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Electrophoresis 2014, 35, 236–244
Engin Karabudak
Review
Mesoscale Chemical Systems Group (MCS), MESA+ Institute for Nanotechnology, University of Twente, The Netherlands
Micromachined silicon attenuated total reflectance infrared spectroscopy: An emerging detection method in micro/nanofluidics
Received May 27, 2013 Revised September 30, 2013 Accepted October 1, 2013
Microfluidics is an emerging field with various applications. In situ analysis of liquids inside microfluidic channels is an important research subject for any of these applications. On the other hand, attenuated total reflectance infrared spectroscopy (ATR-IR) is a strong surface detection method. Recently, there have been various attempts to use ATRIR for detection of liquids inside microfluidic channels. Micromachined silicon ATR-IR (Si-ATR-IR) is an emerging analytical tool for micro/nanofluidics. This paper reviews Si-ATR-IR micro/nanofluidics detection devices and discusses the application fields, capabilities, advantages, and disadvantages of the method. Keywords: Attenuated total reflection / Infrared spectroscopy / Microfluidics / Nanofluidics / Online detection DOI 10.1002/elps.201300248
1 Introduction Microfluidics is a rapidly growing research field, which has various applications in disciplines such as biology [1–5], chemistry [6], life sciences [1, 4, 7, 8], and engineering [9, 10]. In a microfluidic chip, liquid flows inside micron-size channels. Liquids in these channels cannot be analyzed by classical analytical techniques, such as SEM [11], X-ray photoelectron spectroscopy [12], transmission electron microscopy [11], classical UV/Vis spectroscopy [13], or classical infrared (IR) spectroscopy [14]. For this reason, offline detection systems dominate the field. The development of integrated detection systems, however, which can conduct in situ analysis of the channels’ contents, remains an important research topic. Various integrated microfluidic detection methods are reported in the literature [15–20]. Among them, mainly thanks to its simplicity, the fluorescence-based detection [17] technique is the most common method. Surface-enhanced Raman scattering [19], surface plasmon resonance tech-
Correspondence: Dr. Engin Karabudak, Mesoscale Chemical Systems Group (MCS), MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands E-mail: [email protected] Fax: +31-53-4894683
Abbreviations: ATR-IR, attenuated total reflectance infrared spectroscopy; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; EDL, electrical double layer; FET, field effect transistor; IR, infrared; IRE, internal reflection element; LO, longitudinal optical phonon; MIR, multiple internal reflection; Si-ATR-IR, micromachined silicon ATR-IR; TO, transverse optical phonon C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
nique [18], electrochemical detection methods [20], and nuclear magnetic resonance [16] have all been integrated into microfluidic chips, which have advantages as well as disadvantages. This review focuses on micromachined silicon attenuated total reflectance IR spectroscopy (Si-ATR-IR) micro/nanofluidics detection devices [21–25], an emerging method with specific capabilities. In order to explain these capabilities, the following themes are elaborated: general principles of ATR-IR, polished internal reflection element (polished-IRE) coupled ATR-IR micro/nanofluidics, SiATR-IR coupled microfluidics, and recent advances regarding micromachined silicon IREs (Si-IREs) without microchannels.
2 General principles of ATR-IR ATR-IR is a surface characterization technique that was first reported in 1959 [26]. ATR-IR is also known as multiple internal reflection (MIR) FTIR spectroscopy (MIR-FT-IR) [21], multiple internal reflection FTIR spectroscopy (MIR-FTIRS) [23–25], or MIR-IR absorption spectroscopy [27–30], all of which refer to the same technique. To avoid confusion, the term ATR-IR will be used throughout the paper. Before the principles of ATR-IR can be explained, several technical terms such as critical angle, total reflectance, IRE, evanescent wave, and penetration depth must be clearly defined. Light travels at different speeds inside different
Colour Online: See the article online to view Scheme 1 and Figs. 1, 2, and 5–7 in colour.
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Figure 1. Schematics of total reflection, (A) ␣ is angle of incidence, c is the critical angle, dp is penetration depth, E is the evanescent wave, z is the depth; (B) general view of ATR set-up.
materials and the refractive index (n) of a material can be defined as: c (1) n= , v where c is the speed of the light in vacuum and v is the speed of the light in the material. If there is a flat interface between two different materials and light is sent from a material with a high refractive index (n1 ) to a material with low refractive index (n2 ), we can define a critical angle (c ) as: c = sin−1
n2 , n1
(2)
where n2 is the refractive index of the high refractive index material and n1 is the refractive index of the material. If the angle of incidence is bigger than the critical angle, all of the light reflects (total reflection) from the interface. At the point of reflection, the electric field penetrates the interface, whereby it is known as the evanescent wave, to probe the surface just above the IRE as shown in Fig. 1. The decay of the electric field intensity of the evanescent wave can be described as: E = E0e
− dz
p
,
(3)
where E is the evanescent wave strength, E0 is the time averaged electric field intensity at the interface, z is the depth, and d p is the penetration depth. Penetration depth (d p ) is given by the following equation: dp =
n1
2 sin2 −
2 ,
(4)
n2 n1
where is the wavelength of the light and is the angle of incidence. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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IRE is a critical component in an ATR-IR set-up, as the sample is placed on top of IRE and the optical set-up couples IR light to the edge of IRE (see Fig. 1). These systems make sure that the angle of incidence is always bigger than the critical angle, which guarantees that all the light intensity is reflected from the interface. The reflected light forms an evanescent wave at the interface, and this evanescent wave interacts with the sample. This interaction allows spectroscopic information on the sample to be collected. In short, the coupled light travels inside IRE by total reflectance and reaches the detector. IRE must be manufactured from high refractive index material and should be transparent for the measurement of wavelengths. Various IRE materials are used for ATR-IR setups such as germanium, silicon, ZnSe, KRS-5, diamond, and ZnS. Each of these IRE materials has its own properties, such as working spectral range, cut-off wavelength, price, refractive index, hardness, and chemical stability. For detailed discussion on IREs, I direct the reader to the review article by Vigano et al. [31]. Another important issue to be discussed is the production method of IREs. In fact, IRE material needs to be mechanically processable to form a specific shape. The shape is critical for determining the angle of incidence, which is a parameter for determining the penetration depth and evanescent wave. All of the commercial IREs are produced by mechanical polishing in different shapes (polished IRE). Polishing has to be done carefully so that an optical smoothness can be achieved on the reflection side. This requires extra polishing of every single IRE and limits the mass production of commercial IREs. For this reason, commercial IREs are not cost-effective. In addition, extra attention has to be paid while cleaning the IREs, since any roughness at the surface may interfere with the measurements. These are the major disadvantages when conventional IREs are used to collect IR spectra. We will consider how Si-IRE solves this problem in the following two sections. First, for microfluidic applications Si is one of the most attractive IRE materials because silicon processing is well developed in microfabrication [32]. Si-IRE has one disadvantage, however, its working spectral range is unfortunately limited. In fact, Si has a cut-off below 1500 cm−1 because of phonon absorptions [22, 33, 34]. This is a strong drawback, because the region below 1500 cm−1 , the so-called fingerprint region, is the most important aspect of mid-IR spectroscopy. In the fingerprint region, every peak can be a marker of a specific molecule. Therefore, Si-IRE is a common IRE in neither the literature [31,35–40] nor in the market. The following two sections explain how Si-IRE can overcome this disadvantage. Second, in microfluidic applications, the sample does not consist of one single homogeneous material, but of channel walls, channels, and liquids. All these materials have a different refractive index. As shown in the penetration depth formula, the penetration depth also depends on the refractive index. Thus, the penetration depth can be different in a channel wall or a liquid. As discussed in the section of polished IRE-coupled microfluidics, some sources make an average www.electrophoresis-journal.com
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Figure 2. Schematics of ATR-IR imaging set-up. Reproduced from [36] with the permission of the Royal Society of Chemistry.
