al., 2000).

Implanted microchip designed for localized drug delivery
Microfabrication technology has also created a new class of controlled release systems for drug delivery based on programmable devices. These devices are particularly intriguing due to their small size, potential for integration with microelectronics and their ability to store and release chemicals on demand (Santini et al., 2000). With the recent advancements in biosensors and micro-machining, implanted responsive drug release systems are becoming more plausible.
The first experimental demonstration of a microchip with potential application in drug delivery was described in Nature (Santini et al., 1999). The ultimate goal was to develop a microfabricated device devoid of moving parts, but with the ability to store and release multiple chemical substances. The device was fabricated by the sequential processing of a silicon wafer using microelectronic processing techniques including UV photolithography, chemical vapor deposition, electron beam evaporation and reactive ion etching (Santini et al., 1999). The experimental prototype was a 17 mm · 17 mm · 310 lm square silicon device containing an array of 34 square pyramidal reservoirs etched completely through the wafer (Santini et al., 2000(Santini et al., , 1999.

Irreversible metallic valves
The 25 nL reservoirs were sealed at one end by a thin membrane of gold to serve as an anode in an electrochemical reaction. One other electrode was placed on the device to serve as a cathode. The reservoirs were filled through the open end with the chemical to be released by either micro syringe pumps or inkjet printing in conjunction with a computer-controlled alignment apparatus. The open ends of the reservoirs were then covered with a thin adhesive plastic and sealed with waterproof epoxy (Santini et al., 2000(Santini et al., , 1999. When submerged in an electrolyte, ions form a soluble complex with the anode material in its ionic form. An applied electric potential oxidizes the anode membrane, forming a soluble complex with the electrolyte ions (Santini et al., 2000). The complex dissolves in the electrolyte, the membrane disappears, and the chemical is released and allowed to diffuse form the reservoir. The time at which release occurs from each individual reservoir is determined by the time at which the reservoir's anode membrane is removed. Each reservoir, or a group of reservoirs, may be independently addressed by demultiplexing (Santini et al., 2000). This allows each anode to have its own conducting path and electric potential can be applied to any given combination of reservoirs at any given time. However, the rate of release from the reservoir is a function of the dissolution rate of the materials in the reservoir and the diffusion rate of these materials out of the reservoir. Therefore, the rate of release from an individual reservoir can be controlled by proper selection of the materials (e.g. pure drugs, or drugs with polymers) placed inside the reservoir. Using a material that quickly dissolves once the reservoir is opened can be used to achieve pulsatile release whereas a material that dissolves slowly after the reservoir is opened can be used to achieve sustained release (Santini et al., 2000(Santini et al., , 1999.

Reversible polymeric valves
An alternative to the use of irreversible metallic valves is a microchip using reversible polymeric valves. The use of 'artificial muscle' valves in conjunction with silicon micromachined drug release structures can render a microchip responsive to a patient's therapeutic requirements and deliver certain amount of a drug in response to a biological stimulus (Low et al., 2000). 'Artificial muscle' refers to a chemomechanical an actuator consisting of a blend of a hydrogel and an electronically conducting redox polymer. The redox polymer is sensitive to pH, applied potential, and the chemical potential of its microenvironment whereas the hydrogel provides a cross-linked network of hydrophilic homo/copolymers that exhibit dramatic swelling and shrinking upon changes in pH, solvent, temperature, electric field, or ambient light conditions (Madou and Florkey, 2000). By electro-polymerizing these polymers onto electrodes, reservoirs can be opened or closed, and the drug compound released or retained, via the swelling and shrinking processes of the polymer system in response to electrochemical actuation (Fig. 8) (Low et al., 2000).

Nanoporous immunoisolating biocapsules
Diabetes mellitus (DM) represents a serious medical problem. In the US alone, it is the third leading cause of death. While the majority of patients have type-2 diabetes, about 10% of all patients diagnosed with DM are insulin-dependent (type 1). In both cases, disease is caused by decreased circulating concentrations of insulin and decreased response of peripheral tissue to insulin (insulin resistance).
