CA2963807A1 - Microwave enabled portable, label-free, high-throughput detection and content sensing system for lab on a chip platforms - Google Patents

Abstract

This study reports a microwave-microfluidics integrated approach capable of performing droplet detection at high-throughput as well as content sensing of individual droplets without chemical or physical intrusion. The sensing system consists of a custom microwave circuitry and a spiral-shaped microwave resonator that is integrated with microfluidic chips where droplets are generated. The microwave circuitry is very cost effective by using off-the-shelf components only. It eliminates the need for bulky benchtop equipment, and provides a compact, rapid and sensitive tool compatible for Lab-on-a-Chip (LOC) platforms. To evaluate the resonator's sensing capability, it was first applied to differentiate between single-phase fluids which are aqueous solutions with different concentrations of glucose and potassium chloride respectively by measuring its reflection coefficient as a function of frequency. The minimum concentration assessed was 0.001 g ml-1 for potassium chloride and 0.01 g ml-1 for glucose. In the droplet detection experiments, it is demonstrated that the microwave sensor is able to detect droplets generated at as high throughput as 3.33 kHz. Around two million droplets were counted over a period of ten minutes without any missing. For droplet sensing experiments, pairs of droplets that were encapsulated with biological materials were generated alternatively in a double T-junction configuration and clearly identified by the microwave sensor. The sensed biological materials include fetal bovine serum, penicillin antibiotic mixture, milk (2% mf) and D-(+)-glucose. This system has significant advantages over optical detection methods in terms of its cost, size and compatibility with LOC settings and also presents significant improvements over other electrical-based detection techniques in terms of its sensitivity and throughput.

Description

70 Description 71 Title 72 Microwave Enabled Portable, Label-free, High-throughput Detection and Content Sensing System 73 for Lab on a Chip Platforms 74 Field of the Invention 75 This invention lies in the field of microwave and microfluidic engineering.
76 In recent years, there has been growing interest in droplet-based microfluidics because of its promise 77 to facilitate a broad range of scientific research and biological/chemical processes. Potential 78 applications can be found in many areas such as cell ana1ysis,1-4 DNA
hybridization,5detection of 79 bioassays, 6 bio-reactions,7-9 drug screenine and diagnostics. 11,12 Major advantages of droplet-80 based microfluidics versus traditional bioassays include its capability to provide highly uniformed, 81 well isolated environment for reactions with orders of magnitude higher throughput (i.e. kHz). Most 82 droplet-based microfluidic studies rely on high speed imagine-17to provide details of droplet 83 generation and transport, which usually require expensive and bulky high speed camera, and 84 exhaustive post imaging analysis. In addition, in order to differentiate subtle differences in droplet 85 content, fluorescent imaging is often used which, however, tends to lower down the throughput 86 because longer residence time is needed for the droplet to stay in the field of view in order to obtain 87 sufficiently high fluorescent intensity. Although this can be improved by using a pulse solid state 88 laser that is synchronized wiih the camera, which further complicates the system due to the need for 89 precise alignment and fluorescent labelling.
90 In contrast, electrical techniques allow the miniaturization of multiple sensor arrays and their 91 integration into one single microfluidic chip with low power requirement. Of these capacitive, 92 electrochemical and impedance based electrical detection methods are widely available. In 93 electrochemical detection, the measurements are based on the interactions between analytes and 94 electrodes or probes that usually occur in an electrolytic cell. They are not able to distinguish 95 analytes that are not electroactive.18-2 In addition, the detection electrodes are sensitive to variations 96 in temperature, ionic concentration and pH that affect the shelf life of the sensor and shift electrodes' 97 response requiring frequent calibration.18,21,22 Conventional capacitive and impedance detection 98 approaches operate at low frequencies, which causes either low signal-to-noise ratio or long 99 response time and thus limit their applications to droplet microfluidics where droplets are generated 100 at high frequencies. For example, the throughput achieved by a capacitive sensor23 for droplet 101 detection was up to 90 Hz with reasonable sensitivity and for an electrical impedance-based 102 detection' around 10.
103 Microwave technology, as a versatile non-optical method, has the potential to address the above 104 issues because it eliminates the need for chemical modification or physical intrusion of the sample 105 and operates at high frequencies (i.e. GHz). It differentiates materials based on their electrical 106 properties including e1ectric41 conductivity and/or dielectric constant. Previously we demonstrated a 107 microwave sensor that can be integrated with microfluidic devices to differentiate single phase 108 fluids in microchannels and detect the presence of droplets at a very low frequency (i.e. up to 1.25 109 Hz).25 The low detection frequency was mainly restricted by the response time of the vector network 110 analyzer (VNA). In addition, the sensing of droplet content was not achieved because insufficient 111 sampling of droplets did not allow the accurate determination of the time for the droplet to arrive at 112 the capacitive gap, neither differentiation of the content changes.25 Also, in order to get a reliable 113 reading by the microwave sensor, the effect of droplet geometry on sensing performance must be 114 eliminated, and the sensitivity of the microwave sensor must be sufficiently high to detect subtle 115 variations.
116 In this study, we present a sensitive, low-cost, portable microwave circuitry suitable for detection of 117 droplet presence and label-free sensing of individual droplet content in microfluidic devices. More 118 importantly, for future point-of-care application purposes, we limited ourselves to the choices of 119 cost-effective off-the-shelf components for developing the circuitry.
Basically the circuitry that 120 consists of surface mount components is able to generate microwave signal and measure the 121 response of the sensor (reflection coefficient of the sensor) in a very fast manner. We validated that 122 the system has a detection limit of several kilohertz (kHz) itself, and in the experiments we reached 123 over 3 kHz. This microfluidic microwave system might potentially be used as a coulter counter and 124 content analysis in many applications.

