An optofluidic diffusion sensor using laser-induced dielectrophoresis in a device with a sputtered a-Si:H layer is presented. Diffusion sensors enabling high-speed measurement have important potential uses as bio-sensors and for quantitative analysis of nano-sized products. The present sensor was developed for measurement in a few seconds by optic observations of the sample diffusion from transient grating formed by laser-induced dielectrophoresis. As a photoconductive layer for the proposed device, we used a sputtered a-Si:H film. The optical (refractive index and extinction coefficient), structural (Raman and IR spectroscopy), and optoelectronic properties of this film, as well as its applicability to the proposed device are characterized. Nano-sized beads were measured by the fabricated device, and its performance as a diffusion sensor was validated.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Diffusion coefficients, which characterize mass transport phenomena, quantify an important thermophysical property for analyzing the size of nano- and micro-sized samples in solutions, and also for investigating their conformational characteristics interacting with the surroundings. One of the fields that require a diffusion sensing technique is the size-based evaluation of nano-sized biosample. The aggregations of the therapeutic protein products are associated with immunogenicity, and their long-term stability should be evaluated, which ranges from a few nm to several µm [1–3]. Extracellular vesicles, also called exosomes (tens of nm) and microvesicles (several hundreds of nm), act as the cargo in the cell–cell communication. Their size and diffusion property are analyzed for the understanding of intercellular transportation, and studied for the use of biomarkers for cancer with which their size change is associated . The diagnosis of diseases is also an important application, as these devices are highly sensitive to immune responses . Poulsen et al.  performed an assay for detecting the autoantibodies against double-stranded DNA, which is characteristic to patients with systemic lupus erythematosus. The assay requires less than one hour using diffusion sensing, while the current manual procedures, such as ELISA require several hours to complete. The sensitivity to conformation may provide important information about the complex nanoscale conformational changes of biomolecules [6–8], which is also associated with neurodegenerative diseases, such as Parkinson’s disease, resulting from abnormal conformations and aggregations of proteins [9,10]. Beyond these clinical applications, it is important to understand the effect of media characteristics (temperature, pH, etc.) on the diffusion properties dominating mass transport phenomena, which are ubiquitous in the human body. Diffusion sensing, requiring only a small amount of sample, is important for investigating the dynamics of a biosample; in addition, a method that can be performed using a portable compact device in a short time is desired for point-of-care testing (POCT), because it could help realize a diagnostic modality free from spatial restrictions.
Hence, various methodologies to measure the diffusion coefficients of nano- and micro-sized biosamples have been developed, such as the Taylor dispersion analysis (TDA) [3,5], dynamic light scattering (DLS) [11,12], a shear cell method with a phase-shifting interferometry , nanoparticle tracking analysis (NTA) [2,12,14], transient grating (TG) [7,8], fluorescence recovery after photobleaching (FRAP) , and fluorescence correlation spectroscopy (FCS) . Each method has its merits and drawbacks; an ideal diffusion sensing method does not exist, and combinations of several methods are currently the best option for analysis [1,12].
The TG method enables time-resolved diffusion measurement using a lattice-shaped concentration distribution formed by photochemical reactions. The high-speed observation of conformational changes and the intermediate species of the protein was reported [7,8]. FCS and FRAP are also used to investigate the protein dynamics in the sub-ms and ms range, respectively [6,10]. However, these methods are only applicable to samples with reactivity to photo-excitation or fluorescence; otherwise the sample must be prepared in advance [6–8,10]. DLS [11,12] and NTA [2,12,14] are conventional widespread methods because of their simple and pretreatment-free sensing processes using several hundreds of µL samples; thus, many measurements have been obtained using these methods. However, these methods are not applicable to the high-speed conformational event because of the time resolution that is completed within several tens of seconds. The TDA [3,5] also realizes preparation-free, straightforward measurements in several tens of minutes with only nL-scale volumes of sample; however, the TDA is not sensitive to conformational events in a short time. The shear cell method with phase-shifting interferometry realizes precise measurements, but requires longer than a few seconds to perform . As described above, many diffusion-sensing techniques have been developed, but a method applicable to a compact device as small as several cm that can perform sensing in a few seconds with as little as several nL of sample has not been realized.