of refractive indexes and call it an effective refractive index. In addition, not only refractive index of materials, but also periodicity of the channels can be an issue that may affect sensitivity of ATR-IR. If periodicity of any structure on top of IRE is in the range of wavelength of IR light, this can result in a scattering effect. This is also explained in the next section in detail.
3 Polished IRE-coupled microfluidics Companies that produce an ATR-IR set-up usually provide flow cells. These cells consist of a liquid compartment and polished IRE that can be assembled in reverse. In fact, these systems cannot be defined as microfluidic, because their sizes are far above the micron size. On the other hand, researchers have developed a simple method of building microfluidic devices on top of commercial/polished IREs. Usually PDMS devices are integrated with polished/commercial IREs [36, 40]. The precise bonding, leakage problems and limitations of the PDMS material itself can be problematic, however. Moreover, the PDMS material needs to be reassembled and the polished IRE has to be cleaned after each use. Kazarian and colleagues [35–37,39,40] showed that ATRIR can be used for imaging purposes for microfluidics with focal plane array detector. As shown in Fig. 2, commercial/polished ZnSe with a 45o angle is usually used for these purposes and PDMS materials with microchannels are integrated on top of IRE. With this set-up, it is possible to make IR images of the channels with a focal plane array detector. This is a very promising approach. I will not expand on this approach now, because there are already some excellent reviews [37, 40] available. To the best of my knowledge, Si-IREs are not used in this approach, and I believe their usage in the future will contribute to the field of ATR-IR imaging.
4 Si-ATR-IR coupled micro/nanofluidics As mentioned in the previous section, Si IRE has the signal disadvantage of having a cut-off wavelength of 1500 cm−1 . In 1997, Weldon et al. [41] showed that there is a way to overcome this disadvantage. They fabricated homemade polished Si-IRE measuring 1 cm in length from float zone Si wafer and C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. (A) Si-ATR-IR coupled microfluidic chip; (B) background spectra of Si-ATR-IR. Reprinted (adapted) with permission from Herzig-Marx et al. [21]. Copyright 2004, American Chemical Society.
observed that the cut-off wavenumber shifted to 900 cm−1 . It seems that the lower IRE length reduced the phonon absorptions, and the system was able to perform ATR-IR below 1500 cm−1 . The system was originally not designed to work with microfluidics. In fact, Weldon et al. coupled synchrotron IR light source to the IRE and studied oxidation of Si experimentally by using s- and p-polarized light. Theoretical density functional calculations also supported the results. In short, this was the first study to show shorter-length Si IRE can overcome the limited working spectral range of Si-IRE. Another disadvantage of conventional IREs is the requirement for extra polishing of the sidewalls, which limits mass production and therefore has a negative impact on the price. The first Si-ATR-IR microfluidic system in the literature was reported in 2004 [21]. Anisotropic KOH-etching of Si wafer, a guaranteed method to produce optically smooth surfaces with Si wafer [42, 43], was applied instead of polishing Si wafer. This study proved that 16 Si-IREs can be produced from 100-mm Si wafer immediately. Anodic bonding was used to bond the glass wafer with micromachined channels on top of these Si-IREs. Finally, the top surface and connections were produced with PDMS material, as shown in Fig. 3. The whole was put into a commercial FTIR spectrometer without fabrication of any special optical set-up. Fabricated IREs had a length of 1 cm in order to probe the fingerprint region. Thanks to the system, a wavenumber as low as 800 cm−1 could be probed. The study proved for the first time that Si-ATR-IR could probe the chemical reactions inside the channels of microfluidic chips. www.electrophoresis-journal.com
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Figure 4. Si-ATR-IR liquid FET set-up with specific optical set-up, flow connections, nanochannels, and liquid FET voltage connections. Reproduced from Oh et al. [24] with the permission of the Royal Society of Chemistry.
Si has three combination phonon absorption peaks [44, 45] at 1448 cm−1 (3TO), 1378 cm−1 (3TO + LO), and 1302 cm−1 (2TO +LO). TO and LO refer to transverse and longitudinal optical phonons. Optical phonons are the result of out-of-plane movements of atoms in a crystal. These vibrations are quantized in Si and can be seen at specific wave numbers. The mentioned peaks are the result of the combination of some of the TO and LO. In addition to these phonon peaks, below 1200 cm−1 there are various oxide peaks [41] and phonon peaks [44]. These phonon and oxide peaks usually absorb all the IR photons in the commercial systems, because commercial Si-IREs are thicker and longer. These peaks reduce the intensity reaching the detector, but they do not absorb all the IR intensity, because -IRE is shorter and thinner. The background spectra [21] in Fig. 3 experimentally prove that Si-ATR-IR can be used to collect IR spectra below 1500 cm−1 . Even though the absorptions of the IR light by the phonons decrease the sensitivity, it is possible to collect high-quality spectra below 1500 cm−1 . Two different applications of the first Si-ATR-IR microfluidic system were shown in Herzig-Marx et al. [21]. First, researchers studied hydrolysis of ethyl acetate hydrolysis to test Si-ATR-IR-coupled microfluidic chips with different HCl and ethyl acetate concentrations. The system was capable of measuring ethyl acetate concentrations down to 7 mM, including the fingerprint region. Test results were used in the first-order rate approximation model, and rate coefficients of the reaction at different reactant concentrations were obtained. These coefficients were compared with the values in the literature, and a good agreement was achieved between them [21]. Second, reaction of surface-tethered functional groups was monitored with Si-ATR-IR-coupled microfluidic chip. A homemade Teflon holder with two sides was designed in order to increase the sensitivity of the technique. APTES ((aminopropyl)-triethoxysilane) was used to functionalize the Si surface with a previously explained protocol [46]. To prove the concept, the researchers used ATRIR during surface modification within the microfluidic chip
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with a time resolution of 2.5 min. In short, this experiment demonstrated that the Si-ATR-IR-coupled microfluidic chip is useful for probing surface modification of Si wafer, which is a critical factor in sensor applications of Si [47]. The field effect transistor (FET) is a widely known concept in the semiconductor industry. In FET, current flow is controlled by external voltage bias. Fluidic FET [48, 49] is a similar concept, and it is about controlling the liquid flow by applying external voltage. Recently, the behavior of fluidic FET has been studied with the Si-ATR-IR nanofluidic chip [24], as shown in Fig. 4. In fact, the experimental set-up requires complex fabrication steps. Initially, a specific optical set-up for a Si-ATR-IR nanofluidic chip is built. With the optical set-up, light is coupled to Si-IRE more efficiently. Instead of microchannels, nanochannels are fabricated with interferometric lithography on top of Si-IRE. In the next step, Si-IRE is oxidized deeper than nanochannels. Inside the chip, total reflection occurs in the interface between the Si and SiO2 layers, but not in the one between Si and glass. The shapes of nanochannels are shown in Fig. 4. There are 8000 nanochannels in one Si-IRE with the aim of increasing the S/N ratio. The system is sealed with Pyrex glass from the top with anodic bonding. In addition, external voltage sources are coupled to the nanofluidic chip. Various voltages are applied to the chip, and the response of the system is measured with Si-ATR-IR. Furthermore, laser scanning confocal fluorescence microscopy is coupled to the system from above in order to measure the liquid flow speed and direction. There are 8000 nanochannels in one Si-IRE, as shown in Fig. 4. The periodicity of these channels needs to be discussed generally in Si-ATR-IR. If the periodicity is in the same range as the IR light wavelength, scattering can happen, which means that this will in principle result in a significant loss of intensity. In order to prevent scattering and loss of intensity, the periodicity of the structures needs to be ten times lower or ten times higher than the IR light wavelength. This is a general rule for micromachined structures built on Si-IRE. The IR wavelength is between 2.5 m and 15 m
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Figure 5. Si-ATR-IR nanofluidic chip with 71 nL internal volume designed to conduct low-volume mechanistic studies of chemical reactions. Reprinted (adapted) with permission from Karabudak et al. [22]. Copyright 2012, American Chemical Society.
