Lab-on-a-Chip

A Lab-on-a-Chip (LOC) device, also known as a micro-total-analytical system (microTAS) or microfluidics device, is a device that can integrate miniaturized laboratory functions (such as separation and analysis of components of a mixture) on a single microprocessor chip using extremely small fluid volumes on the order of nanoliters to picoliters. From a technology categorization perspective, LOCs can be viewed as a subset of microelectromechanical systems (MEMS) and combine miniaturized or novel sensing systems, fluid flow control concepts from microfluidics, and the suite of fabrication techniques (such as material deposition, material removal, surface patterning, and electrical property modification) used by the semiconductor industry.

Currently, the main commercial applications of LOCs are in the medical and biotechnological fields, where it is anticipated that developments so far are the heralds of a technological revolution. In the same way that miniaturization changed computers from machines of limited capabilities occupying large rooms to small and easily portable yet powerful technology of today, over a period of a few decades, medical, biotechnological, and chemical analysis is expected to move from room-sized laboratories to microchipbased devices housed in hand-held or small portable readout consoles. Figure 28 shows an example of an LOC device that was tested on the International Space Station in 2007.

Figure 28: LOC device tested on the International Space Station in 2007 At the heart of LOC devices are “chips”, ranging in size from a fingernail to a credit card, fabricated using processes adapted from the printed circuit industry such as lithography, chemical etching, and laser machining. Figure 29 illustrates an impression of the size of the chip. Figure 30 provides a functional diagram of LOCs.

Figure 29. A comparison of the size of LOCs

Figure 30. Functional diagram of LOCs In a manner similar to the production of printed circuit boards using techniques such as embossing and molding, microstructures (such as channels for liquid flow and pits for mixing and reactions) are made on the chip by depositing layers of material on top of one another on a surface, then patterning and selectively removing material to form a feature. A flat top surface or lid is attached to enclose the channels or mixing pits, and reagents can be driven around the system by pneumatic, electromotive, or capillary systems. The LOC was first conceived by Michael Widner at Ciba-Geigy (now Novartis) in the 1980s, described conceptually in 1990 (Manz et al. 1990) with a groundbreaking work being published in 1992 (Harrison et al. 1992). Further development occurred as a new area of discovery—microfluidics—was developed in the 1990s. Microfluidics is an interdisciplinary field dealing with the behavior and control of extremely small volumes of fluids and the design of systems that use these small volumes. Though most commonly encountered in ink-jet printers, the vast majority of microfluidics applications have been in biotechnology research, and some experts even regard it as a branch of biotechnology.

In some ways, Microfluidics parallels nanotechnology in that the behavior of fluids at the microscale can differ substantially from the behavior at the macroscale; phenomena such as surface tension, heat conduction, and fluidic resistance start to become important, and issues such as evaporation, absence of turbulent flow, and the threat posed by presence of air bubbles are critical to system design. Initially, much of the impetus for continued development of LOCs came from the Human Genome Project, a 13-year project coordinated by DOE and the National Institutes of Health (NIH) that began in 1990 and was completed in 2003. Currently, much of the impetus for the continued development of LOCs comes from the desire for point-of-care medical diagnostics, whether in the doctor’s office, on a spacecraft, or other remote location. Additionally, development research is driven by the continued need for miniaturization, both to reduce the costs and the environmental impacts of research (green analytical chemistry). The LOC concept, already significant, is still considered to be in its infancy. Development research continues in many areas.

In the area of fabrication materials, LOCs constructed using soft lithography techniques, rather than silicon microchip fabrication processes, are being investigated. Soft lithography is an alternative to silicon-based micromachining that uses replica molding of nontraditional elastomeric materials to fabricate stamps and microfluidic channels. In an extension to the soft lithography approach, multilayer soft lithography, with which devices consisting of multiple layers may be fabricated from soft materials, is being used to build active microfluidic systems containing on/off valves, switching valves, and pumps entirely out of elastomer. The softness of these materials allows the device areas to be reduced by more than two orders of magnitude compared with silicon-based devices. The other advantages of soft lithography (such as rapid prototyping, ease of fabrication, and biocompatibility) are retained (Unger et al. 2000). Environmental LOCs are also being investigated. An environmental LOC project is being funded by EPA with objectives to create a novel, nanomaterial-based submersible microfluidic device, exploiting unique properties of metal nanoparticles and carbon nanotubes for rapidly, continuously, and economically monitoring different classes of priority pollutants.