The disease manifests itself as hyperglycaemia. Insulin remains the mainstay of virtually all type-1 DM and many type-2 DM patients and in most cases is administered subcutaneously. However, the kinetics of insulin administered by this route does not mimic the normal rapid rise and decline of insulin secretion in response to ingested nutrients.
Efforts to address the short-comings of current subcutaneous administration of insulin, including the use of complex multi-dose regimens, have led to the development of other dosage forms and routes of administration such as 'needleless' injectors, constant infusion pumps, and inhaled insulin. These newer approaches still suffer from the same general issue plaguing current subcutaneous administration.

Allotransplantation
A potentially useful approach, which has proven effective in only a handful of cases, is the allotransplantation of islets or whole pancreases from a suitable human donor into a diabetic recipient. Researchers in Canada recently reported successful transplantation of islet cells in type 1 DM patients (Davies, 1997). Although the potential complications of immunosuppressive therapy were reduced by avoiding the use of glucocorticoids, each transplant required two harvestings of islet cells from organ donors. Moreover recipients are still required to take immune suppressing drugs for the rest of their lives. These immunosuppressive drugs are toxic and have potential adverse side effects, including cancer. For this reason, an islet or pancreas allotransplant is normally carried out only in conjunction with a kidney transplant, for which immunosuppression is required in any case.
Because of the toxicity of immune suppressing drugs, and the shortage of organ donors, islet and pancreas allotransplantation appears to hold limited promise as a cure for diabetes. A method then is required to sequester the islets from the body's immune system which is able to recognize and reject these xenogeneic cell grafts. For the past 20 years, investigators have focused on a range of microencapsulation methods most commonly involving sodium alginate and another polycationic substance such as polylysine. These materials have been used in an attempt to create a semipermeable membrane capable of blocking immune molecules such as IgG, cytokines, and cell-secreted antigens from reaching the encapsulated xenogeneic islet cells while allowing glucose and insulin to freely diffuse through the barrier (Lanza and Cooper, 1999). However, this approach has proven generally unsuccessful due to mechanical rupture of the membrane, biochemical instability, incompatibility with islet cell heterogeneity, and broad pore size distributions (Lanza and Cooper, 1999;Lacy et al., 1991;Lanza et al., 1996). When the barrier between the xenogeneic cells and the external bioenvironment is compromised, these foreign cells are subject to various endogenous cells and antibodies as well as complement and a host of cytokines such as tumor necrosis factor, all of which can inflict cell damage. As a result, the use of polymeric microcapsules for allotransplantation has been unsuccessful clinically in the absence of immunosuppression (Lanza and Cooper, 1999;Lacy et al., 1991;Lanza et al., 1996).

Biocapsule design
Microfabrication techniques have been applied to create a biocapsule for effective immunoisolation of transplanted islet cells for treatment of diabetes (Desai et al., 1998). The fabrication of nanochannels in the membrane structure consists of two steps: (1) surface micromachining nanochannels in a thin film on the top of a silicon wafer, and (2) releasing the membrane by etching away the bulk of the silicon wafer underneath the membrane. These nanopore membranes (Fig. 9A) are designed to allow the permeability of glucose, insulin, and other metabolically active products, while at the same time, preventing the passage of cytotoxic cells, macrophages, antibodies, and complement. The membranes are bonded to a capsule that houses the pancreatic islet cells. Because the difference in the size of insulin, which must be able to pass freely through the pores and the size of IgG immunoglobulins, which must be excluded, is only a matter of a few nanometers, the highly uniform pore size distribution provided by micromachine membranes is essential for effective immunoisolation and therapeutic effect.
Control of pore sizes in the tens of nanometers has recently been suggested as probably the most realistic way to achieve immuneisolation (Brissova et al., 1998;Wang et al., 1997). The use of unconventional biomaterials such as silicon and silicon dioxide provides a means to encapsulate pancreatic islet cells in devices that are thermally, chemically, and mechanically stable and retrievable. It is also expected that improved dynamic response of islets can be obtained due to the limited membranes thickness (Fig. 9B) compared with the thickness of conventional polymeric membranes prepared from alginate and polylysine (100-200 lm) (Brissova et al., 1998). It is crucial that rapid secretion kinetics, particularly in the first phase of insulin release, be maintained over time to provide physiologic feedback control of blood glucose concentrations.
