WO2021189048A1

WO2021189048A1 - An electrokinetically-driven microchip for rapid extraction and detection of nanovesicles in situ

Info

Publication number: WO2021189048A1
Application number: PCT/US2021/023490
Authority: WO
Inventors: Leyla ESFANDIARI
Original assignee: University Of Cincinnati
Priority date: 2020-03-20
Filing date: 2021-03-22
Publication date: 2021-09-23

Abstract

A method of characterizing nanovesicles by entrapping and sensing the dielectric properties of the nanovesicles using a Lab-on-a-Chip device is disclosed. The Lab-on-a-Chip device includes a first layer comprising a glass substrate, a second layer comprising a first polyimide, a third layer comprising sensing electrodes, a fourth layer comprising a second polyimide, a fifth layer comprising trapping electrodes, a sixth layer comprising photoresist obstacles, and a seventh layer comprising a PDMS chamber.

Description

AN ELECTROKINETICALLY-DRIVEN MICROCHIP FOR RAPID EXTRACTION AND DETECTION OF NANO VESICLES IN SITU

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application Serial No. 62/992,371, filed March 20, 2020, which application is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates to a novel Lab-on-a-Chip (LOC) device. More specifically, it relates to a device which has the capability of extraction and characterization of sub-micron particles and vesicles in solution.

BACKGROUND OF THE INVENTION

[0003] Electrical Impedance Spectroscopy (EIS) has attracted many interests for the applications in characterization of various biological entities, since it can be used as a label- free method with minimal sample preparation procedures. These techniques have been used to differentiate different cell types and identify the abnormal or tumor cells. One of the most popular design used for EIS is single cell impedance cytometry, in which a microchannel is fabricated with a pair of facing or coplanar electrodes. As the cells pass through a channel one at a time, they disturbed the electric field in the detection volumes and created an electrical signal; this signal was processed to provide the impedance of a single cell. The other strategy was the static state impedance measurement approach, in which a single cell is manipulated to be placed at the center of the measuring electrodes, and thus, the electrical signal in the detection volume is altered due to the presence of the cell. However, the related work on sub micron particles and cell-secreted vesicles (exosomes) with diameters of 30-120 nm has not yet been reported. The main challenge for adapting this system for analysis of single vesicle with heterogeneous size distribution is that the scale of the channel and/or measuring electrodes must be miniaturized to the corresponding size scale of the target vesicle in order to achieve a reliable sensitivity in measurements. Even if the device with miniaturized channel and electrodes could be fabricated, it is still challenging to pass a single nanoparticle through the channel one at a time or manipulate single nanoparticle to the designated position. In addition, the applied high pressure and the blockage of the sub-micron channel in the impedance cytometry design could also be another limitation.

SUMMARY OF THE INVENTION

[0004] To utilize the EIS system as a sensing platform to differentiate between various sub micron vesicles and particles without facing the challenges with regards to measuring a single vesicle, the present invention utilizes a novel microchip to entrap a population of vesicles utilizing insulator-based di electrophoretic (iDEP) system and measure the impedance of a population of vesicles in situ. The present invention involves a Lab-on-a-Chip device including a first layer comprising a glass substrate, a second layer comprising a first polyimide, a third layer comprising sensing electrodes, a fourth layer comprising a second polyimide, a fifth layer comprising trapping electrodes, a sixth layer comprising photoresist obstacles, and a seventh layer comprising a PDMS chamber. In one embodiment, the photoresist obstacles are SU-8 obstacles.

[0005] In another embodiment, the present invention involves a method of characterizing nanovesicles by entrapping and sensing the dielectric properties of the nanovesicles using a Lab-on-a-Chip device. The Lab-on-a-Chip device includes a first layer comprising a glass substrate, a second layer comprising a first polyimide, a third layer comprising sensing electrodes, a fourth layer comprising a second polyimide, a fifth layer comprising trapping electrodes, a sixth layer comprising photoresist obstacles, and a seventh layer comprising a PDMS chamber. In one embodiment, the photoresist obstacles are SU-8 obstacles.