Because of the demand for a high-speed compact sensor with low sample consumption that is free from additives, the micro optical diffusion sensor (MODS) has been developed. Diffusion coefficients can be measured by observing high-speed mass transport phenomena in a micro-scale lattice-shaped concentration distribution. In our method, to form a concentration distribution in our microchannel, a laser-induced dielectrophoresis (LIDEP) using an interference pattern of two laser beams, which is an optoelectronic manipulation technique, is utilized. V-shaped MEMS mirrors, also called micro Fresnel mirrors (MFM), have also been proposed for the formation of the interference pattern in a miniaturized device, and the validity of the proposed measurement method has been demonstrated using the MFM [15–18].
LIDEP is an optoelectronic manipulation technique for nano-sized particles using dielectrophoresis (DEP) force based on optoelectronic tweezers (OET) [19–28]. This technique enables the dynamic and parallel manipulations of microbeads [19–28], cells [19,20,25,26], nanoparticles [15–17,23,27], and DNA [21,23]. Most of the photoconductive layers of these devices consist of an a-Si:H layer deposited by a plasma-enhanced chemical vapor deposition system [21–23,27]. Researchers have shown that the photoconductive layer can be formed differently, as in the case of a-Si:H deposited by an inductively coupled plasma chemical vapor deposition system . Different photoconductive materials have also been used, such as bulk-heterojunction polymer , and titanium oxide phathalocyanine (TiOPC) . In addition to the aforementioned chemical vapor deposition systems, a reactive RF magnetron sputtering method has been used in depositing the a-Si:H [29–34] owing to its low cost and simplicity, although the sputtered films exhibited inferior properties .
Herein, a LIDEP device for diffusion sensing was designed and fabricated using a sputtered photoconductive layer. The optoelectronic manipulation performance of the sputtered photoconductive layer by using the LIDEP had to be evaluated based on the optoelectronic properties; therefore, ellipsometry and photoconductivity measurements were performed. The structural properties associated with the optoelectronic properties had to be evaluated as well; thus, Raman spectroscopy and FT-IR were performed. For the biosample measurement, the measurement in a buffer solution that maintains the viability of the biosample for a longer period is preferable; however, LIDEP is affected by the medium conductivity and the impedance properties [22,26,35]. The applicability of the fabricated device to the sample with various conductivity was investigated.
To confirm the performance of the fabricated device, size-certified nanoparticles (ϕ203 nm), whose diffusion coefficient can be estimated, were measured as a counterpart of the biosamples, such as microvesicles and proteins under aggregation. The performance of the proposed device for diffusion sensing was experimentally confirmed using nanoparticles dispersed in solution.
2. Measurement principle
A schematic diagram of the device structure and setup is shown in Fig. 1. The sensing chip consists of a photoconductive layer of a-Si:H on an ITO-deposited quartz substrate.
The liquid containing the sample of interest is sandwiched between the lower device and the top piece of the ITO-deposited substrate. For the formation of a micrometer-scale lattice-shaped concentration distribution, an AC bias and an interference pattern of two laser beams with a fringe-shaped intensity profile (fringe spacing Λ) are provided. Upon illumination, the amorphous silicon layer’s conductivity increases by many orders of magnitude. Consequently, a localized electric field occurred and a lattice-shaped concentration distribution C(x) = ΔC cos(2πx/Λ) + C0 (C0: initial concentration, ΔC: concentration amplitude) is formed by the DEP force,36,37].
After the formation of the lattice-shaped concentration distribution, the AC bias and the excitation laser are turned off; simultaneously, the probing laser is turned on. The lattice-shaped concentration distribution act as a diffraction grating, and diffracted light is generated.