and Oh et al. [24] made the channel periodicity much lower than the IR wavelength in order to prevent scattering. It is also useful to know about the refractive index of the sample medium in order to calculate penetration depth, critical angle, and evanescent wave. In fact, there is no one single refractive index, because the sample medium consists of periodic micromachined structures with two different refractive indexes. Oh et al. [24] used the formula below to calculate the refractive index of effective medium (nem ). The result was used for calculating the parameters explained earlier: nem =
wSi wSiO2 wH2 O nSi + nSiO2 + n H2 O , p p p
(5)
where, wSi is the width of the Si wall, wSiO2 is the thickness of SiO2 layer, wH2O is width of open channel occupied, and p is channel pitch. In the mentioned study, the micromachining tools were used to fabricate IRE with integrated nanochannels, but polishing was still used for preparing sidewalls. Thus, this became a Si-IRE with polished sidewalls. IRE had a length of 5 cm, so the fingerprint region below 1500 cm−1 could not be measured. Nanochannels were filled with charged fluorescent dye molecules. Si-ATR-IR and laser scanning confocal fluorescence microscopy were applied for an understanding of the behavior and flow of the charged fluorescent dye in the nanochannels depending on the applied potential. It was observed that the flow speed and direction of the charged fluorescent dye could be controlled by applying voltage. Also, fluorescent dyes were irreversibly attached on channel walls at specific voltages, pH, surface charge, and specific conditions. In nanochannels, if the length of the electrical double layer (EDL) is in the order of that of the channel diameter, the number of cations and the number of anions are not equal in the nanochannel. In other words, the nanochannel is no longer electroneutral. Native pH shift is an effect that occurs in this kind of nanochannel. Bottenus et al. [50] studied the native pH shift with Si-ATR-IR-coupled 100 nm nanofluidic chip and fluorescent spectroscopy by using an IR pH indicator and a fluorescent pH indicator. Both Si-ATR-IR and fluorescence measurements produced parallel results, showing that there was a shift of one pH unit in low ionic strengths (longer EDL) and a shift of 0.1 pH in high ionic strengths (shorter EDL). This conclusion was also verified by the theoretical model developed with Comsol Multiphysics. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Fabricating nanochannels coupled with Si-ATR-IR requires complex fabrication techniques. This complexity can be a problem. Karabudak et al. [22] simplified the fabrication method of Si-ATR-IR nanofluidic chips. Instead of using interferometric lithography, optical lithography was used to fabricate nanochannels. SiO2 with a thickness of 660 nm was grown on top of Si-IRE. Then classical optical lithography was used to pattern the SiO2 to form 1D nanochannels. Some 300 nanochannels with dimensions of 660 nm depth, 30 m width, and 1 cm length were fabricated on 1.5 cm × 2 cm Si-IRE. Smaller dimensions produced a higher number of Si-IREs from a single Si wafer. In addition, the smaller length of IRE allowed probing of the fingerprint region below 1500 cm−1 . Periodicity of nanochannels is a critical issue, because periodicity can cause scattering of IR light, as explained before. Previously, Oh et al. [23–25] made the periodicity much lower than IR light wavelength in order to prevent scattering. Through the application of an opposite approach [22], the periodicity has been made much higher than IR light wavelength. Thanks to this strategy, the fabrication has become simpler, and it has allowed the usage of optical lithography instead of interferometric lithography, which is a relatively complex technique. A specific optical set-up was also fabricated for smaller Si-IREs [22] (see Fig. 5). Researchers studied the Knoevenagel condensation reaction between malononitrile and p-anisaldehyde catalyzed by 1,8-diazabicyclo[5.4.0]undec-7ene (DBU) with the chip, which had only 71 nL total volume. The reaction was carried out with eight different DBU concentrations. Unexpectedly, seven different species, including reaction intermediates, were online during the reaction, as shown in Scheme 1. It was found that high DBU concentrations stop the reaction. Repeating experiments in macro scale verified this conclusion. The rate equation and online concentration information were used to determine the rate constants of forward and reverse reaction rates. The results showed that optimum catalyst concentration is 11 mM for DBU, and any higher or lower catalyst concentration decreases the overall rate of the Knoevenagel condensation reaction. The study revealed that mechanistic studies are possible with only 71 nL. This is an important finding for experiments with expensive isotope chemicals or poisonous and explosive chemicals, especially under circumstances where low volume studies are advantageous.
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Scheme 1. Detailed mechanism of the Knoevenagel condensation reaction probed with a Si-ATR-IR nanofluidic chip (Fig. 5), red numbers are the chemicals that could be monitored by SiATR-IR. Reprinted (adapted) with permission from Karabudak et al. [22]. Copyright 2012, American Chemical Society.