Another key feature of an implanted biocapsule system is the role of neovascularization. The membranes proposed for testing have outer openings of 2 by 2 mm while the inner diffusion channels have a pore size of between 10 and 30 nm. Studies have shown that neovascularization at the membrane-tissue interface occurs in membranes having pore sizes large enough to allow complete penetration by host cells (0.8-8 mm) (Brissova et al., 1998). Thus, it is expected that neovascularization can occur at the large openings while not penetrating into the nanometer pores. This phenomenon has two key advantages: (1) the ability to rapidly deliver insulin into the blood stream through new blood vessel growth while (2) limiting pore clogging or fouling.

Applications of biomems devices in analytical techniques
Clinical medicine greatly benefits from MEMS as lab-on-chip technology as it suites for drug tests, tests for observing pandemics, glucose monitoring, diabetic control, diagnosis of diseases and numerous other tests. Lab-on-chip devices enhance numerous biomedical tests that entail mixing, analysis and separation of samples, which usually consist of cell suspensions, nucleic acids, proteins, etc. Analytical, electrical, or optical detection methods are possible. The electrical detection methods depend exclusively on the polar properties of the molecules of the liquid samples. For example, carbon dioxide levels, oxygen levels, or pH values can be measured electrochemically. On the contrary, most analytical or optical techniques require labelling, which entails chemo-luminescence, fluorescence, or radioactive markers. Most separation methods of lab-on-chip systems are miniaturized approaches of larger ones. There are diverse screening methods, which offer high sample throughput, whereas other methods offer reliability and precision. The separation of biomolecules, cells, or nanoparticles can be managed by transportation methods, which are based on the charge and size of the substances. These transportation methods can be the following ones: a. Hydrodynamic manipulation, which employs hydrodynamic pressure (Takagi et al., 2005;Yamada and Seki, 2005;Jaggi et al., 2007;Choi et al., 2007) b. Electrical manipulation which transports electrolytes, suspensions of particles or cells, or entire aqueous volumes like droplets, via the use of electrokinetic mechanisms such as electrophoresis (Zhang and Manz, 2003), dielectrophoresis (Jones et al., 2002(Jones et al., , 2003 and electrowetting (Pollack et al., 2000(Pollack et al., , 2002Washizu, 1998;Moon et al., 2002) c. Magnetophoresis that employs magnetic fields in conjunction with magnetic nanoparticles suspended in the samples (Pamme and Wilhelm, 2006) Optical manipulation that employs laser-light pressure which moves nanoparticles or tiny droplets (MacDonald et al., 2003;Pamme, 2007); alternatively, light actuation can  Brissova et al., 1998).
alter locally the degree of hydrophobicity of a surface and consequently direct aqueous volumes via hydrophilic routes.