[0006] In one embodiment, the sensing electrodes sense nanoparticles using electrical impedance. In another embodiment, the trapping electrodes generate a non-uniform electric field.

[0007] In one embodiment, the nanovesicles are selected from the group consisting of small extracellular vesicles, exosomes, liposomes, viruses and mixtures thereof. In another embodiment, the nanovesicles comprise exosomes.

BRIEF DESCRIPTION OF THE DRAWINGS [0008] FIG 1 A is a picture of an embodiment of the LOC device.

[0009] FIG IB is a schematic of the LOC device of the present invention including the iDEP module for particle trapping and microelectrodes for impedance sensing in situ.

[0010] FIG 2 is an illustration showing a step by step fabrication procedure of an embodiment of the LOC device. [0011] FIG 3 is an equivalent circuit model for the impedance measurement system.

[0012] FIG 4A is a schematic of the impedance measurement system.

[0013] FIG 4B is a bright-field microscopic image of the LOC device.

[0014] FIG 4C is finite element analysis of the distribution of the electric field gradient across the opening created by SU-8. The suspending medium was 10 mM KC1 and the applied voltage was 5V/mm.

[0015] FIG 4D is a fluorescence microscopic image showing the entrapment of 100 nm fluorescently tagged COOH-PS beads with a 5V/mm bias applied across the opening for 5 minutes; the initial particle concentration was 2.3 xlO¹² /mL and the suspending solution was 10 mM KC1.

[0016] FIG 5A is a graph of theoretical modeling and experimental results showing the impedance of solutions with different conductivities as a function of frequency.

[0017] FIG 5B is a graph of theoretical modeling and experimental results showing the impedance of COOH-PS beads suspended in 10 mM KC1. The error bars represented the standard deviation and each experiment was repeated for at least three times.

[0018] FIG 6A is a graph showing the impedance of different particles suspended in electrolytic (10 mM KC1) solution as a function of frequency. The initial concentration of the COOH-PS beads is 1.8x108 /mL.

[0019] FIG 6B is a graph showing impedance of the COOH-PS beads with different initial concentrations suspended in 10 mM KC1 solution. The error bars represented the standard deviation and each experiment was repeated for at least three times.

[0020] FIG 7A is a graph showing the opacity magnitude of 100 nm COOH-PS beads with different entrapped quantities.

[0021] FIG 7B is a graph showing the opacity magnitude of different particles suspended in 10 mM KC1. The error bars represented the standard deviation and each experiment was repeated for at least three times.

[0022] FIG. 8 is a pair of diagrams showing the conformal transformation from physical plane (x,z) to model plane (u,v).

[0023] FIG. 9A is a microscopic image of entrapped fluorescently-tagged liposomes.

[0024] FIG. 9B is a microscopic image of entrapped hTERT Mesenchymal Stem Cell Exosomes. A 5V/mm bias was applied across the channel for 5 minutes and the suspending solution was 10 mM KC1. DETAILED DESCRIPTION OF THE INVENTION [0025] The details of one or more embodiments of the disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided herein.

[0026] The present disclosure may be understood more readily by reference to the following detailed description of the embodiments taken in connection with the accompanying drawing figures, which form a part of this disclosure. It is to be understood that this application is not limited to the specific devices, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting. Also, in some embodiments, as used in the specification and including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” or “approximately” one particular value and/or to “about” or “approximately” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

[0027] While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the disclosed subject matter. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subj ect matter belongs.

[0028] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, size, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0029] The term "nanovesicle" refers to small (diameter between 20-250 nm) vesicles including the lipid bilayer membrane surrounding the interior aqueous space. “Small extracellular vesicles”: “Exosome” means a sub-type of extracellular vesicle arising from the endosomal network and ranging in size from about 40 to 150 nm. The term “liposome” means a particle including lipid-containing molecules arranged to form a unilamellar or multilamellar membrane wall surrounding an interior volume. Many different viruses with similar size range and membrane composition can be characterized using the present invention, including, but not limited to, influenza viruses, coronavirus, adenovirus, and rhinovirus.

[0030] The term "PDMS chamber" refers to a polydimethylsiloxane (PDMS) layer with one or more openings located in the interior of the layer.