5 Recent advances in Si-IREs without microchannels ATR-IR has various advantages in biology, because it does not require tagged samples and the short penetration depth allows performance of studies in water. Detecting various chemicals in situ even in living cells has attracted the interest of several researchers. In particular, cancer research requires this kind of experimental set-up. Usually living cells are put on the IRE, and their IR spectra are monitored by ATRIR. In addition to ATR-IR, Si-ATR-IR has also entered the field of living cell and phospholipid membrane experiments [27–30, 51–53]. In fact, Si-ATR-IR has allowed the usage of Si-IRE with the possibility of working in the fingerprint region. This is an important contribution to the field, because Si is not toxic like other IREs such as germanium, which makes Si-ATR-IR an ideal platform for living cell experiments. Usually biocompatible SiO2 is put on top of Si-IRE and biocompatible PDMS material is embedded on top of Si-IRE. If further information is desired, I recommend the review of ATR-IR usage in living cells in Hirano-Iwata et al. [28]. Recently, it has been shown that Si-ATR-IR can also contribute to the field of artificial photosynthesis and solar fuels [34]. In fact, silicon wafer is commonly used as the photocathode in artificial photosynthesis/solar to fuel devices. When Si absorbs photons, four fundamental steps follow: photoexcitation, photothermalization, transport to surface and surface reaction, as depicted in Fig. 6. The most well-known techniques in chemistry labs can only probe the efficiency of surface reactions. In order to probe photoexcited electrons and phonons inside the Si wafer, timeresolved dedicated optical set-ups are usually required. The study demonstrated that instead of dedicated optical set-ups, Si-ATR-IR alone can be used to detect photoexcited elec C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 6. Top: the four stages of solar to fuel conversion (photoexcitation, thermalization of excited carriers, transport to the surface, and surface reaction); bottom: schematics of Si-ATR-IR to probe photoexcited electrons and phonons inside the Si wafer. Reprinted (adapted) with permission from Karabudak et al. [34]. Copyright 2012, American Chemical Society.
trons and photoexcited phonons inside the Si wafer [34]. The experimentation was rather simple: a laser of 130 mW and 1064 nm is pointed at the Si-IRE to excite electrons via two photon process, and the signal from Si-ATR-IR is analyzed. Photoexcited electrons are seen as a background shift, and phonons are observed at specific wavenumbers (1448 cm−1 (3TO), 1378 cm−1 (3TO + LO), and 1302 cm−1 (2TO + LO)), as mentioned before. This study has proven that Si-ATR-IR can be routinely used in artificial photosynthesis research. Various integrated microfluidic detection methods are described in the literature. Not only capabilities but also the simplicity of the detection method and the fabrication of its chips are critical for broad applications. Therefore, offline detection and online fluorescent detection systems are still dominant in the field today. Recently, the fabrication of Si-IRE has been further simplified [33]. The first disposable Si-IREs have been produced. Instead of KOH etching, researchers experimentally and theoretically showed that cutting classical Si wafer with a dicing machine or even by hand is enough to produce Si-IRE [33]. The schematics of the new disposable Si-IRE in comparison with KOH-etched Si-IRE are shown in Fig. 7. Disposable IREs have less sensitivity [33], because there are a smaller number of reflections, as shown in Fig. 7. The total IR intensity that is coupled to both IREs is the same, however. The angle of incidence is different in each case, and www.electrophoresis-journal.com
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Figure 7. Schematics of KOH-etched Si-IRE and disposable Si-IRE. Reprinted (adapted) with permission from Karabudak et al. [33]. Copyright 2013, American Chemical Society.
the penetration depth is smaller in disposable IRE. The disposable IRE has, however, been successfully tested with thin PS films and composite polymer inorganic material films. Disposable IREs also have advantages in terms of high temperature processes and with chemicals that may irreversibly damage them. More importantly, disposable IREs prove that IR light can be coupled to any Si/glass microfluidic chip if channels are fabricated in a glass side. I believe disposable Si-IRE has potential in microfluidic research, because it is easy to fabricate and use. In fact, it is expected that the usage of Si-ATR-IR microfluidics will grow through the increasingly widespread use of disposable IREs.
6 Miniaturizing of ATR-IR spectrometers Various instrumentation companies are trying to miniaturize IR spectrometers. Every year sees a smaller version of IR spectrometers. Interested readers can read a review of handheld IR spectrometers in Sorak et al. [54]. One commercial hand-held ATR-IR spectrometer is also available from the Pyroes company. The spectral quality and cost efficiency of these systems prevent their mass use, however. It is likely that these systems will become more and more common in analytical labs in the near future if technological development enables the manufacture of much smaller IR spectrometers with better quality and a lower price. In addition, there have been academic attempts to integrate entire ATR-IR systems into a single embedded chip [55–57], as shown in Fig. 8. In these systems, the entire light source and detector are integrated into the same Si wafer. These systems are promising but there are two big disadvantages. First, these systems do not work as efficiently as
commercial IR spectrometers, because the embedded light source and detector are not as efficient as commercial ones. For example, the embedded light source cannot produce high intensity in comparison with commercial light sources. This reduces the quality of the spectra that are obtained. In addition, embedded detectors cannot detect all wavelengths at one time. Normally one or two specific wavelengths can be selected and the entire system can work in these wavelengths, as shown in Fig. 8. Second, fabrication of the device is possible but not straightforward; the fabrication process requires clean room facilities and this increases the price of the device. Price is not a scientific issue, but in terms of the general use of an analytical technique it is important. In short, technologically it is possible to integrate a light source and detector into a single silicon wafer, but the system’s efficiency is quite low and the price is high given today’s technology. If, however, the price of microfabrication and the quality of an embedded light source/detector can be further developed, this kind of system can play a significant role in the field.
7 Conclusion Analyzing liquids inside microfluidic and nanofluidic channels in situ is a valid research challenge. In comparison with other integrated microfluidic detection methods, Si-ATR-IR has specific advantages, because it is an in situ, chemicalspecific, tag-free, and low-volume technique. In comparison with classical Si-IRE ATR-IR systems, Si-ATR-IR has the advantage of working in the fingerprint region providing the possibility of nanochannel integration and mass production. Recent advances in Si-ATR-IR have allowed us to fabricate simpler and more cost-effective Si-ATR-IR elements. With these properties, Si-ATR-IR can become the most
Figure 8. Schematics of light sourceand detector-integrated Si ATR-IR system. Reprinted from Kasberger et al. [55]. Copyright (2011), with permission from Elsevier.
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important analytical tool as regards the analysis of the liquids inside micro/nanofluidic channels. On the other hand, greater simplicity of the entire IR spectrometer is required. In particular, cheaper light sources, detectors, and optical systems could affect the entire future of the field. In addition, the commercialization of ATR-IR-coupled microfluidic platforms could have a significant effect on usage. This project was carried out within the research program of BioSolar Cells, cofinanced by the Dutch Ministry of Economic Affairs, Agriculture and Innovation. Thanks to Han (J.G.E.) Gardeniers and Guido Mul for their support. I thank Recep Kas for enjoyable discussions. The authors have declared no conflict of interest.