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205 Description of Prior Art 206 There have been the development of inventions regarding microwave detection and microfluidics 207 multiphase flows.
208 Patent US 2005/0191708 Al Microwave Microfluidics 209 Patent US 6605454 B2 Microfluidic Devices with Monolithic Microwave Integrated Circuits 210 Patent US 2009/0236330 Al Microwave Heating of Aqueous Samples on a Micro-Optical-Electro-211 Mechanical System 212 Patent US 2008/0277387 Al Use of Microwaves for Thermal and Non-Thermal Applications in 213 Micro and Nanoscale Devices 214 Patent US 2010/0089907 Al Instantaneous In-Line Heating of Samples on a Monolithic Microwave 215 Integrated Circuit Microfluidic Device =

219 Summary of the Invention 221 System overview 222 The system illustrated in Figure 1 consists of a microfluidic chip integrated with a microwave 223 sensor, a pumping unit which could be a pressure controller (Fluigent MFCS-8C) or a syringe pump 224 (Pump33, Harvard Apparatus) depending on the particular study case, an inverted microscope 225 (Eclipse Ti, Nikon) mounted with a high-speed camera (Phantom v210, Vision Research) and the 226 developed microwave custom circuitry. Fluid reservoirs are connected to the microfluidic chip via 227 ethyltrifluoroethylene (ETFE) tubing and connectors (Tefzel, Upchurch Scientific). Two slightly 228 modified configurations (simple flow focusing and double T-junction) were used for droplet 229 generation. For the detection of droplet presence, the simple flow focusing geometry was used while 230 for the sensing of droplet content, the double T-junction geometry was used where droplets with 231 different contents were alternatively generated by the two T-junctions.
Droplet generation and 232 transport were manipulated through the microfluidic channel network design by adjusting the 233 pressures of the inlets or the pumping flow rate of the syringe pump.
The high speed camera was 234 used to record microscopic images and videos through the image processing program ImageJ
235 (National Institute of Health, MD, USA) which were also used to validate the experimental results 236 obtained through the developed circuitry. A data acquisition device and Labview software (National 237 Instruments) were used to control the system and set off the computer interface.
238 Materials 239 Fluorinated oil (FC40 from Sigma-Aldrich) with a 2% custom made surfactant was used as the 240 continuous phase. The surfactant has a chemical structure of PFPE-PEG-PFPE (or Krytox-241 Jeffamine¨Krytox, where Krytox has a molecular weight of 7500 and Jeffamine 900). D-(+)-242 Glucose (Sigma- Aldrich) arid potassium chloride (EMD Millipore) solutions were prepared in ultra-243 pure water. Penicillin¨streptomycin¨ neomycin antibiotic mixture (containing 5000 units penicillin, 244 5 mg streptomycin and 10 mg neomycin per mL), Fetal Bovine Serum (FBS;
Sigma-Aldrich) and 245 milk (contains 2% MF) were used in the sensing of droplet content without further purification or 246 dilution. For further demonstration of the developed system for potential bioapplications, 247 Alzheimer's disease (AD) testing was chosen. Tau-derived hexapeptide (AcPHF6) which is used to 248 model the tau-protein aggregation related to AD was purchased from Celtek Peptides. The assay was 249 carried out with orange G, which is a known inhibitor to the tau-protein aggregation (Sigma-250 Aldrich). AcPHF6 was prepared in ultrapure water at a concentration of

2.5 mg m1-1 as stock 251 solution, and diluted to a final concentration of 0.316 mM. All other solutions were prepared in 252 morpholinepropanesulfonic acid (MOPS) buffer with 0.01% NaN3 and adjusted to pH 7.2, and with 253 assay grade DMSO at 1% (v/v).