After turning off the AC bias and the excitation laser, the concentration distribution decays exponentially with the decay time constant τD, and is expressed as38]:
As light sources, a green excitation laser (λ = 532 nm) and a red probing laser (λ = 638 nm) were utilized to increase the conductivity of the photoconductive layer, and to generate the diffracted light, respectively. The formation of the microscale fringe-shaped concentration distribution (Λ~6 μm) using nano-sized particles (D~10−12 m2/s) can yield a decay time constant shorter than 1 s.
3. Deposition and evaluation of the photoconductive layer
3.1 Deposition of the photoconductive layer
Herein, because the substrate temperature is one of the parameters determining the structural inhomogeneities of an amorphous silicon network in the films , the deposition temperature dependence (room temperature (r.t.), 200 °C, 300 °C) of the a-Si:H layer was investigated. The a-Si:H was deposited using a sputtering system; further, the properties of the deposited film and its applicability to the proposed sensor were evaluated. The deposition was performed with the reactive RF magnetron sputtering system (CFS-4EP-LL [i-miller]; Shibaura Mechatronics, Japan). The depositing conditions are described in Table 1. During the 1-h deposition the substrate temperature was set at r.t., 200 °C, and 300 °C, respectively. The substrates for the sputtered films were 0.525-mm-thick synthetic quartz substrates (Shin-Etsu Chemical, Japan) for the Raman spectroscopy and photoconductivity measurements, and 0.38-mm-thick one-side polishing crystalline Si substrates (KST World, Japan) for the ellipsometry and FT-IR measurements.
3.2 Structural properties by Raman spectroscopy
The structural properties can be investigated by Raman spectroscopy. The broad band centered at 480 cm−1 is caused by the disordered phase (i.e., amorphous network and grain boundaries). A peak at 518 cm−1 would be attributed to a crystalline silicon . The Raman scattering experiments were performed with a confocal Raman microscope (inVia Reflex; Renishaw, UK) using the 532-nm line of a YAG laser for excitation, and the results are shown in Fig. 2. From these spectra no peak at 518 cm−1 was observed; thus, the deposited films were not crystalline but amorphous silicon. The Raman spectra are sensitive to differences in structural properties, and these spectra can be decomposed into four peaks, corresponding to TA (Transverse Acoustical, 150 cm−1), LA (Longitudinal Acoustical, 310 cm−1), LO (Longitudinal Optical, 400 cm−1), and TO (Transverse Optical, 480 cm−1). The ratio of the integrated peak intensity of the TA mode to that of the TO mode (ITA/ITO) can be regarded as the degree of the short-range order [30,32,34], but no obvious differences were observed.
3.3 Optical properties by ellipsometry
The optical constants and thickness were obtained by ellipsometry. The analyzed optical constants (n, k), absorption coefficient, and thickness are shown in Fig. 3. As shown, by increasing the substrate temperature, these values were increased, but no obvious difference in the deposition rate was observed. From the absorption spectra as shown in Fig. 3(c), light sources with shorter wavelength can be used to increase the film conductivity; meanwhile, those with longer wavelengths can be used for transmission observation through the film.
3.4 IR absorption properties sensitive to bonding configuration by FT-IR
IR absorption spectra were obtained by FT-IR to investigate the bonding configuration between hydrogen and silicon atoms (Si-Hx), and are shown in Fig. 4. Strong absorbance peaks were observed at 640 cm−1, and between 2000 and 2100 cm−1. The peak at 640 cm−1 represents Si-H bending and Si-H2 wagging modes, while the stretching broad absorption band between 2000 and 2100 cm−1 can be deconvoluted into two satellite peaks: one at 2000 cm−1, associated with monohydride (Si-H) bonds, and one at 2090 cm−1, allotted to polyhydride (Si-H2, Si-H3, …) bonds . Peaks at 640 cm−1 and 2090 cm−1 were observed in the spectra of all samples; however, the peak around 2000 cm−1 was not observed in the room-temperature spectrum. It was instead observed in the spectra at 200 °C and 300 °C. Therefore, the deposition at higher temperatures results in the formation of monohydrides; meanwhile, that at room temperature would result in the formation of polyhydrides. This result agrees with the finding of a previous report, in that increasing the temperature decreased the polyhydride content and increased the monohydride .
