WO2013012396A1

WO2013012396A1 - A method and apparatus for analyzing interactions of molecules

Info

Publication number: WO2013012396A1
Application number: PCT/SG2012/000259
Authority: WO
Inventors: Cherng-Wen Darren Tan; I. Putu Mahendra WIJAYA; Walter Hunziker; Eva-Kathrin Sinner; Madhavan Nallani
Original assignee: Agency For Science, Technology & Research
Priority date: 2011-07-20
Filing date: 2012-07-20
Publication date: 2013-01-24
Also published as: WO2013012396A9

Abstract

The invention relates to a method and apparatus (500) for analyzing interactions of molecules of interest. Carriers, some of which are embedded with the molecules of interest, are used. The apparatus (500) comprises a microfluidic device (502) which in turn comprises a microfluidic channel (502a) and detectors (502b), and an operations unit (506). First and second samples (respectively comprising the embedded carriers and the remaining carriers) are caused to flow separately through the microfluidic channel (502a). The detectors (502b) are configured to detect presence of the carriers at respective positions along the channel (502a) during the flows. Based on the detections, the operations unit (506) determines rates at which the embedded carriers and the remaining carriers travel through the channel (502a), and obtains, based on a difference in the rates, data indicative of the amount of interactions of the molecules of interest during the flow of the first sample.

Description

A Method and Apparatus for Analyzing Interactions of Molecules

Field of the Invention The present invention relates to a method and apparatus for analyzing interactions of molecules of interest.

Background of the Invention Cells are the building blocks of higher organisms. For example, tissues and organs in the human body are made up of cells.

Fig. 1 illustrates a typical cell structure in the human body. As shown in Fig. 1 , each cell comprises a complex collection of microstructures and is enveloped by a sac-like lipid, bilayered membrane that isolates its contents from the external environment. Within each cell, a different network of membranes separate micro-volumes of the cell content (organelles) from the rest of the cytoplasm. The organelles include the nucleus, endoplasmic reticulum, Golgi apparatus, and various endocytic and secretory vesicles. As with the cell, these organelles are membrane-bound to preserve their internal environment.

While it is important to isolate the contents of a cell from its external environment, the survival of the cell also depends very much on its ability to sense its external environment. The critical function of sensing the cell's external environment is performed by membrane proteins embedded in the lipid bilayer of the cell membrane. Besides performing the sensing function, the membrane proteins also allow the cell to interact with its external environment by serving as points of interaction. There are many different types of membrane protein as shown in Fig. 1 , with each type of membrane protein performing a different function. One particular type of membrane protein are the integral membrane proteins which have regions spanning the entire lipid bilayer and which are in contact with both the external environment of the cell as well as the internal environment of the cell comprising the cytoplasm. As such, these membrane proteins are able to sense the external environment of the cell and relay information acquired via this sensing to the internal environment of the cell.

Other types of membrane protein, such as cell adhesion proteins, bind to other cells or to a substrate. Cell-cell binding achieved by the membrane proteins allows the formation of cell sheets, and in turn, the formation of the epithelial layer for many organs. For instance, the blood-brain barrier, the kidney tubules and the lining of the digestive tract are formed from the cell sheets.

The epithelial layer serves to regulate the transport of materials, such as ions and proteins, between one side of itself to the other. In particular, the epithelial layer allows the passage of these materials across itself via different routes, including routes through the epithelial cells and routes through the intercellular spaces between the epithelial cells. Fig. 2 shows a schematic diagram illustrating different routes materials can take across the renal epithelium of a kidney to produce a filtrate of blood. These routes include (i) transport through the cytoplasm of a cell, (ii) transport through a cell by transcytosis and (iii) intercellular transport through the intercellular spaces and gap junctions between adjacent cells. Regulation of material transport across the epithelial layer of an organ is crucial for the organ to perform its function. For example, failure of the kidney tubule epithelium will almost certainly result in the loss of renal function and ultimately, renal failure.

The epithelial layer also serves as a crucial protective barrier to protect the underlying tissue of the organ. For instance, a breach in the blood-brain barrier will cause the brain to be vulnerable to infection by parasites and even the body's own immune system. As such, the failure of the blood-brain barrier gives rise to various pathological conditions such as meningitis and epilepsy. For the epithelial layer to perform its protective function well, it is necessary to maintain the seal between the epithelial cells. This is achieved by strips of membrane proteins mainly circumscribing abutting cells. These strips of membrane proteins between adjacent cells interact and form tight junction strands, each serving as an almost impenetrable obstacle to solutes, except those which are to be transported across the epithelial layer (the epithelial tissue functions to regulate this transport). Fig. 3 shows a diagram illustrating how a tight junction strand (TJ strand) is formed between the plasma membranes of two adjacent cells.