255 Microwave Sensor 256 The designed microwave sensor works essentially as a resonator. The sensor structure is made of 257 two concentric copper loops similar to the one presented previously25.
Microwave signal is excited 258 by the outer coplanar transmission line loop, which supplies a time-varying oscillating current 259 circulating around the loop and a magnetic field passing through the loop. The inner loop with a 260 small gap constructs the resonator and the microchannel where droplets are passing through is 261 aligned on top of this gap. When materials with different electrical properties (permittivity, 262 conductivity) pass by the gap region, the capacitance of the gap changes and the resonance 263 frequency shifts which can be used to characterize the materials. Take water-in-oil emulsion as an 264 example, water droplets have a much higher dielectric constant (-80) than the carrier fluid, oil (-2-265 3). When a water droplet passes by the resonator, the resonance frequency will be shifted which can 266 be used to detect droplet's presence. Similarly, when droplets with different materials pass by the 267 resonator, the magnitude of shift in the resonance frequency can be used to characterize the droplet 268 content. The resonance frequency shift caused by a perturbation in the permittivity of the medium is 269 described by26.
At"¨ f AEit Eodv 270 (1) f f(Eg.Eo + pH. H o)dv 272 where E0 and E are the electric fields before and after the perturbation, HO and H are the magnetic 273 fields before and after the perturbation, f is the resonance frequency before the perturbation, E is the 274 permittivity of the medium and itt is the permeability of the medium.
In this study, a spiral-shaped 275 capacitive region is designed for sensing purposes because it allows the system to operate at lower 276 frequencies compared to T-shaped designs25, which thus allows inexpensive off-the-shelf 277 components to be chosen for the circuitry design.

279 Fabrication 280 The microfluidic chip consists of two main components, a glass base with the microwave 281 components and a polydimethylsiloxane (PDMS) mold with the designed microchannels for droplet 282 generation and transport, which are fabricated separately and then bonded together. Thus, the device 283 fabrication consists of two stages: microchannel and microwave component fabrication. Microwave 284 component. The electrical traces for the microwave components are fabricated using a combination 285 of photolithography and electroplating. Briefly, the positive photoresist, S1813 (Rohm-Haas), is 286 spin-coated at 1500 rpm for 60 s onto a 50 nm thick copper film (EMF
Corporation) that is pre-287 deposited on a glass slide and then baked at 95 C for 120 s. The design is patterned into the 288 photoresist via UV lithography and subsequently developed with MF-319 (Rohm- Haas) for 2 min.
289 The patterned slide is then immersed in an acidic copper electroplating solution (0.2 M CuSO4, 0.1 290 M H3B03, and 0.1 M H2504) and electroplated at 2 mA for 4 min and then 7 mA for 20 min. After 291 electroplating, the photoresist is removed with acetone leaving an electroplated copper film 292 approximately 5 jim thick. Next, the base layer of predeposited copper is removed by etching with 293 dilute ferric chloride (5%) (MG Chemicals). A passivation layer of a mixture of PDMS (Sylgard 294 184, Dow Corning) and toluene (1 : 1 (w/w) PDMS/toluene) is spin-coated at 4000 rpm for 60 s 295 followed by 1 h curing at 95 C to protect the electrical traces. A
subminiature version A (SMA) 296 connector (Tab Contact, Johnson Components) is then soldered to the electrodes of the microwave 297 components to provide an external connection to the microwave circuitry. Microchannel.
298 Microchannels are fabricated from PDMS using standard soft-lithography techniques. The PDMS is 299 mixed in a 10: 1 ratio of base to curing agent, degassed and molded against SU-8/silicon masters 300 which are fabricated using the same procedure developed previously23 and then cured at 95 C for 2 301 h. The molds are then peeled off from the masters and fluidic access holes are made using a 1.5 mm 302 biopsy punch. Both the finished microwave components and the PDMS mold are then treated with 303 oxygen plasma at 29.7 W, 500 mTorr for 30 s. The plasma treatment process renders PDMS
304 hydrophilic; however, for water in oil droplets, the PDMS channels should be hydrophobic to form 305 droplets. For this purpose, the walls of the microfluidic channels are coated with Aquapel (PPG
306 Industries) to ensure that they are preferentially wet by the fluorinated oil.