[0031] The present invention involves a novel Lab-on-a-Chip (LOC) device which has the capability of extraction and characterization of sub-micron particles and vesicles in solution. The impedance of electrolyte solution with different ionic concentration with and without suspended sub-micron particles were analyzed with both the numerical modeling and empirical measurements. The results showed that the sub-micron particles are entrapped by the insulator- based Dielectrophoresis (iDEP) module and detected by an integrated impedance measurement system in situ. This technique provides a label-free, non-invasive, and fast method for classification of sub-micron particles and vesicles based on their unique dielectric properties. The device of the present invention can be used for characterization and detection of extracellular vesicles (exosomes) from different cellular origins, and thus, it has potential as a simple, yet powerful tool for early diagnosis in liquid biopsy.

[0032] The LOC device of the present invention comprises multiple layers: a first layer comprising a glass substrate, a second layer comprising a first polyimide, a third layer comprising sensing electrodes, a fourth layer comprising a second polyimide, a fifth layer comprising trapping electrodes, a sixth layer comprising photoresist obstacles, and a seventh layer comprising a PDMS chamber. In one embodiment, the photoresist obstacles are SU-8 obstacles.

[0033] In one embodiment, the sensing electrodes sense nanoparticles using electrical impedance. In one embodiment, the sensing electrodes comprise two or more electrodes that apply an AC field across the trapped nanovesicles. In another embodiment, the AC field applies a field in the range of from about 500 KHz to about 50 MHz. In another embodiment, the AC field is altered in magnitude and the results of the magnitude changes are analyzed to identify one or more biophysical dielectric properties of the nanoparticles. In yet another embodiment, the AC field is altered in phase and the results of the phase changes are analyzed to identify one or more dielectric properties of the nanoparticles. In one embodiment, the AC field is altered in magnitude and phase and the results of the magnitude and phase changes are analyzed to identify one or more dielectric properties of the nanoparticles.

[0034] In one embodiment, the trapping electrodes comprise two or more electrodes to produce an electric field. The resulting electric field may act on particles in the suspending medium by way of three forces: an electrophoretic force, a di electrophoretic force; and an electro-osmotic force that is due to an electro-osmotic flow of the suspending medium.

[0035] In one embodiment, the present invention is a LOC device which is able to entrap a population of particles and vesicles utilizing insulator-based dielectrophoretic (iDEP) system and measure the impedance of a population of vesicles in situ to differentiate between various sub-micron vesicles and particles without facing the challenges with regards to measuring a single vesicle. In this embodiment of the device, SU-8 obstacles were used to create micron- openings and thus, particles are entrapped at the openings due the present of the electrokinetic force balance when a DC voltage applied. The entrapped particles and vesicles could be further measured by an integrated co-planar impedance sensor. The examples below reveal that the sub-micron polystyrene beads, liposomes, and extracellular vesicles are successfully isolated by the device. Their impedances measurement results show that the device is able to differentiate different particles based on their dielectric properties. An equivalent circuit model is established to study the working principle of our device utilizing the MATLAB. The equivalent circuit model matches the experimental results closely with different ionic solutions and particles. The experimental and simulation results reveal that the device could distinguish the solution with different ionic strength at 1 MHz as a result of the double layer capacitance predominate at low frequency range (f < 0.1 MHz), and parasitic capacitance predominate at high frequency range (f> 6 MHz). After adding COOH-PS beads to the system, the impedance of the system increased due to the high resistivity of COOH-PS beads compared to pure 10 mM KC1 solution. However, extracellular vesicles showed an opposite trend probably due to both the shell and inner lumen of the extracellular vesicles were more conductive than suspending solution. To rule out the effect of the number of particles and demonstrate the differences of the particles based on their accumulated dielectric properties we have normalized the measured impedance data based on the ‘opacity’ concept. The experimental results prove that the opacity magnitude only relies on the property of measured particles and the device is able to differentiate different sub-micron particles, including 100 nm COOH-PS beads, 510 nm COOH-PS beads, and 100 nm liposomes and extracellular vesicles.