8 References [1] Dong, Y., Skelley, A. M., Merdek, K. D., Sprott, K. M., Jiang, C. S., Pierceall, W. E., Lin, J., Stocum, M., Carney, W. P., Smirnov, D. A., J. Mol. Diagn. 2013, 15, 149–157. [2] Kim, P., Kwon, K. W., Park, M. C., Lee, S. H., Kim, S. M., Suh, K. Y., Biochip J. 2008, 2, 1–11. [3] Lee, J., Soper, S. A., Murray, K. K., Anal. Chim. Acta 2009, 649, 180–190. [4] Mu, X., Zheng, W. F., Sun, J. S., Zhang, W., Jiang, X. Y., Small 2013, 9, 9–21. [5] Park, J. W., Kim, H. J., Kang, M. W., Jeon, N. L., Lab Chip 2013, 13, 509–521.
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Engin Karabudak
Review
Mesoscale Chemical Systems Group (MCS), MESA+ Institute for Nanotechnology, University of Twente, The Netherlands
Micromachined silicon attenuated total reflectance infrared spectroscopy: An emerging detection method in micro/nanofluidics
Received May 27, 2013 Revised September 30, 2013 Accepted October 1, 2013
Microfluidics is an emerging field with various applications. In situ analysis of liquids inside microfluidic channels is an important research subject for any of these applications. On the other hand, attenuated total reflectance infrared spectroscopy (ATR-IR) is a strong surface detection method. Recently, there have been various attempts to use ATRIR for detection of liquids inside microfluidic channels. Micromachined silicon ATR-IR (Si-ATR-IR) is an emerging analytical tool for micro/nanofluidics. This paper reviews Si-ATR-IR micro/nanofluidics detection devices and discusses the application fields, capabilities, advantages, and disadvantages of the method. Keywords: Attenuated total reflection / Infrared spectroscopy / Microfluidics / Nanofluidics / Online detection DOI 10.1002/elps.201300248
1 Introduction Microfluidics is a rapidly growing research field, which has various applications in disciplines such as biology [1–5], chemistry [6], life sciences [1, 4, 7, 8], and engineering [9, 10]. In a microfluidic chip, liquid flows inside micron-size channels. Liquids in these channels cannot be analyzed by classical analytical techniques, such as SEM [11], X-ray photoelectron spectroscopy [12], transmission electron microscopy [11], classical UV/Vis spectroscopy [13], or classical infrared (IR) spectroscopy [14]. For this reason, offline detection systems dominate the field. The development of integrated detection systems, however, which can conduct in situ analysis of the channels’ contents, remains an important research topic. Various integrated microfluidic detection methods are reported in the literature [15–20]. Among them, mainly thanks to its simplicity, the fluorescence-based detection [17] technique is the most common method. Surface-enhanced Raman scattering [19], surface plasmon resonance tech-
Correspondence: Dr. Engin Karabudak, Mesoscale Chemical Systems Group (MCS), MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands E-mail: [email protected] Fax: +31-53-4894683
Abbreviations: ATR-IR, attenuated total reflectance infrared spectroscopy; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; EDL, electrical double layer; FET, field effect transistor; IR, infrared; IRE, internal reflection element; LO, longitudinal optical phonon; MIR, multiple internal reflection; Si-ATR-IR, micromachined silicon ATR-IR; TO, transverse optical phonon C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
nique [18], electrochemical detection methods [20], and nuclear magnetic resonance [16] have all been integrated into microfluidic chips, which have advantages as well as disadvantages. This review focuses on micromachined silicon attenuated total reflectance IR spectroscopy (Si-ATR-IR) micro/nanofluidics detection devices [21–25], an emerging method with specific capabilities. In order to explain these capabilities, the following themes are elaborated: general principles of ATR-IR, polished internal reflection element (polished-IRE) coupled ATR-IR micro/nanofluidics, SiATR-IR coupled microfluidics, and recent advances regarding micromachined silicon IREs (Si-IREs) without microchannels.
2 General principles of ATR-IR ATR-IR is a surface characterization technique that was first reported in 1959 [26]. ATR-IR is also known as multiple internal reflection (MIR) FTIR spectroscopy (MIR-FT-IR) [21], multiple internal reflection FTIR spectroscopy (MIR-FTIRS) [23–25], or MIR-IR absorption spectroscopy [27–30], all of which refer to the same technique. To avoid confusion, the term ATR-IR will be used throughout the paper. Before the principles of ATR-IR can be explained, several technical terms such as critical angle, total reflectance, IRE, evanescent wave, and penetration depth must be clearly defined. Light travels at different speeds inside different
Colour Online: See the article online to view Scheme 1 and Figs. 1, 2, and 5–7 in colour.
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Figure 1. Schematics of total reflection, (A) ␣ is angle of incidence, c is the critical angle, dp is penetration depth, E is the evanescent wave, z is the depth; (B) general view of ATR set-up.
materials and the refractive index (n) of a material can be defined as: c (1) n= , v where c is the speed of the light in vacuum and v is the speed of the light in the material. If there is a flat interface between two different materials and light is sent from a material with a high refractive index (n1 ) to a material with low refractive index (n2 ), we can define a critical angle (c ) as: c = sin−1
n2 , n1
(2)
where n2 is the refractive index of the high refractive index material and n1 is the refractive index of the material. If the angle of incidence is bigger than the critical angle, all of the light reflects (total reflection) from the interface. At the point of reflection, the electric field penetrates the interface, whereby it is known as the evanescent wave, to probe the surface just above the IRE as shown in Fig. 1. The decay of the electric field intensity of the evanescent wave can be described as: E = E0e
− dz
p
,
(3)
where E is the evanescent wave strength, E0 is the time averaged electric field intensity at the interface, z is the depth, and d p is the penetration depth. Penetration depth (d p ) is given by the following equation: dp =
n1
2 sin2 −
2 ,
(4)
n2 n1
where is the wavelength of the light and is the angle of incidence. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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IRE is a critical component in an ATR-IR set-up, as the sample is placed on top of IRE and the optical set-up couples IR light to the edge of IRE (see Fig. 1). These systems make sure that the angle of incidence is always bigger than the critical angle, which guarantees that all the light intensity is reflected from the interface. The reflected light forms an evanescent wave at the interface, and this evanescent wave interacts with the sample. This interaction allows spectroscopic information on the sample to be collected. In short, the coupled light travels inside IRE by total reflectance and reaches the detector. IRE must be manufactured from high refractive index material and should be transparent for the measurement of wavelengths. Various IRE materials are used for ATR-IR setups such as germanium, silicon, ZnSe, KRS-5, diamond, and ZnS. Each of these IRE materials has its own properties, such as working spectral range, cut-off wavelength, price, refractive index, hardness, and chemical stability. For detailed discussion on IREs, I direct the reader to the review article by Vigano et al. [31]. Another important issue to be discussed is the production method of IREs. In fact, IRE material needs to be mechanically processable to form a specific shape. The shape is critical for determining the angle of incidence, which is a parameter for determining the penetration depth and evanescent wave. All of the commercial IREs are produced by mechanical polishing in different shapes (polished IRE). Polishing has to be done carefully so that an optical smoothness can be achieved on the reflection side. This requires extra polishing of every single IRE and limits the mass production of commercial IREs. For this reason, commercial IREs are not cost-effective. In addition, extra attention has to be paid while cleaning the IREs, since any roughness at the surface may interfere with the measurements. These are the major disadvantages when conventional IREs are used to collect IR spectra. We will consider how Si-IRE solves this problem in the following two sections. First, for microfluidic applications Si is one of the most attractive IRE materials because silicon processing is well developed in microfabrication [32]. Si-IRE has one disadvantage, however, its working spectral range is unfortunately limited. In fact, Si has a cut-off below 1500 cm−1 because of phonon absorptions [22, 33, 34]. This is a strong drawback, because the region below 1500 cm−1 , the so-called fingerprint region, is the most important aspect of mid-IR spectroscopy. In the fingerprint region, every peak can be a marker of a specific molecule. Therefore, Si-IRE is a common IRE in neither the literature [31,35–40] nor in the market. The following two sections explain how Si-IRE can overcome this disadvantage. Second, in microfluidic applications, the sample does not consist of one single homogeneous material, but of channel walls, channels, and liquids. All these materials have a different refractive index. As shown in the penetration depth formula, the penetration depth also depends on the refractive index. Thus, the penetration depth can be different in a channel wall or a liquid. As discussed in the section of polished IRE-coupled microfluidics, some sources make an average www.electrophoresis-journal.com
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Figure 2. Schematics of ATR-IR imaging set-up. Reproduced from [36] with the permission of the Royal Society of Chemistry.
