Optofluidic liquid sensing on electromicrofluidic devices

Electromicrofluidic (EMF) devices are used to handle and move tiny amounts of liquids by electrical actuation, including electrowetting-on-dielectric (EWOD) and dielectrophoresis (DEP). Monitoring the liquid characteristics in one of these devices requires suitable sensing transducers incorporated within the microfluidic structure. In the present work, we describe the incorporation of an optofluidic photonic transducer in an EMF device to monitor the refractive index of a liquid during its manipulation. The incorporated transducer consists of a responsive porous Bragg Microcavity (BM) deposited via physical vapor oblique angle deposition. Besides reporting the manufacturing procedure of the sensing-EMF device combining liquid handling and monitoring, the performance of the BM is verified by infiltrating several liquids dripped on its surface and comparing the responses with those of liquid droplets electrically moved from the delivery part of the chip to the BM location. This study proved that modified EMF devices can incorporate photonic structures to analyze very low liquid volumes (∼0.2 μL) during its handling.


Introduction
Measuring the Refractive Index (RI) of a liquid sample is one of the most common biological/chemical analysis methods using optofluidic sensors. Although this method only reveals differences in liquids with different RI, the availability of procedures relying on a microfluidic approach (i.e., handling minute amounts of liquid) may be of interest for certain niche applications (e.g., to monitor solute concentration in solutions or mixture of liquids) where the scarcity of samples is a bottleneck for a conventional RI detection [1]. Optofluidic RI sensors based on plasmonics [2][3][4][5][6], photonic crystals [7][8][9][10][11] or photonic crystals fibers [12] are primarily composed of periodic metallic or dielectric structures that can be used to confine and guide light. However, even if these sensing techniques enable detection of liquids with extremely small volumes, in practice, the rudimentary character of the currently available liquid delivery systems makes that still relatively large liquid volumes are required for detection. Not to mention the difficulty of selectively and precisely positioning various analytes at the detection spot where the light-matter interaction is the strongest [13].
Microfluidic devices offer a wide variety of methods for handling small amounts of liquids in a controlled way. The so-called electromicrofluidic (EMF) method, operating by changing the interfacial properties of the driven liquids using electric forces, e.g., electrowetting-on-dielectric (EWOD) and dielectrophoresis (DEP), is one of the most popular and successful procedures to generate and move droplets on an array of electrostatically actuated electrodes [14][15][16][17][18][19]. EMF offers precise and programmable liquid positioning and is more reliable than other microfluidics procedures. It consumes very little power and is easily scalable [20]. Moreover, an EMF platform is ideal for accommodating and incorporating complex sensing modules. In recent years, EMF systems have been used for biosensing applications that provide high throughputs of the parallel processing of multiple samples on a single chip. Several works have already demonstrated the potential use of EMF devices for liquid handling [21][22][23][24][25][26][27][28][29] and sensing [30]. In particular, EMF devices integrating optical refractive index procedures, such as surface plasmon resonance [31,32] and microrings [33][34][35], have been applied with promising results. However, these systems usually involve time-consuming and expensive fabrication methods, which call for more practical alternatives. In the present work we prove the feasibility of a new and simple approach to incorporate a RI detecting sensor to a EMF device. The easiness of the preparation method, its solventloss character and its compatibility with any kind of substrate, including polymers, make this approach very interesting to incorporate of a miniaturized optofluidic sensor onto a selected part of the EMF chip. Moreover, the reported experiments prove that the selective deposition of the sensor BM does not affect the electrical response of the EMF chip.
The procedure relies on a recently reported results about the manufacturing by physical vapor oblique angle deposition (PV-OAD) [36][37][38] of porous multilayers in the form of Bragg Microcavities (BMs). This system consists of the successive stacking of porous layers of two transparent materials with different refractive indices and was successfully tested for the detection of glucose solutions. Due to light interference processes, the optical spectra of these multilayers present a resonant peak in the middle of a band gap that can be used for monitoring the refractive index of liquids or solutions filling the pores. An outstanding characteristic of these porous BMs as optofluidic sensor device is their capacity to monitor very low analyte volumes (~1 nL) [36][37][38]. The present work constitutes an attempt to combine in a single chip system with an EMF device for handling small droplets of liquids and with a porous BM chip, thus providing the double capacity of moving liquids at the microscale and proceeding to their RI analysis. For this purpose, we firstly describe the fabrication of a porous BM onto an EMF chip. Secondly, we prove that the thus modified EFM chip retained its capacity of moving liquids between their driving electrodes. Finally, to test the performance of this system, we compare the optofluidic response of the BM incorporated in the EMF device electrically actuated with EWOD or DEP forces to move the liquid droplet till the measurement point with the measurements of liquids manually dripped onto the BM surface. The equivalent results obtained in the two cases prove the feasibility of the approach to integrate in a single chip the capacity to move liquid droplet and to proceed to their analysis in an electrically operated way. Figure 1(a) shows that the EMF device consists of two parallel plates with an unpatterned ground electrode on the top plate, the patterned driving electrode on the bottom plate and a spacer of 50 μm between them. We used a top glass plate (thickness 0.7 mm) coated with transparent indium tin oxide [39] (ITO, thickness 200 nm) to enable optical analyses and direct observation during the experiments. To facilitate liquid handling, the top ITO glass was spin-coated with a hydrophobic layer (Teflon ® AF 1600, DuPont, thickness 120 nm). The bottom black BT (Bismaleimide Triazine) resin plate with Cu driving electrodes patterns was covered with a dielectric layer. It was manufactured by Chip Win Technology Co., Ltd, Taiwan. A Teflon layer (thickness 120 nm) was also coated on the bottom plate to make the surface hydrophobic. Before assembly of the two plates, the liquids were manually pipetted on the bottom plate; the top plate was then carefully placed and adhered onto appropriate spacers (thickness 50 μm) attached to the bottom plate. The multiple driving electrodes on the bottom plate were individually activated through single pole double throw (SPDT) relays (LU-5, Rayex Electronics). The electric potential of the electrodes was switched between the electric ground and high potentials. AC electric signals with a frequency of 1 kHz were generated by a function generator (33210 A, Agilent Technologies) and amplified through an amplifier (A-304, A. A. Lab Systems). The relays were switched by the digital output signals of a data-acquisition device (USB-6509, National Instruments) programmed with LabVIEW software.