[0036] The LOC device of the present invention has the ability to differentiate particles with different dielectric properties. It has the potential to be a simple, yet powerful tool for rapid, label-free and non-invasive characterization and differentiation of sub-micron vesicles, such as extracellular vesicles secreted from different cellular origins. Preparation of sub-micron particles

[0037] Electrolyte solutions containing different potassium chloride (KC1) concentrations (1 mM, 10 mM, 500 mM) were prepared at pH 7.0. The conductivity of KC1 solutions were measured utilizing a conductivity meter (Oakton Cond 6+) as: 0.3 S/m for 10 mM KC1, 1.4 S/m for 100 mM KC1, and 5.9 S/m for 500 mM KC1.

[0038] 100 nm COOH-PS beads were re-suspended into 10 mM KC1 to the final concentration of 1.8x108 /mL and 2.3x1012 /mL. 100 nm liposomes were re-suspended into 10 mM KC1 at a final concentration of 1.9x1011 /mL. 146 nm hTERT Mesenchymal Stem Cell Exosomes were distributed in 10 mM KC1 with the concentration of 6.1x109 /mL. The zeta potential of COOH-PS beads, liposomes, and exosomes dispersed in 10 mM KC1 at 25°C were measured at least 3 times using the Zetasizer-NanoBrook Omni (Brookhaven Instruments, NY, USA).

Device layout and fabrication

[0039] The LOC device was designed with AutoCAD 2018. A picture of an embodiment of the LOC device is shown in Fig. 1 A, and a cross-sectional view of the LOC device is shown in FIG. IB. Referring to FIG. IB, the device 10 was fabricated on a glass slide 30 , a first polyimide (PI) layer 40 to improve the adhesion strength of the substrate, the sensing electrodes layer 50, a second PI layer 60 to avoid short circuit of different electrode layers, the trapping electrode layer 70, the SU-8 obstacle layer 80, and the PDMS chamber layer 90.

[0040] In this embodiment of the device, the first PI layer was deposited to increase the adhesion between gold and the glass substrate. Prior to the deposition of PI, adhesion promoter VM652 was spin-coated at 2000 rpm for 30 seconds. PI2610 was then spread at 500 rpm for 5 seconds followed by 5000 rpm for 30 seconds to form a 1 mih thin film (Fig. 2a). To fabricate the sensing electrodes, a layer of metal (10 nm Cr and 200 nm Au) was deposited on the PI- coated substrate using the E-beam evaporator (Fig. 2b). The deposited metal was patterned using the photolithography technique with AZ5214E as the positive photoresist and MIF 917 as the developer. A pair of digital sensing electrode array was then created by etching the redundant Au and Cr on the first metal layer. Afterwards, the photoresist residual was removed by acetone (Fig. 2d). Prior to the deposition of trapping electrodes, adhesion promoter VM652 and PI2610 were spin-coated to insulate the sensing electrodes (Fig. 2d). 10 nm Cr and 200 nm Au were then deposited (Fig. 2e) and patterned (Fig. 2f). EVG620 mask aligner was used to align the trapping and the sensing electrodes. The width and the length of each trapping electrode was designed to be 0.25 mm and 26 mm, and the distance between the trapping electrodes was 2 mm. In order to connect the sensing electrodes with the digital impedance analyzer (HF2LI, Zurich Instrument), the PI film that covered the corresponding area were removed by a reactive ion etching (RLE) process with the photoresist AZ5214 as the shadow mask. After the pattern was properly defined, two large rectangular windows (9 mmx 8.5 mm) on the sides and a narrow rectangular window (34 mih^c 23 mm) in the middle of the device were etched utilizing RTF process (Technics 85 Reactive Ion Etcher, 190 mTorr, 150W, 6 minutes) to expose the tails and tips of the sensing electrodes respectively (Fig. 2g).