of refractive indexes and call it an effective refractive index. In addition, not only refractive index of materials, but also periodicity of the channels can be an issue that may affect sensitivity of ATR-IR. If periodicity of any structure on top of IRE is in the range of wavelength of IR light, this can result in a scattering effect. This is also explained in the next section in detail.
3 Polished IRE-coupled microfluidics Companies that produce an ATR-IR set-up usually provide flow cells. These cells consist of a liquid compartment and polished IRE that can be assembled in reverse. In fact, these systems cannot be defined as microfluidic, because their sizes are far above the micron size. On the other hand, researchers have developed a simple method of building microfluidic devices on top of commercial/polished IREs. Usually PDMS devices are integrated with polished/commercial IREs [36, 40]. The precise bonding, leakage problems and limitations of the PDMS material itself can be problematic, however. Moreover, the PDMS material needs to be reassembled and the polished IRE has to be cleaned after each use. Kazarian and colleagues [35–37,39,40] showed that ATRIR can be used for imaging purposes for microfluidics with focal plane array detector. As shown in Fig. 2, commercial/polished ZnSe with a 45o angle is usually used for these purposes and PDMS materials with microchannels are integrated on top of IRE. With this set-up, it is possible to make IR images of the channels with a focal plane array detector. This is a very promising approach. I will not expand on this approach now, because there are already some excellent reviews [37, 40] available. To the best of my knowledge, Si-IREs are not used in this approach, and I believe their usage in the future will contribute to the field of ATR-IR imaging.
4 Si-ATR-IR coupled micro/nanofluidics As mentioned in the previous section, Si IRE has the signal disadvantage of having a cut-off wavelength of 1500 cm−1 . In 1997, Weldon et al. [41] showed that there is a way to overcome this disadvantage. They fabricated homemade polished Si-IRE measuring 1 cm in length from float zone Si wafer and C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. (A) Si-ATR-IR coupled microfluidic chip; (B) background spectra of Si-ATR-IR. Reprinted (adapted) with permission from Herzig-Marx et al. [21]. Copyright 2004, American Chemical Society.
observed that the cut-off wavenumber shifted to 900 cm−1 . It seems that the lower IRE length reduced the phonon absorptions, and the system was able to perform ATR-IR below 1500 cm−1 . The system was originally not designed to work with microfluidics. In fact, Weldon et al. coupled synchrotron IR light source to the IRE and studied oxidation of Si experimentally by using s- and p-polarized light. Theoretical density functional calculations also supported the results. In short, this was the first study to show shorter-length Si IRE can overcome the limited working spectral range of Si-IRE. Another disadvantage of conventional IREs is the requirement for extra polishing of the sidewalls, which limits mass production and therefore has a negative impact on the price. The first Si-ATR-IR microfluidic system in the literature was reported in 2004 [21]. Anisotropic KOH-etching of Si wafer, a guaranteed method to produce optically smooth surfaces with Si wafer [42, 43], was applied instead of polishing Si wafer. This study proved that 16 Si-IREs can be produced from 100-mm Si wafer immediately. Anodic bonding was used to bond the glass wafer with micromachined channels on top of these Si-IREs. Finally, the top surface and connections were produced with PDMS material, as shown in Fig. 3. The whole was put into a commercial FTIR spectrometer without fabrication of any special optical set-up. Fabricated IREs had a length of 1 cm in order to probe the fingerprint region. Thanks to the system, a wavenumber as low as 800 cm−1 could be probed. The study proved for the first time that Si-ATR-IR could probe the chemical reactions inside the channels of microfluidic chips. www.electrophoresis-journal.com
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Figure 4. Si-ATR-IR liquid FET set-up with specific optical set-up, flow connections, nanochannels, and liquid FET voltage connections. Reproduced from Oh et al. [24] with the permission of the Royal Society of Chemistry.