310 Microwave custom circuitry 311 Vector Network Analyzers (VNAs) are widespread tools for microwave characterization due to their 312 accuracy and user-friendly interface. However, VNAs are expensive normally which has driven the 313 development of inexpensive alternatives27-29. Regular VNAs have limitations on the data sampling 314 rate and thus throughput which only allowed a very low throughput (i.e.
up to 1.25 Hz for droplet 315 detection). Another major disadvantage of such bulky benchtop setups is their size which makes it 316 difficult to be widely applied for point-of-care applications. In this regard, it is necessary to develop 317 portable yet affordable microwave circuitries that have comparable accuracy and sensitivity as 318 commercially available VNAs. In this study, such a microwave circuitry for label-free detection and 319 content sensing of droplets in microfluidic devices is developed.
Considering the microwave 320 structure used in detecting and sensing droplets, a microwave circuit that measures the reflection 321 coefficient from a one port network is designed since the change in the resonance frequency can be 322 monitored in the reflection cocefficient. The microwave circuitry is mainly composed of three sub-323 systems: i) signal generator, ii) power coupling unit, and iii) gain detector as shown in Figure 2.

326 This subsystem consists of a voltage controlled oscillator (VCO) (Mini-Circuits, ROS-2350-519+), a 327 voltage regulator (Rohm Semiconductor, BA17805FP-E2), a data acquisition system (DAQ) 328 (National Instruments), an op-amp (Texas Instruments, LM358DR), and a power supply (24V
329 battery) that supplies voltage to the op-amp and voltage regulator. The VCO provides the required 330 microwave frequency by converting the input analog voltage, which consists of two components:
331 one voltage source provided by the 24V power supply but regulated by the voltage regulator to the 332 maximum of 5V and the other by the DAQ (0 - 10V) for tuning purposes.
Tuning voltages are 333 amplified by the op-amp with a gain factor of 2. The total amplified voltage ranging between 0 to 334 20V controls the tuning voltage of the VCO which is measured by the DAQ
and LabView program 335 and characterized by a spectr.um analyzer (Agilent, E4440A). Serial capacitors are used in order to 336 reduce the parasitic effects and filter the signal for the op-amp and VCO input. The microwave 337 signal generator subsystem facilitates the sweeping over the desired frequency range (1.9 GHz to 2.6 338 GHz). We designed the sensor operating below 3 GHz at which the corresponding microwave 339 components are widely available and inexpensive that allow the total cost of the electronic 340 components below $200. Wider frequency ranges can be achieved by adjusting the tuning voltage of 341 the VCO.

343 The primary function of the power coupling unit is to provide proper microwave signals to the 344 sensor and the gain detector which would require careful isolation of signals without sacrificing the 345 useful power. The high operating frequency at the GHz range brings in more challenges in the 346 design and fabrication of the microwave components. First, the impedance of the transmission lines 347 in the printed circuit board must match to the characteristic impedance of 50 1/ because any 348 mismatch between the transmission line traces causes reflections and reduces the performance27. For 349 this purpose, 0.79 mm thick FR-4 PCB material (c=4.34) is used and microstrip impedances are 350 carefully calculated considering the trace width, thickness and substrate height. The copper ground 351 (at the bottom of the PCB) and ground planes (on the top of the PCB) are connected with vias closer 352 to 1/20th X where X is the wavelength of the signal flowing through it to reduce noise'. In order to 353 minimize parasitic coupling to the transmission lines, separations between the ground planes and all 354 other traces are designed to be at least three times larger than the substrate thickness. Second, 355 reflections between different components must be controlled well which include the reflection from 356 the attenuator, VCO, and directional coupler, to the microwave sensor which is connected to the 357 circuitry through coaxial cables, and that from the microwave sensor back to the VCO which have 358 disturbing effects on robust frequency generation. The above concerns are taken into consideration 359 in the design of the power coupling and isolator subsystem.
Specifically, a high directivity bi-360 directional coupler (Mini-Circuits, SYBD-16-272HP+) with 16 dB coupling is used to regulate 361 microwave power to the resonator. Additionally, a 20 dB resistive attenuator is used as an isolator 362 network which is chosen to isolate the reflected signal because of the mismatch of the sensor while 363 not reducing the useful power significantly.