[0041] Moreover, to develop the obstacles, as trapping zones, a layer of negative photoresist SU-8 2050 was spin-coated at 3000 rpm for 30 seconds to obtain a 50 mih film (Fig. 2h). Prior to SU-8 coating, a thin layer of OmniCoat was spin-coated at 3000 rpm for 30 seconds to allow easy stripping of SU-8 and improve the adhesion. The SU-8 layer was exposed under 160 mJ/cm2 ultraviolet light with a mask and developed with SU-8 developer to create triangular obstacles with 10 mih width separation (Fig. 2i). RLE was then performed to remove the residual OmniCoat (Fig. 2j). Polydimethylsiloxane (PDMS) chamber was created by pouring the mixture of silicone elastomer base and curing agent (volume ratio 10 to 1) on a glass slide and heating up to 70°C for 4 hours. After the PDMS was fully crosslinked, it was peeled off from the glass slide and cut into rectangular pieces with 2 cm in width and 4 cm in length. Six holes with diameter of 3.5 mm were punched as the inlets and outlets. At the final stage, the PDMS chamber was adhered on the device to cap the SU-8 obstacles and create the opening with the dimension of 10 mih ^c50 mih. A heat seal connector was used to connect the tail of the electrodes on the microchip to a home-designed PCB board. The PCB board was then connected to the power supply and the digital impedance analyzer to apply voltage and conduct the impedance measurement.

Particle trapping and Impedance Measurement

[0042] 25 mί of electrolyte solution containing different particles including 1.8X10⁸ /mL and 2.3xl0¹² /mL COOH-PS beads, 1.9X10¹¹ /mL liposomes, and 6.1X10⁹ /mL exosomes were injected in to different device chambers. A 5V/mm DC bias was applied across the trapping electrodes using a Keithley 2220G-30-1 voltage generator for 5 minutes. The microscopic images were recorded using an inverted microscope, Olympus 1X71, equipped with a high-resolution camera, AndorNeoZyla 5.5.

[0043] Impedance measurement was conducted utilizing the digital impedance analyzer (HF2LI, Zurich Instrument) as an AC field with a peak amplitude of 100 mV swept from 1 kHz to 10 MHz to record the magnitude and phase components at each frequency. Afterwards, the data was processed with a custom script written in MATLAB (MathWorks Inc., Natick, MA, USA) for statistical analysis. The impedance signals were recorded at a sampling rate of 225 sample/sec. Each measurement was repeated at least 3 times. Furthermore, to rule out the effect of the particles concentration and to demonstrate the difference between the particles’ dielectric properties, the impedance was normalized based on the ‘opacity’ concept which was reported by Gawad et. al. (Eqn. 1).

0 - ^{z( )} (Eqn. 1) f Z( 0.5 MHz)

[0044] where Z(/) and Z(0.5 MHz) are the impedance magnitude measured at frequencies higher than 0.5 MHz and at 0.5 MHz respectively. This has been widely applied in cell cytometry to normalize the impedance with respect to the cell size and position since the impedance at 0.5 MHz typically reflects the particle size information.

[0045] The impedance sensitivity has been analyzed based on the following equation:

(Eqn. 2)

[0046] where DZ is the impedance change due to the presence of particles, Z_m is the complex impedance of the detection volume containing medium and Z_mix is the complex impedance of the mixture (the medium and the particles) in the detection volume.

[0047] Statistical analysis was performed using the student’s t-test and two-way analysis of variance. Difference with p-values less than 0.05 were considered significant.

[0048] After impedance measurements, the device was cleaned by the established Lab-on-Chip device cleaning protocol. Specifically, the device was injected with DI water to push most of the particles out of the channel. Afterwards, the device was soaked in the mild detergent solution, methanol, acetone, and DI water for five minutes each with an ultrasonic bath environment to completely remove the residue.

Finite element analysis

[0049] Finite-element software, COMSOL Multiphysics 5.2a (COMSOL Inc, Burlington, MA, USA), was utilized to determine the distribution of the electric field gradient as 5V/mm DC was applied across the gap which was created by SU-8 obstacles. The height of the SU-8 obstacles was 50 mih and the gap distance between a pair of triangular SU-8 obstacles was 10 mih. The conductivity and relative permittivity of the suspending solution in the model was set as 0.3 S/m and 80 to mimic the conductivity of 10 mM KC1 solution. The temperature and pressure were assumed to be 298 K and zero Pa, respectively.