Si has three combination phonon absorption peaks [44, 45] at 1448 cm−1 (3TO), 1378 cm−1 (3TO + LO), and 1302 cm−1 (2TO +LO). TO and LO refer to transverse and longitudinal optical phonons. Optical phonons are the result of out-of-plane movements of atoms in a crystal. These vibrations are quantized in Si and can be seen at specific wave numbers. The mentioned peaks are the result of the combination of some of the TO and LO. In addition to these phonon peaks, below 1200 cm−1 there are various oxide peaks [41] and phonon peaks [44]. These phonon and oxide peaks usually absorb all the IR photons in the commercial systems, because commercial Si-IREs are thicker and longer. These peaks reduce the intensity reaching the detector, but they do not absorb all the IR intensity, because -IRE is shorter and thinner. The background spectra [21] in Fig. 3 experimentally prove that Si-ATR-IR can be used to collect IR spectra below 1500 cm−1 . Even though the absorptions of the IR light by the phonons decrease the sensitivity, it is possible to collect high-quality spectra below 1500 cm−1 . Two different applications of the first Si-ATR-IR microfluidic system were shown in Herzig-Marx et al. [21]. First, researchers studied hydrolysis of ethyl acetate hydrolysis to test Si-ATR-IR-coupled microfluidic chips with different HCl and ethyl acetate concentrations. The system was capable of measuring ethyl acetate concentrations down to 7 mM, including the fingerprint region. Test results were used in the first-order rate approximation model, and rate coefficients of the reaction at different reactant concentrations were obtained. These coefficients were compared with the values in the literature, and a good agreement was achieved between them [21]. Second, reaction of surface-tethered functional groups was monitored with Si-ATR-IR-coupled microfluidic chip. A homemade Teflon holder with two sides was designed in order to increase the sensitivity of the technique. APTES ((aminopropyl)-triethoxysilane) was used to functionalize the Si surface with a previously explained protocol [46]. To prove the concept, the researchers used ATRIR during surface modification within the microfluidic chip
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with a time resolution of 2.5 min. In short, this experiment demonstrated that the Si-ATR-IR-coupled microfluidic chip is useful for probing surface modification of Si wafer, which is a critical factor in sensor applications of Si [47]. The field effect transistor (FET) is a widely known concept in the semiconductor industry. In FET, current flow is controlled by external voltage bias. Fluidic FET [48, 49] is a similar concept, and it is about controlling the liquid flow by applying external voltage. Recently, the behavior of fluidic FET has been studied with the Si-ATR-IR nanofluidic chip [24], as shown in Fig. 4. In fact, the experimental set-up requires complex fabrication steps. Initially, a specific optical set-up for a Si-ATR-IR nanofluidic chip is built. With the optical set-up, light is coupled to Si-IRE more efficiently. Instead of microchannels, nanochannels are fabricated with interferometric lithography on top of Si-IRE. In the next step, Si-IRE is oxidized deeper than nanochannels. Inside the chip, total reflection occurs in the interface between the Si and SiO2 layers, but not in the one between Si and glass. The shapes of nanochannels are shown in Fig. 4. There are 8000 nanochannels in one Si-IRE with the aim of increasing the S/N ratio. The system is sealed with Pyrex glass from the top with anodic bonding. In addition, external voltage sources are coupled to the nanofluidic chip. Various voltages are applied to the chip, and the response of the system is measured with Si-ATR-IR. Furthermore, laser scanning confocal fluorescence microscopy is coupled to the system from above in order to measure the liquid flow speed and direction. There are 8000 nanochannels in one Si-IRE, as shown in Fig. 4. The periodicity of these channels needs to be discussed generally in Si-ATR-IR. If the periodicity is in the same range as the IR light wavelength, scattering can happen, which means that this will in principle result in a significant loss of intensity. In order to prevent scattering and loss of intensity, the periodicity of the structures needs to be ten times lower or ten times higher than the IR light wavelength. This is a general rule for micromachined structures built on Si-IRE. The IR wavelength is between 2.5 m and 15 m
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Figure 5. Si-ATR-IR nanofluidic chip with 71 nL internal volume designed to conduct low-volume mechanistic studies of chemical reactions. Reprinted (adapted) with permission from Karabudak et al. [22]. Copyright 2012, American Chemical Society.
and Oh et al. [24] made the channel periodicity much lower than the IR wavelength in order to prevent scattering. It is also useful to know about the refractive index of the sample medium in order to calculate penetration depth, critical angle, and evanescent wave. In fact, there is no one single refractive index, because the sample medium consists of periodic micromachined structures with two different refractive indexes. Oh et al. [24] used the formula below to calculate the refractive index of effective medium (nem ). The result was used for calculating the parameters explained earlier: nem =
wSi wSiO2 wH2 O nSi + nSiO2 + n H2 O , p p p
(5)
where, wSi is the width of the Si wall, wSiO2 is the thickness of SiO2 layer, wH2O is width of open channel occupied, and p is channel pitch. In the mentioned study, the micromachining tools were used to fabricate IRE with integrated nanochannels, but polishing was still used for preparing sidewalls. Thus, this became a Si-IRE with polished sidewalls. IRE had a length of 5 cm, so the fingerprint region below 1500 cm−1 could not be measured. Nanochannels were filled with charged fluorescent dye molecules. Si-ATR-IR and laser scanning confocal fluorescence microscopy were applied for an understanding of the behavior and flow of the charged fluorescent dye in the nanochannels depending on the applied potential. It was observed that the flow speed and direction of the charged fluorescent dye could be controlled by applying voltage. Also, fluorescent dyes were irreversibly attached on channel walls at specific voltages, pH, surface charge, and specific conditions. In nanochannels, if the length of the electrical double layer (EDL) is in the order of that of the channel diameter, the number of cations and the number of anions are not equal in the nanochannel. In other words, the nanochannel is no longer electroneutral. Native pH shift is an effect that occurs in this kind of nanochannel. Bottenus et al. [50] studied the native pH shift with Si-ATR-IR-coupled 100 nm nanofluidic chip and fluorescent spectroscopy by using an IR pH indicator and a fluorescent pH indicator. Both Si-ATR-IR and fluorescence measurements produced parallel results, showing that there was a shift of one pH unit in low ionic strengths (longer EDL) and a shift of 0.1 pH in high ionic strengths (shorter EDL). This conclusion was also verified by the theoretical model developed with Comsol Multiphysics. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Fabricating nanochannels coupled with Si-ATR-IR requires complex fabrication techniques. This complexity can be a problem. Karabudak et al. [22] simplified the fabrication method of Si-ATR-IR nanofluidic chips. Instead of using interferometric lithography, optical lithography was used to fabricate nanochannels. SiO2 with a thickness of 660 nm was grown on top of Si-IRE. Then classical optical lithography was used to pattern the SiO2 to form 1D nanochannels. Some 300 nanochannels with dimensions of 660 nm depth, 30 m width, and 1 cm length were fabricated on 1.5 cm × 2 cm Si-IRE. Smaller dimensions produced a higher number of Si-IREs from a single Si wafer. In addition, the smaller length of IRE allowed probing of the fingerprint region below 1500 cm−1 . Periodicity of nanochannels is a critical issue, because periodicity can cause scattering of IR light, as explained before. Previously, Oh et al. [23–25] made the periodicity much lower than IR light wavelength in order to prevent scattering. Through the application of an opposite approach [22], the periodicity has been made much higher than IR light wavelength. Thanks to this strategy, the fabrication has become simpler, and it has allowed the usage of optical lithography instead of interferometric lithography, which is a relatively complex technique. A specific optical set-up was also fabricated for smaller Si-IREs [22] (see Fig. 5). Researchers studied the Knoevenagel condensation reaction between malononitrile and p-anisaldehyde catalyzed by 1,8-diazabicyclo[5.4.0]undec-7ene (DBU) with the chip, which had only 71 nL total volume. The reaction was carried out with eight different DBU concentrations. Unexpectedly, seven different species, including reaction intermediates, were online during the reaction, as shown in Scheme 1. It was found that high DBU concentrations stop the reaction. Repeating experiments in macro scale verified this conclusion. The rate equation and online concentration information were used to determine the rate constants of forward and reverse reaction rates. The results showed that optimum catalyst concentration is 11 mM for DBU, and any higher or lower catalyst concentration decreases the overall rate of the Knoevenagel condensation reaction. The study revealed that mechanistic studies are possible with only 71 nL. This is an important finding for experiments with expensive isotope chemicals or poisonous and explosive chemicals, especially under circumstances where low volume studies are advantageous.
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Scheme 1. Detailed mechanism of the Knoevenagel condensation reaction probed with a Si-ATR-IR nanofluidic chip (Fig. 5), red numbers are the chemicals that could be monitored by SiATR-IR. Reprinted (adapted) with permission from Karabudak et al. [22]. Copyright 2012, American Chemical Society.