365 An integrated circuit (Analog Devices, AD8302) is employed in the gain detection subsystem, 366 which communicates with the microwave sensor and the power-coupling unit. The signal traveling 367 from the signal generator is coupled by the bi-directional coupler to the gain detector as a reference 368 signal and to the resonator, which is aligned with microchannel. Then the gain detector enables the 369 amplitude and phase difference between the signal reflected from the sensor and the reference signal 370 to be measured which is described by the reflection coefficient.

=

372 RL = ___________ Reflected Voltage (ZR ¨ Zo) (2) Incident Voltage (ZR + Zo) 374 where ZR is the frequency dependent input impedance of the device presented in Figure 1(b) that 375 includes the resonator and the excitation structure and Zo is the characteristic impedance of the 376 transmission line used for feeding the structure. The gain detector converts the microwave signals to 377 DC signal, and this magnitude ratio of electronic signal is post-processed and used to relate 378 nanoliter-sized droplet detection and sensing of its content. For example, if different materials pass 379 by the sensor, the reflected signal would be different even though the incident voltage would be the 380 same which can be used for detection and sensing of materials. The system is able to detect ac-381 coupled input signals from -60 dBm to 0 dBm. The output reflection coefficient range can be 382 accurately measured between -30 dB to +30 dB which is scaled to 30mV/dB. The system is also 383 able to measure the phase over a range of 0 -180 . The minimum and maximum levels of the 384 detection limits are characterized by the limit that each individual log amp can detect as well as the 385 finite directivity of the coupler.
386 The LabVIEW program is used as an interface to collect and convert the measurement data, and 387 control the system real time. Meanwhile, a calibration algorithm is used to correlate the measured 388 data readings of the gain detector to the reflection coefficient of the microwave sensor which carry 389 the information of the physical droplet system.

391 Results and Discussion 392 Prior to the droplet detection and sensing using the developed microwave circuitry, its sensitivity 393 and accuracy is first evaluated by comparing its measurement results with that obtained using a 394 commercial VNA (MS2028C, Anritsu). Table 1 below shows the comparison of the measured 395 resonance frequencies for FC-40 (c=1.9), air (8=1), and water (c=78.54) between the custom-made 396 circuitry and VNA. The developed microwave circuitry has very similar performance to the 397 commercial VNA with the maximum difference of 1.283% found for water.

400 Table 1 Comparison of the resonance frequencies between custom microwave design and the VNA.
Material (Liquid) f@Sii min using f@Sii min using Variation Percentage VNA (MHz) Custom Design (%) (MHz) Air 2588 2580 0.309 FC-40 2582 2573 0.349 Water 2417 2386 1.283 402 Then the detection and counting function of the developed circuitry was thoroughly evaluated by 403 measuring the reflection coefficient of the resonator for various fluids in microchannels as a 404 function of frequency. In order to prevent potential contamination caused by the residual of the 405 previous sample; the microchannels were primed with the solution to be tested for at least 15 min 406 prior to each test, and flushed with oil for 10 min. The tubing was cleaned twice before 407 measurements by purging air and then with isopropanol.
408 As shown in Figure 3, the circuitry is able to differentiate between fluids with permittivity effects 409 dominant (Figure 3a) and conductivity effects dominant (Figure 3b).
Different concentrations of D-410 (+)-Glucose and potassium chloride solutions (KCI) were prepared with ultra-pure water. The 411 minimum assessed concentration is 0.001 g/ml for KCI and 0.01 g/ml for glucose. The frequency 412 step size was 0.1 MHz in the analysis. The lower detection limit was achieved for the KCI solutions 413 because of the combined effects of permittivity and ionic conductivity (dominant effect). For 414 example, the conductivity of potassium chloride increase from 17.84 mS/cm to 60.8 mS/cm when its 415 concentration increases from 0.01g/m1 to 0.05 g/ml, which were experimentally measured using a 416 conductivity meter kit (Thermo Scientific, Orion 5-Star) after calibration of the probe with three 417 different calibration solutions. While increasing KCI concentration causes a decrease in the 418 resonance frequency, increase in the glucose concentration results in higher resonance frequencies.
419 As well, concentration changes cause sharp decline in the reflection coefficient (S11) so that the 420 change in resonance frequency can be monitored in the reflection coefficient. The differentiation of 421 fluids with small differences in electrical properties validated the dynamic performance of the 422 customized microwave circuitry along with the microwave sensor integrated with the 423 microchannels.