The project also seeks to understand the relationship between the physical and chemical properties of these nanomaterials and their observed behavior. The challenge addressed is to help transform the LOC concept to an effective environmental monitoring system, and involves the examination of nanoparticle and nanotube materials for the separation and detection processes, respectively (Wang 2007). In addition, the NIH is supporting the development of a point detection disposable LOC with built-in mercury precursor electrodes for heavy metal detection (Ahn 2006). Within these development efforts, it is also recognized that for novel and innovative technologies, even those that have an established market presence, close communication between developers and future users is essential. For example, in the United Kingdom, research collaboration between five leading universities in the healthcare technology assessment arena and a group of industrial partners—Multidisciplinary Assessment of Technology Centre for Healthcare (MATCH) —is conducting a survey of LOC point-ofcare device manufacturers.

Point-of-care in this context is defined as “analytical testing performed outside the central pathology laboratory using a device or devices that can be easily transported to the vicinity of the patient” (MATCH 2006). The aim is to assess the value of LOC for the diagnosis of cardiac-related problems using case studies, and to develop methods to shorten the time and decrease the costs of LOC development. 3.2.1 Summary of Environmental Potential The LOC has great potential for addressing environmental needs. The technology platform is mature and well-established, and as other nano-enabled sensing technologies are developed, integration into the LOC should be facile.

The twin features of rapid sample throughput and field portability should make the LOC a valuable tool in filed operations, particularly in circumstances such as the EPA Triad approach, where realtime monitoring is required to guide the progress of remedial work. 3.2.2 References Ahn, C.H. 2006. A Point Detection Disposable Lab-on-a-Chip With Built-in Mercury Precursor Electrodes For Heavy Metal Detection. Website. Accessed December 2007. www.biomems.uc.edu/sponsors/_index.html. Harrison, D.J., A. Manz, Z. Fan, H. Lüdi, H. M. Widmer. 1992. Capillary Electrophoresis and Sample Injection Systems Integrated on a Planar Glass Chip. Analytical Chemistry. 64: 1926–1932. Manz, A., N. Graber, H.M. Widmer. 1990. Miniaturized total chemical analysis systems: A novel concept for chemical sensing. Sensors and Actuators B: Chemical. 1(1-6): 244248.

Microcantilever Sensors

Microcantilever sensors are a technology which may develop into sensing systems for radionuclides, or is at least an example showing the potential and emergence of a new generation of highly sophisticated but flexible sensors. From a mechanical engineering perspective, a cantilever is simply a beam supported at one end and capable of defined bending and vibrational behavior. A microcantilever is simply a very small cantilever, the properties of which can be understood from basic engineering principles. The microcantilever was first developed in 1986 (Binnig et al. 1986) for use in Atomic Force Microscopy (AFM), the premier tool for nanoscale imaging and measuring.

In AFM, an extremely sharp microscale tip, (with a tip radius of a few nanometers) connected to the end of a microcantilever (up to 10 nm thick, about 500 nm wide and about 2,500 nm long, and fabricated from Si or Si3N4), is positioned extremely close to the surface of a sample and the sample, is then moved beneath the tip. Interactions between the surface of the sample and the end of the tip arising from atomic forces (such as Van der Waals force, electrostatic force, magnetic force, or capillary force) attract or repel the tip (depending on mode of operation) and bend the microcantilever. Reflecting a laser beam off the cantilever and monitoring the beam’s deflection with photodiode arrays measures the amount of bending. A graphical representation of the AFM sensing components is provided in Figure 31, and a Scanning Electron Micrograph of a microcantilever biosensor used for DNA detection is shown in Figure 32.

Figure 31. Principle of operation of the atomic force microscope

Figure 32. Scanning electron micrograph of fabricated microcantilever biosensor used for DNA detection The microcantilever is the simplest MEMS device. Early work soon demonstrated the versatility of the device in AFMs, with the microcantilever able to perform in air, liquid, or vacuum and across a range of temperatures. It also demonstrated the extreme sensitivity of the AFM to environmental effects and impurities, and the need to control these in making accurate measurements. However, studies on overcoming these issues also showed that the sensitivity of AFM to these environmental factors could be turned around, allowing the AFM to be a sensor for these same factors. For example, a microcantilever fabricated from silicon and coated with an aluminum surface for reflection of the laser beam can act as a “bimetallic strip” and respond to temperature changes. If a thermal event takes place on the cantilever surface, the silicon and aluminum expand to different extents and the cantilever bends, allowing the cantilever to act as a calorimeter of near-ultimate sensitivity.