Fabrication of porous Bragg Microcavities
To incorporate the porous BM onto the EMF chip we proceeded to its deposition by PV-OAD on the desired driving electrode (from now on the BM-electrode) of the chip by covering the rest with an aluminum shadow mask. Simultaneously, deposition by electron beam evaporation was also performed on ITO substrates and on silicon wafers. As reported in previous works alternating layers were stack deposited by evaporation at a zenithal angle of deposition (α) of 70° [36,37]. The BM stack consisted of 15 alternating layers of TiO 2 and SiO 2 , starting and ending with TiO 2 , each of them with a thickness of about 85 nm except for the central layer that, acting as optical defect, had a thickness of ca. 200 nm.

Optical monitoring of the BM-electrode
Reflectance-visible spectra of the different parts of the device including the BM-electrode were taken using a home-made optical setup consisting of an extensive source of light (range 350-900 nm, Halogen Reflector Lamp Decostar Osram ® ) collimated to a spot of around 1 mm 2 and a camera connected to a computer with a detector placed in the center of the camera. The impinging and reflected beam directions were 20°apart (incidence 10°o ff normal). Spectra were recorded before and after infiltrating the BM with liquids either dripped manually with a pipette or moved to the BM-electrode by EWOD or DEP actuation. The room temperature during the experiments was 20°C.
A silicon wafer covered with the BM was diced to take Scanning Electron Microscopy (SEM) cross section micrographs in a Hitachi S4800 field emission microscope working at 2 keV primary beam energy. Both detecting modes, Secondary Electron (SE) and Back Scattered Electron (BSE) were used to highlight either topographic or compositional information, respectively.