424 High-Throughput Droplet Detection 425 The performance of the microwave circuitry for droplet detection and counting is performed using a 426 flow focusing generator as shown in Figure 4. The channel height is 50 pm and assumed to be 427 uniform across the entire chip. The channel width smoothly narrows down from 300 pm to 80 m at 428 the intersection which is the same as that of the dispersed phase for generating droplet stably. The 429 wider channels were designed to lower down the hydrodynamic resistance for easy pumping while 430 the uniform channel widths near the generator is the most stable design for droplet generation31.
431 Initially, the droplet generation and transport were visualized and characterized with the optical 432 microscope (Eclipse Ti, Nikon) integrated with a high speed camera which captured images at a 433 frame rate of 9000 fps. Figure 4(a) shows the image of the generator and generated droplets which 434 are around Inl considering the droplets are fully confined by the channel (50um high, 80um wide) 435 with a length varying from 1-3 channel widths (80um 240um). Figure 4(b) compares the droplet 436 generation frequencies measured by the optical imaging and microwave sensor. When a droplet 437 passes the capacitive region (gap) of the resonator, the electromagnetic field is disturbed by the 438 presence of the droplet and the dielectric change (from the oil phase to the aqueous droplet phase) 439 causes a peak in the collected signal. The perturbation in the EM field can be used to determine the 440 droplet generation frequency by counting the number of the perturbations over a specific time 441 period. As shown in Figure 4(c), the signal peaks correspond to droplet presence while the valleys 442 correspond to the carrier fluid (oil). The resonator was operated at 2.59 GHz which was the 443 resonance frequency for FC40 oil, and at this frequency the signal change in reflection coefficient is 444 used to determine the droplet presence. It is worthy to mention that the sinusoidal look of peaks at 445 very high droplet formation frequencies is caused by shorter and unstable droplet spacing which are 446 likely due to the use of syringe pump which cause unpredictable uncertainties31. Due to the 447 limitation of the maximum pressure that the pressure controller can provide which limits the 448 throughput of droplet generation, a syringe pump was used in order to evaluate the detection 449 performance of the microwave system. With carefully cleaning and preparation of microfluidic 450 chips we reached as high flow rates as 4000 M1/hr for water and 4750 1/hr for the continuous phase 451 (i.e. oil). Correspondingly, we were able to generate droplets at the maximum rate of 3.33 kHz 452 which can be detected with the microwave system successfully. Further high frequencies can be 453 achieved by increasing the flow rate, which however tends to break chip made of PDMS32.
454 Ideally, in order to detect 'a droplet, at least one signal level needs to be sampled from carrier 455 fluid and one from dispersed (droplet) phase. This will result to give a minimum and a maximum 456 value, and maximum detection limit can be estimated to be the half of the signal generation rate 457 provided that the data sampling rate of the system is equal or larger than signal generation rate. In 458 our system, since the signal generation frequency is at microwave range (i.e., GHz), which is 459 extremely higher than data sampling that the data sampling rate basically determines the maximum 460 detectable limit. However, since the droplet spacing is low in our very high droplet generation 461 frequencies because of the droplet generation limitations explained above, the collected data gives a 462 sinusoidal look. For a clearly resolved droplet detection data for very high-throughput scenarios, the 463 droplet spacing should be at least one droplet length or higher. Here, with a data sampling rate of 10 464 i_ts and well-spaced droplets, the theoretical droplet detection limit of the developed microwave 465 system is 50 kHz.
466 Over a period of ten minutes, two million droplets were counted without missing any droplet. In 467 order to assess the minimum sensible dielectric variation for detectable droplets, it is important to 468 evaluate the resolution of the circuitry. An RMS noise level of 0.78 mV
has been calculated over an 469 interrogation time of 20 s. With this noise level, a resolution threshold of 0.026 dB in reflection 470 coefficient was obtained. Since a resonant microwave sensor is designed and used in the study, 471 electromagnetic energy into small region is accumulated, which is extremely sensitive to small 472 changes. Likewise, utilizing the characteristic feature of microwaves, i.e., operation at GHz 473 frequencies, allows working at shorter time scales. This gives a great opportunity and advantages 474 over other detection techniques such as capacitive and electrochemical which operate at lower 475 frequencies.