Electrical and mechanical micropumps are largely employed in microfluidic manipulation (Laser and Santiago, 2004). Electric micropumps utilize electrokinetics (Gascoyne and Vykoukal, 2002), piezoelectric (Van Lintel et al., 1988;Nisar et al., 2008) or magnetohydrodynamics (Zhong et al., 2002). While mechanical micropumps utilize hydrodynamic pressure (Xu et al., 2001), thermal expansion (Yokoyama et al., 2004), osmotic pressure (Salimi-Moosavi et al., 1997), or other transducer or induced forces. Electrocapillary, an important electrokinetic pumping mechanism, boosted the development of lab-on-chip devices (Harrison et al., 1992;Li and Harrison, 1997;Manz et al., 1991). This achievement caused the realization of miniaturized analytical instruments, namely on-chip chromatographic systems (Terry et al., 1979). Other pumping mechanisms employ thermal gradients, or magnetophoresis. Fluidic lab-on-chip devices can facilitate cell screening (Oh et al., 2006;Choi et al., 2007;Tan et al., 2006). This works on the basis of forcing cells into specific streamlines, separates them by size, and drives them into specific outlets (Yamada et al., 2004). This finds applications in blood cell separation, where erythrocytes flow right in the centre of a microcapillary and the leukocytes, due to their differing mass, pursue streamlines along the capillary wall (Jaggi et al., 2007;Shevkoplyas et al., 2005). Besides hydrodynamic separation, microvalves can sort suspensions of cells or nanoparticles, usually in conjunction with electrokinetic actuation methods (Gomez-Sjoberg et al., 2007;Nevill et al., 2007;Melin and Quake, 2007). Electrically actuated microvalves are made of electroactive elastomers or piezoelectrics, which can be embedded easily in fluidic chips. Piezoelectric micropumps are activated when voltage applies on a piezoelectric material causing expansion. The expansion can be several micrometres, but in the microscale this causes significant pressure, which pushes the fluid adequately. Magnetically actuated microvalves also exist, made of magnetically inductive materials. The magnetic actuation field can be produced on-chip by means of current patterns, integrated on the chip. Lab-on-chip devices are efficient in mixing samples in a controllable and precise manner due to the use of tiny liquid volumes. Examples of mixers include distributive mixers, static mixers, T-junction mixers and vortex mixers. Active mixers consume power in order to increase the interfacial area between the mixing fluids. Examples of active mixers include electrokinetic mixers, chaotic advection mixers and magnetically driven mixers. Passive mixing can be achieved in microchannels with structures called twists that cause turbulence. Some mixers are more efficient at faster flow rates, whereas others work more efficiently at slower flow rates (Squires and Quake, 2005).
Temperature control is important in microfluidic devices. Temperature gradients can be generated inside microfluidic channels by means of miniaturized heating elements, such as heat exchangers, heaters or coolers (Talary et al., 1998). The efficiency of micro-heat exchangers depends on their ability to regulate the temperature of the transported fluid, in conjunction with reservoirs that buffer rapid changes of temperature. The micro-heaters are assembled in the form of coils. Due to the inherently small dimensions of the microfluidic devices, the thermal coupling between a micro-heater and a reservoir is very efficient. Coolers can be made out of heatsinks that induct the heat to the environment. The combination of small fluidic volumes and the precision of the heatsinks allow cooling to ambient temperature with ease. Repeatable temperature cycles are essential in the process for nucleic acid amplification and find application in the polymerase chain reaction (PCR) arrays-on-chips (Talary et al., 1998;Kopp et al., 1999;Wildinga et al., 1998;Shoffner et al., 1996;Cheng et al., 1996). PCR replicates small amounts of nucleic acids into large amounts. PCR involves a three-step thermal cycle that promotes the replication: heating (90-95°C), cooling (50°C), and slight warming (60-70°C). PCR chips include various temperature zones whose benefit is that the cycle time no longer relies on the time required to heat or cool the sample. The benefit of performing PCR in microfluidic chips is due to the very small liquid volumes used, for which temperature homogeneity and diffusion efficiency are higher in comparison with ordinary laboratory vessels. However, thermal cycles may induce undesirable effects, especially regarding evaporation and bubble formation. The droplet-based biochips eliminate most of these defects during PCR processes due to the isolation of the droplets by a second phase oleic fluid (Wang et al., 2005;Beer et al., 2007). Fully automated PCR biochips that contain mixers, heaters, and microarray sensors are capable of performing sequential functions like hybridization, preconcentration, purification, lysis, and