[0050] The migration mobility of ionic species (ii) was computed using the Nemst-Einstein relation (Eqn. 3):

(Eqn. 3)

[0051] in which, I); is the diffusion coefficient, R is the molar gas constant and T is the absolute temperature. For 10 mM KC1, the value of D_L was set as 2x 10⁹ mV¹.

[0052] Boundary conditions corresponding to the solution obtained from the Poisson-Boltzmann equation for electric potential were applied. The boundary conditions established that the electric potential was not diverged and the gradient of this potential on the SU-8 surface varied with the change in surface charge density.

Theoretical modeling and equivalent circuit

[0053] A simplified equivalent circuit model (Fig 3) was used to demonstrate the physical principle of the impedance measurement system. In this model, the channel impedance Z_Ch is in series with an electrical double layer capacitance C_di and is in parallel with a stray capacitance C _stray. In addition, a lead inductance (Li_d) is included in the equivalent circuit, which is associated to the electrodes and the cables connecting the device to the impedance analyzer. The values of C_di, C _stray and Li_d were obtained via measurements on electrolyte solutions with well-known electrical properties, followed by fitting into the combination of constant phase element and Cole- Cole model. Fitting parameters that were used throughout this theoretical modeling were C_di = 10 pF, C s_tray = 2.2 pF, and Li_d = 6 mH, respectively.

[0054] Channel impedance Z_Ch was calculated based on Maxwell’s mixture theory (Eqn. 4).

(Eqn.4)

[0055] where e_mix is the equivalent complex permittivity of the mixture of particles and the medium, w is the angular frequency, and G is the geometrical constant of the system.

[0056] The equivalent complex permittivity of mixture of homogeneous spherical particles in suspension can be calculated as:

(Eqn. 5)

[0057] where f is the volume fraction (the volume ratio between the particle and the suspending system), which is estimated as 0.1 for COOH-PS based on the estimated size of entrapped particles cluster under the microscopy; f_CM is the complex Clausius-Mossotti factor, which is defined as:

[0058] where i_m and i_p are the complex permittivity of the suspending medium and particle ί_s respectively; and e = e - where j = —1, e and s are permittivity and conductivity. The

CO relative permittivity and conductivity of the 100 nm polystyrene beads are set as 2.55 and 7.2 mS/m, respectively.

[0059] The geometrical constant Gf in Equ.4 can be presented as Gf = KW, where w is the width of the electrode and k is the correction factor describing the fringing field. The value of k was derived analytically using the conforming mapping method. Utilizing this method, k and geometric constant Gf were calculated as 0.73 and 7.3 mih, respectively (the details of the derivation is provided in supplementary information (Fig. 8)).

EXAMPLES

Example 1

[0060] To integrate the trapping mechanism with the sensing module on a single chip (Fig.4A), SU-8 triangular obstacles were developed with 10 mih width separation to generate a non- uniform electric field at the openings to entrap the particles. Furthermore, a pair of co-planar electrodes were used to measure the impedance of the trapped particles (Fig. 4B). A finite element simulation was carried out to study the E-field gradient distribution under DC bias (Fig. 4C). The results illustrated that the highest E-field gradient was localized at the narrowest part of the openings.

Example 2

[0061] A series of experiments were conducted with fluorescently-tagged COOH-PS beads, fluorescently-tagged liposomes, and exosomes suspended in 10 mM KC1 (pH 7.0). 25 mί_^ solution containing various particles were injected separately into different chambers of the device and 5V/mm DC bias was applied across the opening for 5 minutes. Fig. 4D and Fig. 9 show that the particles were trapped at the narrowest region of the opening as expected.