3.5 Photoconductivity evaluation
Co-planar-type electrodes were formed on the sputtered film to measure the conductivity under the laser illumination. The electrodes were patterned by the vacuum evaporation (BHCU-8P-40; Shibaura Mechatronics, Japan) of Au through an Al stencil mask. The width and the length of the electrode gap were 500 µm and 50 µm, respectively. The thickness of the a-Si:H layer obtained by ellipsometry was used to calculate the electrical conductivity from the resistance, which was measured by providing a voltage using an electrometer (6517A; Keithley Instruments, USA). The optical illumination was provided by a 532-nm optically pumped semiconductor laser (Sapphire 532 SF; Coherent, USA), and the irradiation diameter was ϕ1.4 mm.
Electric current–voltage (I–V) curves were recorded at various illumination intensities as shown in Fig. 5(a) and 5(b). The linearity of the I–V curve represents the correct electrical connection between the electrode and the photoconductive film. An obvious difference in the sensitivity to the light illumination was not observed, but the films deposited at r.t. and 300 °C exhibited lower electrical conductivities than that deposited at 200 °C. Regarding this non-monotonic increase, Moustakas et al.  found a transition around 250 °C in the network organization, and in the relation between the electrical conductivity and the substrate temperature.
The high sensitivity of the electrical conductivity to light illumination is required to induce a strong DEP force. In addition, in terms of its applicability to biosamples, the photoconductive layer with a higher conductivity is preferable from the viewpoint of impedance properties. Hence, the device was designed using a-Si:H deposited at 200 °C.
4. Device characterization and fabrication
4.1 Numerical simulation for device design
Based on the results of the photoconductivity evaluation, a numerical simulation was performed using a commercial finite element method (FEM) package (COMSOL Multiphysics 4.2a; Sweden) to estimate the DEP force in the device, and to characterize the thickness of the photoconductive layer. The simulation model is illustrated in Fig. 6.
The conductivity distribution in the photoconductive film is assumed to follow the power approximate expression to the light intensity based on the measurement result of the conductivity described in Fig. 5(b) (written as σ = AIB, where σ is the electrical conductivity of the film, I is the optical intensity, and A and B are the fitting parameters). The intensity distribution of the interference pattern of the two laser beams is expressed by
An electric-field-gradient profile with this conductivity distribution was subsequently extracted via FEM, and the DEP force was calculated. In this study, the simulations were performed at within the medium conductivities of 5 × 10−3 S/m and 1 S/m, which correspond to the sample dissolved in DI water and 1x buffer solution, respectively. The thickness of the photoconductive film was also varied from 100 nm to 1 µm. All subsequent simulations assumed that the relative permittivities of the medium, the polystyrene particle, and the a-Si:H were 80, 2.56, and 11 [20,22], respectively. In addition, the sample was ϕ200-nm polystyrene spherical beads with a plain surface (the surface conductivity Ks = 1.65 nS ), and the thickness of the microchannel was 20 µm. As an excitation condition, a 1-mW laser was focused on a 400-µm spot size with 6-µm fringe spacing, and 10 Vpp at 10 kHz was supplied.