5 Recent advances in Si-IREs without microchannels ATR-IR has various advantages in biology, because it does not require tagged samples and the short penetration depth allows performance of studies in water. Detecting various chemicals in situ even in living cells has attracted the interest of several researchers. In particular, cancer research requires this kind of experimental set-up. Usually living cells are put on the IRE, and their IR spectra are monitored by ATRIR. In addition to ATR-IR, Si-ATR-IR has also entered the field of living cell and phospholipid membrane experiments [27–30, 51–53]. In fact, Si-ATR-IR has allowed the usage of Si-IRE with the possibility of working in the fingerprint region. This is an important contribution to the field, because Si is not toxic like other IREs such as germanium, which makes Si-ATR-IR an ideal platform for living cell experiments. Usually biocompatible SiO2 is put on top of Si-IRE and biocompatible PDMS material is embedded on top of Si-IRE. If further information is desired, I recommend the review of ATR-IR usage in living cells in Hirano-Iwata et al. [28]. Recently, it has been shown that Si-ATR-IR can also contribute to the field of artificial photosynthesis and solar fuels [34]. In fact, silicon wafer is commonly used as the photocathode in artificial photosynthesis/solar to fuel devices. When Si absorbs photons, four fundamental steps follow: photoexcitation, photothermalization, transport to surface and surface reaction, as depicted in Fig. 6. The most well-known techniques in chemistry labs can only probe the efficiency of surface reactions. In order to probe photoexcited electrons and phonons inside the Si wafer, timeresolved dedicated optical set-ups are usually required. The study demonstrated that instead of dedicated optical set-ups, Si-ATR-IR alone can be used to detect photoexcited elec C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 6. Top: the four stages of solar to fuel conversion (photoexcitation, thermalization of excited carriers, transport to the surface, and surface reaction); bottom: schematics of Si-ATR-IR to probe photoexcited electrons and phonons inside the Si wafer. Reprinted (adapted) with permission from Karabudak et al. [34]. Copyright 2012, American Chemical Society.
trons and photoexcited phonons inside the Si wafer [34]. The experimentation was rather simple: a laser of 130 mW and 1064 nm is pointed at the Si-IRE to excite electrons via two photon process, and the signal from Si-ATR-IR is analyzed. Photoexcited electrons are seen as a background shift, and phonons are observed at specific wavenumbers (1448 cm−1 (3TO), 1378 cm−1 (3TO + LO), and 1302 cm−1 (2TO + LO)), as mentioned before. This study has proven that Si-ATR-IR can be routinely used in artificial photosynthesis research. Various integrated microfluidic detection methods are described in the literature. Not only capabilities but also the simplicity of the detection method and the fabrication of its chips are critical for broad applications. Therefore, offline detection and online fluorescent detection systems are still dominant in the field today. Recently, the fabrication of Si-IRE has been further simplified [33]. The first disposable Si-IREs have been produced. Instead of KOH etching, researchers experimentally and theoretically showed that cutting classical Si wafer with a dicing machine or even by hand is enough to produce Si-IRE [33]. The schematics of the new disposable Si-IRE in comparison with KOH-etched Si-IRE are shown in Fig. 7. Disposable IREs have less sensitivity [33], because there are a smaller number of reflections, as shown in Fig. 7. The total IR intensity that is coupled to both IREs is the same, however. The angle of incidence is different in each case, and www.electrophoresis-journal.com
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Figure 7. Schematics of KOH-etched Si-IRE and disposable Si-IRE. Reprinted (adapted) with permission from Karabudak et al. [33]. Copyright 2013, American Chemical Society.
the penetration depth is smaller in disposable IRE. The disposable IRE has, however, been successfully tested with thin PS films and composite polymer inorganic material films. Disposable IREs also have advantages in terms of high temperature processes and with chemicals that may irreversibly damage them. More importantly, disposable IREs prove that IR light can be coupled to any Si/glass microfluidic chip if channels are fabricated in a glass side. I believe disposable Si-IRE has potential in microfluidic research, because it is easy to fabricate and use. In fact, it is expected that the usage of Si-ATR-IR microfluidics will grow through the increasingly widespread use of disposable IREs.
6 Miniaturizing of ATR-IR spectrometers Various instrumentation companies are trying to miniaturize IR spectrometers. Every year sees a smaller version of IR spectrometers. Interested readers can read a review of handheld IR spectrometers in Sorak et al. [54]. One commercial hand-held ATR-IR spectrometer is also available from the Pyroes company. The spectral quality and cost efficiency of these systems prevent their mass use, however. It is likely that these systems will become more and more common in analytical labs in the near future if technological development enables the manufacture of much smaller IR spectrometers with better quality and a lower price. In addition, there have been academic attempts to integrate entire ATR-IR systems into a single embedded chip [55–57], as shown in Fig. 8. In these systems, the entire light source and detector are integrated into the same Si wafer. These systems are promising but there are two big disadvantages. First, these systems do not work as efficiently as
commercial IR spectrometers, because the embedded light source and detector are not as efficient as commercial ones. For example, the embedded light source cannot produce high intensity in comparison with commercial light sources. This reduces the quality of the spectra that are obtained. In addition, embedded detectors cannot detect all wavelengths at one time. Normally one or two specific wavelengths can be selected and the entire system can work in these wavelengths, as shown in Fig. 8. Second, fabrication of the device is possible but not straightforward; the fabrication process requires clean room facilities and this increases the price of the device. Price is not a scientific issue, but in terms of the general use of an analytical technique it is important. In short, technologically it is possible to integrate a light source and detector into a single silicon wafer, but the system’s efficiency is quite low and the price is high given today’s technology. If, however, the price of microfabrication and the quality of an embedded light source/detector can be further developed, this kind of system can play a significant role in the field.
7 Conclusion Analyzing liquids inside microfluidic and nanofluidic channels in situ is a valid research challenge. In comparison with other integrated microfluidic detection methods, Si-ATR-IR has specific advantages, because it is an in situ, chemicalspecific, tag-free, and low-volume technique. In comparison with classical Si-IRE ATR-IR systems, Si-ATR-IR has the advantage of working in the fingerprint region providing the possibility of nanochannel integration and mass production. Recent advances in Si-ATR-IR have allowed us to fabricate simpler and more cost-effective Si-ATR-IR elements. With these properties, Si-ATR-IR can become the most
Figure 8. Schematics of light sourceand detector-integrated Si ATR-IR system. Reprinted from Kasberger et al. [55]. Copyright (2011), with permission from Elsevier.
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important analytical tool as regards the analysis of the liquids inside micro/nanofluidic channels. On the other hand, greater simplicity of the entire IR spectrometer is required. In particular, cheaper light sources, detectors, and optical systems could affect the entire future of the field. In addition, the commercialization of ATR-IR-coupled microfluidic platforms could have a significant effect on usage. This project was carried out within the research program of BioSolar Cells, cofinanced by the Dutch Ministry of Economic Affairs, Agriculture and Innovation. Thanks to Han (J.G.E.) Gardeniers and Guido Mul for their support. I thank Recep Kas for enjoyable discussions. The authors have declared no conflict of interest.
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