Example 3

[0062] To study the capability of the device to differentiate between solutions with different ionic strengths, and understand the physical principle of the impedance measurement, an equivalent circuit model was constructed and the theoretical and experimental results were compared. Fig. 5 A demonstrates both the theoretical and experimental results of the impedance when solutions with different conductivities were tested. The theoretical results were closely matched with the experimental measurements, which implies that the established equivalent circuit model was reliable for predicting the impedance of the system. In addition, the results were in line with the previously reported observations and suggested that as the frequency increased, the absolute value of impedance decreased for all solutions. This is due to the fact that the reactive part of the impedance was predominately capacitive and thus, the co-planar impedance sensor acted as a capacitor, storing electrochemical energy. Statistical data obtained from the experimental measurements show in Table 1 indicates that the impedance of the solutions were significantly different from each other (p <0.05) at a wide frequency spectrum, and thus, the device is capable of differentiating solutions with different ionic strengths. However, the results also indicate that the impedance of the solutions with 1.4 S/m and 5.9 S/m conductivities at frequency > 10 MHz were not significantly different from each other. This could be justified since the stray capacitance is dominated at frequency > 10 MHz which resulted in the reduction of the difference in their impedance . In addition, to further investigate the capability of the circuit model to predict the impedance of the particles, theoretical results and experimental measurements were compared utilizing the well-defined 100 nm COOH-PS beads suspended in 10 mM KC1. Fig. 5B demonstrates that the theoretical results were closely matched with the experimental measurements, which proves that the established equivalent circuit model is reliable for predicting the impedance of the system with added beads. [0063]

Table 1

Example 4

[0064] To investigate the impedance response of different sub-micron particles, COOH-PS beads, liposomes, and exosomes, suspended in 10 mM KC1 were injected into different chambers of the device. The particles were trapped at the triangular trapping zones by applying DC bias, and their impedance were recorded under AC field. The impedance of the entrapped liposomes and COOH-PS beads were increased when compared to the solution containing no particles (Fig. 6A). This result could be justified since the lipid bilayer in liposome and the bulk polystyrene materials in COOH-PS beads have lower conductivities when compared to the surrounding medium, and thus, resulting in the enhancement of the channel resistance. However, as exosomes were incorporated into the system, a lower impedance was measured which suggested that exosomes were more conductive than the suspending medium, which is because proteins with a relatively high conductivity are embedded on exosomes’ membrane.

Example 5

[0065] To further study the impedance response of the particles with different concentration, COOH-PS beads with different initial concentration (1.8x108 /mL and 2.3x1012 /mL) were injected into different chambers of the LOC device and trapped by applying 5 V/mm DC field for 5 minutes. The microscopic images in Fig. 6B show that as the initial concentration of COOH-PS beads was increased, more beads were trapped at the triangular trapping zone. Consequently, the impedance of the system significantly increased due to the enhancement of the channel resistance and the reduction of channel capacitance (Fig. 6B and Table 2). The impedance sensitivity calculation is shown in Table 3 which indicates that the sensitivity of the device is in the range of 0.03 to 0.55. In addition, as the initial particle concentration of COOH- PS beads increased from 1.8x108 /mL to 2.3x1012 /mL, the impedance sensitivity was significantly increased due to the increase of the mixture impedance (the medium and the particle ) in the detection volume.

[0066]

Table 2

Table 3

Example 6

[0067] To rule out the effect of the particles’ concentration on their impedance and only show the effect of their dielectric properties by impedance measurements, the results were normalized based on the opacity concept. The impedance of the COOH-PS beads with different initial concentration (1.8X10⁸ /mL and 2.3X10¹² /mL) were normalized based on opacity magnitude and plotted in Fig. 7A and summarized in Table 4. The results demonstrate that there were no significant differences (p >0.05) between the opacities of COOH-PS beads with different initial concentrations.

[0068]

Table 4

Example 7

[0069] To further investigate the capability of the system to differentiate between particles with different dielectric properties, the opacity magnitude of three types of particles with different compositions were analyzed and plotted in Fig. 7B. A detailed representation of the data with statistical analysis are shown in Table 5. The results show that COOH-PS beads and exosomes were differentiated at frequency range > 1 MHz, and COOH-PS beads and liposomes were differentiated at the frequency range > 2 MHz. These results indicate that the dielectric properties of the COOH-PS beads are vastly different from the nanovesicles due to the difference of composition and surface charge (Table 6). In addition, liposomes and exosomes could be differentiated at the frequency range > 6 MHz, which most likely reflects on their membrane capacitance differences due to the presence of proteins on exosomes’ membrane. [0070]