Figure 7 shows the simulated x component of the gradient distribution of the electric field around the beam center in a device using 100-nm a-Si:H. The estimated maximum value of FDEP in the solution with conductivity ranging from 5 × 10−3 S/m to 1 S/m is illustrated in Fig. 8, and the utilized Re[K*(ω)] values at 10 kHz under each condition are represented in the inset of Fig. 8. As the conductivity increases, the induced FDEP decreases, because the voltage drop in the sample layer decreases, resulting in a decrease in E2; in addition, Re[K*(ω)] decreases from a positive value to a negative value, and the absolute value of FDEP assumes a minimum value. These values are compared to the minimum force required to overcome the effect of diffusion and friction. One can define the observable threshold force [35,40] as35], and that of ϕ200-nm beads under r.t. can be estimated as 2.1 × 10−12 m2/s; thus, assuming that the induction time of the DEP force is 1 s, F0 is calculated as 3.9 × 10−15 N. These simulation results and the F0 shown in Fig. 8 suggest that as the medium conductivity increases, the DEP force decreases; in addition, the device with a thinner photoconductive layer sustains a relatively stronger DEP force. The device was fabricated using 100-nm a-Si:H, which can be perform the optoelectronic manipulation in the broad conductivity range.
4.2 Device fabrication
The fabrication process is described in Fig. 9. A transparent ITO (200 nm) electrode was deposited on a synthetic quartz substrate (Shin-Etsu Chemical, Japan) using the reactive RF magnetron sputtering system (CFS-4EP-LL [i-miller]; Shibaura Mechatronics, Japan); a photoconductive a-Si:H thin film (100 nm) was deposited on top of it by the same sputtering system. The depositing conditions are described in Table 1 (the substrate temperature was 200 °C); the deposition time was adjusted based on the deposition rate. Photolithography with a negative photoresist (SU-8 3025; MicroChem, USA) was performed to form a microchannel (20 μm). On another synthetic quartz substrate, through-holes as inlets and outlets were formed by water jet processing (Abrasive jet Cutter Varuna; Sugino, Japan); subsequently, ITO was deposited. These wafers were diced into small chips (7.5 mm × 15 mm) using a dicing saw (DAD-522; Disco, Japan), and thermally bonded on a hotplate (190 °C). The measurement sample was injected into the microchannel of the fabricated device.
5. Experiments using the proposed diffusion sensor
5.1 Experimental setups
The experimental setups and the timing chart are illustrated in Fig. 10(a) and 10(b), respectively. As an excitation laser to increase the conductivity of photoconductive layer, we used a 532-nm optically pumped semiconductor laser (Sapphire 532 SF; Coherent, USA). The excitation laser was split by a non-polarized beam splitter and focused by lens 1 (L1) to form a sinusoidal interference pattern on the photoconductive film. The probing laser was a diode laser (OBIS 637LX; Coherent, USA), the beam width was expanded by L2 and L3, and focused by L4 on the same area as the excitation laser through a dichroic mirror (FF552-Di02-32x44-FX; Semrock, USA) to generate the diffracted light, which was subsequently collimated by L5. The iris obstructs the transmitted (0th-order) light but allows the first-order diffracted light pass to the photodetector (2001-FS-M; Newport, USA) through a bandpass filter (FF01-637/7-25; Semrock, USA).
The trigger signal to isolate the excitation timing and that of probing were generated by FG1 (WF1946B, NF, Japan). When the trigger for the DEP excitation (CH2) is high, the optical beam shutter system (shutter 1, SH05 & SC10; Thorlabs, USA) on the excitation laser line is opened, and FG2 (AFG 3252; Tektronix, USA) to apply an AC bias is operated to induce a DEP force. Soon after shutting the AC bias and the excitation laser off, shutter 2 (the same product as shutter1) is opened for the observation of the diffusion phenomenon. The detected signal is recorded by a digital phosphor oscilloscope (MDO3014; Tektronix, USA) and analyzed using a personal computer, which controls the measurement equipment. The experimental conditions are described in Table 2.
5.2 Experimental results
As a solution containing the measurement sample, NISTTM traceable-size standard beads (certified mean diameter: 203 nm ± 5 nm, k = 2) dispersed in water (3200A; Thermo Fisher Scientific, USA) were used. To validate the adequacy of the buffered solution, non-buffered sample (beads + DI water, 6 × 10−3 S/m) and buffered sample (beads + DI water + phosphate buffer saline (166-23555; Wako Pure Chemical Industries, Japan), 3 × 10−2 S/m) were prepared. The solution concentration prepared was 0.1 vol%. The medium conductivity was measured using a conductivity meter (B-173; Horiba, Japan).