Similarly, if the microcantilever is given an absorbent coating that can attract water and is allowed to vibrate in dry air, it will have a natural frequency of vibration; changes in humidity change the mass of the microcantilever, and thus change the frequency of vibration, allowing it to act as a humidity sensor. From this, it is a short step to the concept of coating the microcantilever with a chemical functionality that binds selectively with a target analyte. In the absence of the analyte, the microcantilever vibrates with one frequency, while in the presence of the analyte, binding with the microcantilever coating occurs and vibration takes place at a frequency directly related to the degree of analyte binding—hence concentration. A related sensing mode is that the surface of the microcantilever can be coated with a layer of material that contains the chemical functionality able to bind selectively with a target analyte. If this layer expands or contracts as the analyte is bound, then bending of the microcantilever occurs in a manner analogous to that of the bimetallic strip used for thermal sensing. The degree of bending is directly related to analyte concentration.

This behavior was the basis of a microcantilever radionuclide sensor development project at ORNL. As described in Section 2.3 on SAMMS, if a head group at the end of a long organic chain molecule can interact strongly with a substrate, then a closely packed molecular monolayer of the chain molecules aligned with the chains pointing outwards from the substrate surface can form spontaneously when the two are brought into contact. The tail end of the organic chain can be functionalized prior to, or after, self-assembly using chemical functionalities selective for a targeted species. Thiol (S-H) head groups and a gold substrate form an excellent pairing for this type of behavior, and the self assembly of alkanethiols has been observed to produce surface stress in the gold substrate (Berger et al. 1997). Silicon and closely-related materials have been the main materials of construction for microcantilevers. Silicon has been recognized as an outstanding mechanical material for over a quarter of a century (Petersen 1982). However, silicon is not the only fabrication material under investigation. Chemical sensing with micromolded plastic cantilevers and production issues have been explored (McFarland and Colton 2005). In addition to microcantilevers based on silicon, plastics; and related materials, nanostructures (such as single-walled carbon nanotubes (SWCNT)) have been explored for gas-sensing applications (Hsu 2007).

The approach uses the fact that SWCNTs are capable of interacting with the gaseous species—either directly through surface adsorbtion, or indirectly by using a polymer analyte coated on its surface—and the higher surface-to-bulk ratio available with a nanostructure leads to higher sensitivity and shorter response time. As with other microcantilevers, the effect of bound or adsorbed species is manifested either as a change in resonant frequency (which can be detected by using a Wheatstone bridge circuit), or as an increased surface stress (which can be detected by measuring the change in the capacitance value through comparison with a specific reference capacitor). Hsu’s work involved successful simulation, fabrication, and manipulation of the SWCNT; development and simulation of a capacitive sensing circuit layout; and consideration of packaging and integration issues, including use of a “nanoglue” developed at Rensselaer Polytechnic Institute, based on the processing of a self-assembled molecular monolayer and capability of bonding completely dissimilar materials.

Among the drivers for further development was the widespread need for portable, realtime, in-situ chemical, physical, and radiological sensors in a variety of applications including the characterization and monitoring of mixed waste, ground water, contaminated soil, and process streams. Microcantilever-based sensors were recognized as a potential solution for this need. They also provide excellent sensitivity for important metal ions in solution such as Hg2+, CrO42-, Sr2+, and TcO4-. The ability of the microcantilever to detect cesium (Thundat et al. 1999, Ji et al. 2000a) (though irreversibility problems were observed) and chromate (Ji et al. 2000b) was demonstrated; concentrations below the parts per billion level were obtained with exceptional selectivity even in the presence of other interferences. Fundamental microcantilever research at ORNL showed that adsorption-induced changes in the spring constant of a cantilever, leading to errors in the calculation of adsorbed mass from shifts in resonance frequency (Thundat 2002). Simultaneous measurement of resonance frequency and adsorption-induced bending was shown to allow the change in spring constant to be determined.

A silicon microcantilever with gold coating on one side was found to respond selectively and sensitively to Hg(II) ions in solution, and while modification of the Si surface with a silane reagent did not change the response to Hg, modification of the gold surface with octanethiol greatly retarded the rate of deflection, indicating the Hg(II) is reacting with the gold surface. The surface charge on the goldsolution interface is postulated to reduce Hg(II) to a surface amalgam. Modification of the gold surface with a monolayer 1,6-hexanedithiol makes the surface sensitive and selective for (CH3)Hg+ adsorption-induced deflection. Na+, K+, Pb2+, Zn2+, Cd2+, Ca2+, and Ni2+ in solution do not interfere with the response of the microcantilever to Hg. Gold coated cantilevers with chemically modified surfaces respond sensitively to Ca2+ ions at a concentration of 10-9 M. The sorption of a monolayer of 2-(4-mercaptophenoxy)-N, Ndiethyl-acetamide, as well as the agent bis (11-mercaptoundecyl) phosphate were shown to detect Ca2+ ions, although the former was more selective. A self-assembled monolayer of L-cysteine on a cantilever coated with gold on one side was shown to be effective for the detection of a concentration of 10-10 M Cu2+.