477 Droplet Content Sensing 478 Microwave sensing of droplet content was also carried out with the spiral resonator design. The 479 spiral resonator was placed 8 mm away from the generator intersection.
The microfluidic channel 480 network and droplet generators are shown in Figure 5(a). Fluid pumping and droplet generation 481 were controlled using a microfluidic pressure controller system (Fluigent MFCS-8C) which can 482 provide more stable droplet generation29. For this set of experiments, a double T-junction generator 483 was used to alternatively generate droplet pairs with different materials encapsulated33 such as type 484 A and type B. The alternating generation works as follows. When one droplet (i.e. with content type 485 A) is being generated in one of the T-junctions, it obstructs the main channel as it is growing and 486 thus restricts the flow of the continuous phase, which causes a dramatic increase in the pressure 487 upstream of the T-junction intersection. When the pressure increases to a critical value, it drives the 488 continuous phase to neck and then pinch off the droplet34-36. After pinch off the remaining interface 489 recoils back to the T-junction inlet. While this process is taking place, the other T-junction generator 490 repeats similar droplet (type .13) formation process. By well-tuned applied pressures, two alternating 491 droplets can be formed sequentially. During the formation of droplets, although two pairs come to 492 close proximity, they do not coalesce or cross-contaminate at certain operating regimes33. This 493 configuration has advantages over a simple Y-channel design in terms of operation of the two 494 droplet generators and robustness. In addition, with this configuration there is no need to add a 495 dilution stream in order to increase droplet spacing.
496 To demonstrate the sensitivity of the sensor and its potential to be applied in the area of biosensing 497 with appealing features of no chemical and physical intrusion to the sample, some materials were 498 strategically chosen. In particular, aqueous based solutions with slight differences in their 499 concentration such as the potassium chloride solutions and glucose solutions used here, which result 500 in similar dielectric constant and/or electric conductivity values, were chosen to demonstrate the 501 sensitivity of the sensor. Two biochemical materials, fetal bovine serum that is a widely used serum-502 supplement for in vitro cell culture of eukaryotic cells and penicillin-streptomycin-neomycin 503 antibiotic mixture (contain 5,000 units penicillin, 5 mg streptomycin and 10 mg neomycin/mL), that 504 is widely used to prevent bacterial contamination of cell cultures due to their effective combined 505 action against gram-positive and gram-negative bacteria, were chosen to demonstrate its potential 506 for biosensing. Thawing fetal bovine serum and penicillin solution started in the fridge at 8 C, then 507 completed at room temperature while the bottles was swirled gently to mix the solution during the 508 thawing process. D-(+)-Glucose and potassium chloride solutions were prepared in ultra-distilled 509 water, and 2% fat content of milk was used.
510 In order to ensure that the microwave system can differentiate droplets with small difference in 511 dielectric properties, the experiments were carefully designed to eliminate the droplet size effect. As 512 can be seen in Eq. (1), the frequency shift is a function of permittivity difference and the relative 513 size of the droplet over the resonator. Considering that the electromagnetic field is accumulated in 514 the sensing region, and the droplet width and height is confined with the channel, droplet size has no 515 effect on the reflection coefficient as long as its length is longer than the sensor region. This 516 consideration ensures that the response of the sensor to different droplets is caused by dielectric 517 property variation, namely by the specific droplet content.