A detected single-shot signal using the non-buffered sample (6 × 10−3 S/m) under 1-s excitation, and one using the buffered sample (3 × 10−2 S/m) under 10-s excitation are shown in Fig. 11(a) and 11(b), respectively. Both signals exhibited a generation of the first order diffracted light, and the formation of the lattice-shaped concentration distribution by LIDEP was confirmed. In comparison to the detected signal using the non-buffered sample, lower signal-to-noise ratio of the buffered sample was observed.
The insets show the semilogarithmic plot of each signal. As shown in the inset of Fig. 11(a), the exponential decay is observed; thus, the mass diffusion phenomenon described in Eq. (5) is confirmed. The deviation between each detected signal and the exponential curve fitting using the non-buffered sample and the buffered sample are also shown in the lower part of Fig. 11(a) and 11(b), respectively. There is a small deviation in the case of the non-buffered sample; meanwhile, a large deviation is observed in the case of the buffered sample. The large deviation in Fig. 11(b) can be attributed to the small FDEP due to the small voltage drop in the sample layer and the small absolute value of Re[K*(ω)] at this solution conductivity. Another obstructing effect specific to the high-conductivity solution, such as the electrothermal effect, is also suspected, because these effects become stronger as the solution conductivity increases [22,26,35].
Owing to the large deviation shown in Fig. 11(b), the signals using the buffered sample (3 × 10−2 S/m) were insufficient to calculate the diffusion coefficient. The averaged value and the standard deviation (N = 8) of the measured diffusion coefficient using the non-buffered sample (6 × 10−3 S/m) are listed in Table 3. Those of the certified diameter (ϕ203 nm) at 20 °C can be calculated based on the Stokes-Einstein equation, and is listed in Table 3. There was a 3.3% deviation between the measured value and the calculated value. The deviation could be from the unconsidered element in the calculation using the Stokes-Einstein equation, and the difference between the hydrodynamic diameter and the certified diameter. The deviation in the temperature-dependent parameters is also suspected because of the lack of precise temperature control in the present measurement system.
We conclude that the proposed optofluidic device performed as designed; moreover, the performance of the fabricated device as a diffusion sensor for a nano-sized sample was experimentally demonstrated.
We have designed an optofluidic diffusion sensor using a LIDEP composed of a sputtered photoconductive layer of a-Si:H. In this work, the designed sensing device was fabricated and tested. The signal detection using the fabricated device proved that the a-Si:H layer deposited using a reactive RF magnetron sputtering system is a viable photoconductive layer for a LIDEP device. The concentration distribution of nano-sized samples formed by LIDEP was confirmed using both of the non-buffered sample (ϕ203 nm at 6 × 10−3 S/m) and buffered sample (ϕ203 nm at 3 × 10−2 S/m). Moreover, the analysis of the detected signals using non-buffered sample (ϕ203 nm at 6 × 10−3 S/m) showed good agreement with the sample specification, confirming the utility of the device as a diffusion sensor for nano-sized samples.
In our future study, measurements using biosamples, such as microvesicles and proteins under aggregations as small as the beads utilized in this paper are planned. The time-resolved measurement of these nano-sized biosamples will provide significant information to understand the nanoscale dynamics. Further development of the proposed device will realize lab-on-a-chip devices that will enable the multi-sample, sequential sensing device to be integrated with a sorting system using LIDEP.
JSPS KAKENHI Grant Numbers JP18H01388, and JP17J04445; Keio Leading-edge Laboratory of Science and Technology (KLL) specified research projects.
The fabrication was performed in the clean room at the Global Nano Micro Technology Business Incubation Center (NANOBIC), Kawasaki, Japan with support from the Academic Consortium for Nano and Micro Fabrication of Four Universities (Keio University, Waseda University, Tokyo Institute of Technology, and the University of Tokyo).
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