Both the Ca2+ and the Cu2+ were relatively free from interference by each other and Na+, K+, Pb2+, Zn2+, Cd2+, and Ni2 in solution (Thundat 2002). Subsequent work showed that electrochemically-active metal ions (Cu, Cr, Hg and Pb) could be detected by the novel approach of using a cantilever as a working electrode since electrodeposition of electro-active metal ions on cantilever surface results in cantilever bending. Together with the observation that that the cantilever bending is extremely sensitive to electrochemical current in the solution, this has led to the development of a technique where the cantilever serves as a reference/counter electrode for electrochemical reactions occurring on another working electrode (Thundat et al. 2006), with work continuing towards the development of field-deployable, miniature sensors with extremely high sensitivity, exceptional selectivity, and the ability to be integrated into a wireless communication system that will allow real-time data to be provided on concentration and speciation of multiple contaminants and their variation with time. Work has also continued on microcantilevers functionalized with metal-binding moieties. Gold-coated sides of silicon nitride microcantilevers functionalized with the metalbinding protein AgNt84-6 have been demonstrated to be sensors for the detection of heavy metal ions, such as Hg2+ and Zn2+ (Cherian et al. 2003).

On exposure to HgCl2 and ZnCl2 solutions, the microcantilevers underwent bending corresponding to an expanding gold side, while exposure to MnCl2 solution did not result in a similar bending, indicating a weak or lacking interaction of Mn2+ ions with the AgNt84-6 protein. The microcantilever bending data were consistent with data from electrophoresis that showed protein interaction with Zn2+ ions but not with Mn2+ ions, demonstrating that microcantilever bending can be used to discriminate between metal ions that bind and do not bind to AgNt84-6 protein in real time. 3.3.1 Summary of Environmental Potential Microcantilever sensors are one of the few nano-sensor technologies that have already been investigated with the detection of radioactive species in mind. The technology is very flexible; all that is needed for detection of a species is a coating with a chemical functionality capable of binding the target species. The technology is well-established, reliable, and sensitive—its origin in the AFM makes it a gateway technology for the nanotechnology age—and is easily integrated into the LOC platform. The versatility of the technique is further expanded by the fact that many materials, including innovative nanomaterials such as carbon nanotubes, may be usable as the cantilever.

3.3.2 References

Berger, R., E. Delamarche, H.P. Lang, Ch. Gerber, J.K. Gimzewski, E. Meyer, H.-J. Güntherodt. 1997. Surface stress in the self-assembly of alkanethiols on gold. Science. 276: 2021-2024. Binnig, G., C.F. Quate, C. Gerber. 1986. Physics Review Letters. 56: 930–933. Cherian, S., R. K. Gupta, B. C. Mullin and T. Thundat. 2003. Detection of heavy metal ions using protein-functionalized microcantilever sensors. Biosensors and Bioelectronics. 19(15) 411-416. Hsu. J.C. 2007. Fabrication of Single Walled Carbon Nanotube (SW-CNT) Cantilevers for Chemical Sensing. Thesis for Master of Science in Electrical Engineering (etd11082007-103811), Louisiana State University, May 2007. Ji, H.-F., E. Finot, R. Dabestani, T. Thundat, G. M. Brown, P. F. Britt. 2000a. A Novel Self-Assembled Monolayer (SAM) Coated Microcantilever for Low Level Cesium Detection. Chemical Communications. 457-458. Ji, H-F., T. G. Thundat, R. Dabestani, G. M. Brown, P. F. Britt, P. V. Bonnesen. 2000b. Ultrasensitive Detection of CrO42- Using a Microcantilever Sensor. Analytical Chemistry. 73: 1572-1576. McFarland, A.W. and J.S. Colton. 2005. Chemical sensing with micromolded plastic microcantilevers. Journal of Microelectromechanical Systems. 14: 1375-85. Petersen, K. E. 1982. Silicon as a mechanical material. Proceedings of IEEE. 70: 420– 457.

Thundat, T., E. Finot, H-F. Ji, R. Dabestani, P. F. Britt, P. V. Bonnesen, G. M. Brown, R. J. Warmack. 1999. Highly Selective Microcantilever Sensor for Cesium Ion Detection. Proceedings of Electrochemical Society. 99(123) 314-319. Thundat, T.G. 2002. Microsensors for In situ Chemical, Physical, and Radiological Characterization of Mixed Waste. EMSP-73808-2002. U.S. DOE Environmental Management Science Program Report, Washington DC. Thundat, T.G., Z. Hu, G.M. Brown, B. Gu. 2006. Microcantilever Sensors for In situ Subsurface Characterization. 2006 ERSD Annual Report. Oak Ridge National Laboratory, Tennessee.