Results and discussion
3.1. Characterization of the BM deposited onto EMF device Figure 1(b) shows SEM micrographs of the BM deposited onto a silicon wafer, together with an overprinted scheme of the layers stack. The BSE micrograph reveals the composition of each layer of the stack with the TiO 2 corresponding to the brighter layers and the SiO 2 to the darker ones. Meanwhile, the SEM micrograph shows the typical slanted nanocolumnar microstructure of the PVD-OAD films and highlights the high porosity of these layers that in previous works we have estimated around 50%. Figure 1(c) presents the EMF device with the BM deposited on a pre-selected driving electrode (the BM-electrode) that remained uncovered by the mask during deposition. This part presented an orange color that was taken as hint of the successful deposition of the BM, providing a straightforward way to localize it onto the device. An optical characterization of the different parts of the EMF device in the form of reflectance spectra is shown in figure 1(d), together with an equivalent spectrum of a reference BM sample deposited onto an ITO substrate which is included for comparison. This reference spectrum depicts the typical wide reflectance gap and resonance peak of this type of 1D photonic structure (blue line in Figure 1(d)). As reported in previous works [36][37][38], this resonant peak at around 460 nm can be used to monitor changes in the BM when it is liquid-infiltrated. The reflectance spectrum recorded for the BM deposited onto the EMF chip depicts a similar spectrum where the resonant peak was less well defined and appeared slightly shifted to shorter wavelengths (red line in Figure 1(d)). We attributed this slight loss of optical quality for the BM deposited onto the EMF device to the roughness of the driving electrode. Optical spectra of the black background and of a driving electrode of the EMF, also reported in Figure 1, were recorded to identify their possible contributions to the BM spectrum in case of misalignment of the incident beam. The high reflectivity of the BM-electrode zone in comparison with the black background and a driving electrode of the DMF device could be appreciated with the bare eyes in the dashed blue square of Figure 1(c) and was characterized by the spectrum presented in Figure 1(d). Figure 2 presents the optofluidic characterization of the BM-electrode when delivering the liquids directly onto the BM with a pipette. Dripping liquids onto this zone should fill the pores of the BM and, according to previous analysis, would induce changes in the optical response of the BM transducer [36][37][38], Effectively, Figure 2(a) shows that the infiltration with different liquids led to a shift of the optical spectra to larger wavelengths (i.e., a redshift), that in the case of mineral oil reached up to 21 nm. The representation of the magnitude of these shifts as a function of the refractive index of the infiltrating liquid in Figure 2(b) shows a clear dependence that can be used to differentiate a specific liquid from others with different refractive index. The plot shows that below a RI of 1.38 the accuracy of the sensor decreases preventing to discriminate liquids below this point.

Liquid movement in the EMF-device
Isopropanol (mainly driven by EWOD) and silicone oil (mainly driven by DEP) were used to determine the working parameters of the EMF device to move liquid droplets from one driving electrode to the other on the chip. Droplets of these liquids were moved by electrical actuation and the displacement from one electrode to another took around 0.2 s. A square wave signal of voltage 360 V pp and a frequency of 1 kHz were supplied to move the liquid droplets, as well as to split the liquid into small droplets of volume  0.2 μl. A succession of pictures showing the formation of specific silicone oil patches in the EMF-device is presented in Figure 3.

Optofluidic response of the BM-electrode infiltrated with silicone oil by dielectrophoretic forces
Main objective of this work was to investigate the ability of liquid droplet manipulation on an EMF device incorporated with a liquid transducer. To that end, a big silicone oil droplet was split into smaller droplets and one of them was delivered to the BM-electrode by means of the DEP forces by electrical actuation on the device. Figure 4 is the optofluidic response of the BM-electrode showing that the resonant peak of the BM experienced the same redshift (i.e., 15 nm) as that found when dripping the silicone oil by a pipette (cf Figure 2). This evidence proved the successful integration of an optofluidic BM transducer onto the EMF device and the successful driving of liquid droplets by DEP actuation. These results showed that the porous BM has worked acceptably well in combination with the EMF optofluidic device and we attributed the lesser shift observed in comparison with the reference BM to some microstructural changes induced by the growth of the porous thin films on the Teflon coating covering the ITO layer of the EMF device.

Conclusions
In this work, an optofluidic device combining a porous thin film photonic structure on an EWOD/DEP-based EMF device is presented for the first time. For this aim, porous BMs have been successfully deposited onto one driving electrode of an EMF device. The thin film deposition process did not affect the electromicrofluidic features of the EMF device, an outstanding result that proves that optofluidic transducers and EMF systems can be integrated in a unique device. The optofluidic transducer preserved its performance when incorporated onto  the EMF device, as proved by manually delivering the liquid onto the sensing region and by electrical driving the liquid analyte droplet to the zone containing the BM. An additional advantage of integrating the BM into an EMF device is the lower volume needed for analysis in comparison with the amount required when using other rudimentary sample delivery systems.