electrochemical assessment of complex biological solutions . In bio-analysis, biosensors, integrated in microfluidic devices, offer possibilities for electrochemical or optical analysis of samples. Enzyme-based electrochemical biosensors can measure oxygen consumption or pH production by means of enzymatic reactions. The enzymes catalyse the reactions of the analytes and the sensor measures the products (Urban, 2009). In the field of bioanalytical separation, the capillary chromatographic chips adopt fluorescent polystyrene nanoparticles or labelled macromolecules to enhance cell or protein separation by image recognition (Kutter, 2000;Chmela and Tijssen, 2002;Blom et al., 2002). In analogy, the ion exchange chromatographic chips separate electrokinetically analytes such as polar molecules, proteins, nucleotides and amino acids according to their ionic charge. Genomic research requires analysis, which can be utilized by means of specialized fluidic chips called microarrays, which are arrangements of open planar arrays that each contains thousands of specific oligonucleotides, covalently bonded on glass or silicon substrate (Niemeyer and Blohm, 1999;Blohm and Guiseppi-Elie, 2001;Pirrung, 2002;Jung, 2002). These oligonucleotides function as probes, which hybridize the DNA fragments that contact the microarray, by forming hydrogen bonds between the DNA fragments and the complementary nucleotide base pairs of each probe. A microarray can accomplish many genotype tests in parallel. The detection method in microarray chips is based on microscopy analysis via the use of fluorophore or chemoluminescence labels that determine abundance of nucleic acid sequences. The lab-on-chip microarrays can be distinguished between cDNA-microarrays and oligonucleotidemicroarrays. The latter can perform genotyping and resequencing (Saliterman, 2005). The microarray chips can be employed in nucleic acid analysis, which includes DNA extraction and purification, amplification, hybridization, sequencing, gene expression analysis, genotyping and DNA separation. Moreover, microarrays can recombine nucleotide fragments by employing endonucleases to cleave and rebind the fragments with DNA ligase enzymes. Fluorescence microscopy is mainly employed in microarrays because of its challenging limits of detection, due to its sensitivity, which approaches concentrations of Pico-moles. Detection sensitivity of Picomoles is appropriate in most applications that require high sensitivity, such as DNA sequencing and many immunoassays (Fan and Ricco, 2006). There are great potential applications of microarray chips in biotechnology as well as of DNA-inspired lab-on chips in nanotechnology (Wagenknecht, 2008). The oxidative type of DNA-mediated charge transport has significant results in mutagenesis, apoptosis, or cancer studies. On the other hand, excess electron transport plays a growing role in the development of electrochemical DNA chips for the detection of DNA base mutations (Drummond et al., 2003). Knowledge about excess electron transport in DNA has potentials for nanotechnological applications, such as supramolecular electronics. In this field, the research focuses on the role of DNA as supramolecule that features important properties for nanowires (Porath et al., 2004).
Lab-on-chip devices support cell detection by means of capillary electrophoresis, electroporation, cytometry or electrical impedance. The cells can be cultivated within aqueous microreactors inside the chips (Nevill et al., 2007;Melin and Quake, 2007;Martin et al., 2003). With lab-on chip devices one can measure accurately chemical stimuli on cells, as cellular signals are weak and not easily detected with conventional analytical methods. Cytometers allows cell counting and analysis of cellular parameters like size distribution or growth rate. Cytometry commonly employs electrical impedance methods (Sun et al., 2007), but also employs fluorescence microscopy; however, fluorescence microscopy requires cell labelling with dyes or fluorescent nanoparticles and external optical apparatuses, such as optical fibres and illuminators (Fu et al., 1999). Other electrokinetic methods rely on contactless conductivity detection of organic ions or electrolytes where typically two on-chips embedded tubular electrodes, away from each other, measure changes of the ohmic resistance of the solution that flows inside the microfluidic channel Laugere et al., 2003;Pumera et al., 2002;Lee et al., 2006) Capillary electrophoresis performs electroseparation of the ionic compounds, based on the size tocharge ratio of the ions, which are transported electrokinetically in the carrier liquid. The electrocapillary forces drag the ions into the capillary channel while the fluidic sample approaches the inlet of the microchannel (Gorbatsova et al., 2009;Abad-Villar and Kuban, 2005). Capillary electrophoresis is capable of separating particulate substances like cells, amino acids, proteins, peptides, mitochondria, or bacteria (Cabrera and Yager, 2001). In microfluidic channels the surface-to-volume increases significantly due to reduced dimensions, and electrophoresis force becomes more efficient due to the shortening of the distance. In capillary electrophoresis devices the electric fields range to some hundreds V/cm with pulse durations of milliseconds. Capillary electrophoresis separation is fast and robust and high throughput can be obtained. Electroporation is an electrical method that accesses the interior of cells. Applying short and intense electric pulses increases the conductivity and transiently permeabilizes the lipid membrane of a cell and introduces substances into the cytoplasm. Electroporation is used for inserting drugs or genes into cells and is highly efficient when performed in vitro in lab-on-chip devices . Electrokinetic manipulation of suspended particles by means of electric fields is widely used in labon-chip devices for in vitro separation of particles or cells (Li et al., 2007). Dielectrophoresis manipulates and separates cells, beads or nanoparticles by means of inhomogeneous electric fields that can be produced with electrodes of specific shapes (Jones et al., 2003;Jones, 2002). The cells can be collected or directed away from the electrodes under the influence of the dielectrophoresis force, which is directed towards the field gradient, and originates from the permittivity difference between the carrier fluid and cells (Huang and Pethig, 1991). The possibility of using dielectrophoresis for in vitro separation of blood cells, based on their conductivity characteristics, can automate partition of white blood cells from erythrocytes. Several human cancer cells have been successfully studied and dielectrophoretically sorted Wang et al., 1997;Cen et al., 2004). Dielectrophoresis can further measure dielectric differences of cells or particles by means of electro-rotational spectra (Jones, 1995). Cancerous cells are different from healthy erythrocytes and lymphocytes. Their dielectric diversification can be exploited for removing metastatic cancer cells from healthy blood cells. Other electrokinetic means with growing applications in lab-on-chips technology is electro-osmotic flow, a coulomb effect that results in transportation of ions of an electric double layer (Kirby, 2010;Cahill et al., 2004), and its reverse effect, streaming potential, that is induced voltage on an electrode due to the flowing motion of counter-ions of an electric double layer (Chun et al., 1996).
Today, magnetic separation devices are available and magnetic cell separation offers diagnostic and therapeutic values (Estes et al., 2009). Magnetophoretic sorters can capture cells that are bonded with magnetic beads, or nanoparticle aggregates, and force them flow into the carrier fluid with increased selectivity (Pamme and Wilhelm, 2006;Gijs, 2004). Moreover, magnetic nanoparticles can be used to destruct targeted cells by penetration, or to release carried drug into the cell's cytoplasm (Ogiue-Ikeda et al., 2003). The magnetic field can be generated by current patterns on the chip. An important advantage of magnetic actuators in comparison with the electric ones is due to the magnitude of the magnetic driving force that is significantly larger in comparison to electrokinetic forces. This highly improves the control of the liquid flow. The possibility of incorporating magnetic nanoparticles into cells as inert tracers for cell monitoring, allows measuring cytoskeleton associated cell functions (Valberg and Butler, 1987;Wang et al., 1993). Within cytoplasm the magnetic nanoparticles may equilibrate, but intracellular transports disorient the magnetic nanoparticles resulting in the decay of magnetization which can be recorded by magneto-impedimetric sensor, embedded in the chip. Magnetic actuators can transport aqueous droplets that enclose magnetic nanoparticles (Egatz-go´mez et al., 2006). Under the influence of the magnetic field the magnetic nanoparticles can drag the droplet and execute all the usual microfluidic operations such as displacement, merging, mixing and separation (Lehmann et al., 2006).

Conclusion
Microfabrication techniques have been adapted as BioMEMS to create physiologically relevant materials and devices that mimic the scale cells experience in vivo and have found wide biomedical applications, including drug delivery, tissue engineering and analytical techniques. BioMEMS devices must be made of materials that are biocompatible, chemically inert, reliable and useable. The race to find effective diagnostic and the therapeutic tools is under way, as BioMEMS are getting closer to the clinical application of intelligent drug delivery devices and significantly enhanced the analytical devices ever before. Though a danger exist in proclaiming this technology a panacea for a large portion of biomedical problems such assertions are doomed to failure; entrenched, mature conventional technology is meant to supplement this established technologies.