Table 5

Table 6

Methods

[0071] All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted. 100 nm carboxylic acid polystyrene (COOH-PS) beads were obtained from Phosphorex Inc. (Hopkinton, MA, USA). N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)-l,2- dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-DHPE) fluorescently labeled 100 nm liposomes were purchased from FormuMax Scientific Inc. (Sunnyvale, CA, USA). Tel om erase reverse transcriptase (hTERT) Mesenchymal Stem Cell Exosomes with the average diameter of 146 nm were purchased from ATCC (Manassas, VA, USA). The zeta potential and the size distribution for all the particles have been shown in Table 6. Silicone elastomer base and curing agent were purchased from Dow Corning (Elizabethtown, KY). Gold etchant (Type TFA) and chromium etchant (1020AC) were obtained from Transene Company Inc. (Danvers, MA, USA). Photoresist AZ5214E and developer AZ917 MIF were purchased from Integrated Micro Materials (Argyle, TX, USA). SU-8 2050, SU-8 developer and OmniCoat were obtained from Microchem Corp. (Westborough, MA, USA). Polyimide PI2610 and adhesion promoter MV652 were obtained from Hitachi DuPont MicroSystems LLC. (Parlin, NJ, USA). Heat seal connectors were obtained from Elform Inc. (Reno, NV, USA). The printed circuit board (PCB) was fabricated by PCB Universe (Vancouver, WA, USA). Glass slides were purchased from Ted Pella Inc. (Redding, CA, USA).

[0072] Table 1: The statistical data for the impedance measurement of different electrolyte solutions p-values were obtained from two-tails unpaired student t-test. The highlighted data are significantly different. Table 2: The statistical data for the impedance measurement of different particles suspended in 10 mM KC1. p-values were obtained from two-tails unpaired student t-test. The highlighted data are significantly different. Table 3. The statistical data for the impedance sensitivity of different particles p-values were obtained from two-tails unpaired student t-test. The highlighted data are significantly different. Table 4: The statistical data for the opacity magnitude of COOH-PS beads with different concentration suspended in 10 mM KC1. p-values were obtained from two-tails unpaired student t-test. Table 5: The statistical data for the opacity magnitude of different particles suspended in 10 mM KC1. p-values were obtained from two-tails unpaired student t-test. The highlighted data are significantly different. Table 6: Zeta potential and size of COOH-PS beads, liposomes, and exosomes. The zeta potential of particles were measured in 10 mM KC1.

[0073] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0074] All documents cited are incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention. [0075] It is to be further understood that where descriptions of various embodiments use the term “comprising,” and / or “including” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of’ or "consisting of.”

[0076] While particular embodiments of the present invention have been illustrated and described, it would be obvious to one skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

What is claimed is:

1. A Lab-on-a-Chip device comprising: a. a first layer comprising a glass substrate, b. a second layer comprising a first polyimide, c. a third layer comprising sensing electrodes, d. a fourth layer comprising a second polyimide, e. a fifth layer comprising trapping electrodes, f. a sixth layer comprising photoresist obstacles, and g. a seventh layer comprising a PDMS chamber.

2. The device of claim 1 wherein the photoresist obstacles are SU-8 obstacles.

3. A method of characterizing nanovesicles comprising entrapping and sensing the dielectric properties of said nanovesicles using a Lab-on-a-Chip device comprising: a. a first layer comprising a glass substrate, b. a second layer comprising a first polyimide, c. a third layer comprising sensing electrodes, d. a fourth layer comprising a second polyimide, e. a fifth layer comprising trapping electrodes, f. a sixth layer comprising photoresist obstacles, and g. a seventh layer comprising a PDMS chamber.

4. The method of claim 3 wherein the photoresist obstacles are SU-8 obstacles.

5. The method of claim 3 wherein the sensing electrodes sense nanoparticles using electrical impedance.

6. The method of claim 3 wherein the trapping electrodes generate a non-uniform electric field.

7. The method of claim 3 wherein the nanovesicles are selected from the group consisting of small extracellular vesicles, exosomes, liposomes, viruses and mixtures thereof.

8. The method of claim 3 wherein the nanovesicles comprise exosomes.