518 In order to verify the sensing performance of the microwave system, ultra-pure water droplets were 519 generated from both T-junction generators with FC40 oil as the continuous phase. The same sized 520 droplets and different sized droplets were sensed with the same signal magnitude and the longer 521 droplets resulted in wider signals due to their longer residence time in the sensing region.
522 Subsequently, a set of droplet pairs of the same size were sensed which include a pair of Fetal 523 bovine serum and penicillin-strep.-neomycin, a pair of D-(+)-glucose(0.2g/m1) and milk(2%mf), and 524 a pair of potassium chloride(0.03 g/m1) and water droplets. Figure 5(a) shows the coordinated 525 optical imaging and microwave sensing results while Figure 5(b) and (c) shows the microwave 526 sensing results.
527 The reflection coefficient difference between the fetal bovine serum droplets and penicillin droplets 528 is -1.61 dB, which is 1.16 times lower, while -9.01 dB difference with the baseline of carrier oil 529 FC40. As well, the difference between glucose and milk droplets is -4.02 dB, and between KCI and 530 water droplets -3.45 dB. It is worthwhile that very low (-5 dBm) output excitation power was used 531 in order to avoid any heating effect on droplets. These results show that our microwave module is 532 very sensitive to nanoliter droplet permittivity contrast and can easily distinguish various droplet 533 contents. Very high reproducibility is accomplished. This microwave system can also be used with 534 other bio-materials for content analysis or for synthesis and reaction monitoring. It should be noted 535 that the demonstrated throughput of sensing is not high; however, it is limited by the throughput of 536 droplet generation for the particular scenarios considered here rather the sensor which has been 537 demonstrated for high throughput sensing as shown in Figure 4.
538 To demonstrate that this platform has the potential to be used as a tool for pharmaceutical 539 applications, it is applied to perform a similar assay developed to screen inhibitors for tau-540 aggregation that is linked with neurodegenerative disorders such as the Alzheimer's disease (AD)37-541 38. The tau-derived hexapeptide (AcPHF6) which is normally considered as a model for tau-protein 542 aggregation in many assays was used as the peptide and orange G which is one of the common 543 inhibitors used in the traditional assay was chosen for this preliminary testing.
544 Figure 6 shows that the microwave sensor is able to differentiate the droplets with and without the 545 mixture of peptide and inhibitor and the droplets with different concentrations of the inhibitor 546 (orange G), which are 0.665 mM and 0.332 mM respectively (inhibitor I
and II respectively in the 547 figure). The peptide concentration was kept at 0.316 mM and all droplets contain Thioflavin S (0.05 548 mg m1-1), which is a fluorescent indicator dye normally used in tau-aggregation assays. The 549 samples were prepared in 4-morpholinepropanesulfonic acid (MOPS) buffer of 20 mM with a pH of 550 7.2. There is only one set of droplets containing no mixture of orange G and AcPHF6, which is used 551 as a base similar to the negative control in the traditional assay37.
552 It should be noted that the sensing shown in Figure 6 only demonstrates that the developed 553 microwave and microfluidic platform has the potential to serve as a tool for drug discovery or 554 pharmaceutical applications:These results are not a quantitative measure of the effects of the 555 inhibitor on tau-aggregation because it is difficult to judge whether the signal difference is caused by 556 the concentration of the inhibitor or the degree of peptide aggregation induced by the different 557 inhibitor concentrations. To perform such an assay to quantitatively compare with the traditional 558 assay would require systematic design of the microfluidic chip and microwave sensor and require 559 further improvements on the sensor fabrication protocol as well to improve its sensitivity, which is 560 beyond the scope of this study. However, with a calibration process of drug assays, the platform 561 developed herein presents promising and insightful results for Alzheimer's disease drug screening 562 assays.

564 \
565 List of figures 566 Figure 1. (a) A schematic description of the microwave-microfluidics integrated device, (b) 567 schematic of microwave sensor with a spiral resonator design and an excitation loop, (c) and (d) a 568 closer view of droplet formation channels and spiral capacitive gap, respectively.
569 Figure 2. Schematic description of the microwave circuitry.
570 Figure 3. Reflection coefficient of the resonator for a series of glucose¨water (a) and KC1¨water (b) 571 mixtures for testing the circuitry.
572 Figure 4. (a) Image of the generator and generated droplets, (b) comparison of the droplet generation 573 frequencies; optical imaging vs. microwave sensor, (c) high-throughput droplet detection.
574 Figure 5. Label-free content sensing of individual droplets. (a) FBS¨penicillin, (b) glucose (0.2 g 575 m1-1)¨milk, (c) water¨potassium chloride (0.03 g m1-1) droplets.
576 Figure 6. Demonstration of sensing of droplets involving AcPHF6 and orange G which are the 577 model peptide and inhibitor respectively used in traditional tau-aggregation assays that is linked to 578 neurodegenerative disorders such as the Alzheimer's disease.

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625 Detailed Description 633 .
634 Potential Application 635 The integrated microwave and microfluidics platform has application of detection of E.Coli bacteria 636 in food and drinking water, detection and sensing in dairy product industry, pharmaceutical drug 637 discovery and biomedical diagnosis.

=

Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A system capable of, but not limited to, biomaterial sensing and E.Coli bacteria detection The system comprises of the following subsystems:
a. Microwave miniaturized coplanar sensor design (resonator) operating at GHz range.
b. Microfluidic platform that is able to carry single phase flow and droplet-based flows c. Microwave custom circuitry design that is able to generate and analyze microwave signal which is correlated to bacteria, cells or any biomaterials in the fluidic medium.
d. The microwave custom circuitry design includes, microwave signal generation, power coupling and gain detection sub-units.

2. The microwave custom system has better high-throughput analysis

3. The microwave custom system has improved sensitivity and portability

4. The microwave custom system is cost-effective.