Claudins

In 1998, Furuse et al. identified claudins as an essential membrane protein in the formation of tight junction strands. In particular, the impenetrable seal between the cells is achieved via the interactions between the claudin molecules.

A member of the claudin family of proteins is the Claudin-2. Fig. 4 (S. Amasheh, N. Meiri, A. H. Gitter, T. Schoneberg, J. Mankertz, J. D., Schulzke and M. Fromm. Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. (2002) 1 15: 4969 - 4976) is a micrograph showing transfected MDCK cells and the locations of fluorescent antibodies raised against the Claudin-2 (Cldn2) molecules of these cells. The fluorescent antibodies are shown in white in the micrograph and their locations correspond to the locations of the Claudin-2 molecules. As shown in Fig. 4, the Claudin-2 molecules lie along the cell membranes of the cells.

Studying membrane proteins Membrane proteins make up more than half the drug targets in the pharmaceutical industry. In particular, membrane-bound cell adhesion proteins are implicated in many pathological conditions. This has led to interest in understanding their functions and how their failure contributes to diseases. For example, understanding how the tight junction strands work and how they may fail can help to achieve significant progress in the treatment of barrier-failure diseases. Of interest is the identification of those molecules that disrupt, or preserve, the interaction between the membrane proteins forming the tight junction strands.

The molecular mechanism of membrane protein functions is usually studied using cell-based assays. For instance, to study claudins, it is often necessary to either examine those cells which endogenously express the claudins (such as kidney epithelia cells), or to use claudin-free cells transfected with DNA encoding the claudins. However, cell-based assays are usually performed well only by researchers having special skills at handling mammalian cell cultures and specialized facilities. This therefore hinders the process of drug discovery and screening. Thus, it is preferable to use in vitro alternatives to study membrane protein functions. An added advantage of using in vitro alternatives is that in vitro samples are relatively free of contaminating membrane proteins ubiquitous in cells, thus facilitating quantitative analyses of assay results. However, membrane proteins have been notoriously difficult to produce in quantity and purity using conventional in vitro synthesis. One reason for this is that the native structure of membrane proteins includes exposed hydrophobic regions. When membrane proteins are in their native environment, the proteins retain their native structure (i.e. conformation) because the lipid bilayer in which they are embedded interacts with and stabilizes these exposed hydrophobic regions. However, when the proteins are exposed to a hydrophilic environment, they lose their native conformation and in turn, their function. Therefore, to produce membrane proteins in vitro that are correctly folded and are able to interact with other membrane proteins, it is necessary to mimic the native lipid microenvironment of the membrane proteins. It is possible to synthesize membrane proteins in vitro using synthetic membranes as a carrier platform. In particular, a cell-free extract may be used to transcribe and translate complementary DNA encoding the membrane proteins in the presence of membrane-mimics, such as lipid or polymer vesicles and tethered membranes. This method has been shown to be able to express the membrane proteins and insert them into the membrane-mimic scaffolds. In fact, recent data have shown that the membrane proteins are not only inserted into the membrane-mimics, they are also correctly oriented and are able to maintain their native structure within the membrane-mimics.

In order to study the membrane protein functions using the in vitro synthesized membrane proteins, it is first necessarily to establish that the functions of these synthesized proteins (and not just their structures) have been retained. However, functional characterization of the in vitro synthesized membrane proteins remains a challenge to date. This is elaborated below.

Functional characterization of binding proteins is usually done using a binding assay. Such an assay probes for the interaction between binding molecules and typically involves, first, the exposure of a binding protein to its binding partner, such as an antibody to its ligand, and then rigorous washing to remove non- binding partner molecules (note however, that the washing step is not required in homogeneous techniques). Rigorous washing is necessary since less stringent washing would leave the assay vulnerable to non-specific binding and in turn, proteins interacting specifically cannot be accurately distinguished from those associating randomly through adsorption.

For the binding assay to be effective, the interactions between the binding molecules have to be strong enough to withstand the rigorous washing. Weak protein-protein interactions, such as those occurring between tight junction cell adhesion proteins cannot be effectively probed using conventional protein- binding assays. An example of such weak interactions is the claudin-claudin interactions. These interactions are not as strong as the interactions between other binding partners, such as receptors and their ligands. More specifically, although the cumulative effect of multiple claudin-claudin interactions can result in the effective seal typical of tight junctions between cells, the individual claudin-claudin interactions tend to be transient and weak. As such, conventional protein-binding assays are not effective for the study of in vitro synthesized claudins.

Note that in this document, by "weak interactions", it is meant that the interactions have a rate of dissociation of about 10^"3 second^"1 (sec ¹) at a loading rate of 10² - 10³ pN/sec (S.L Tong et al. 2008, J Mol. Biol. 381 : 681 - 691 ).

Summary of the invention The present invention aims to provide a new and useful method and apparatus for analyzing interactions of molecules of interest.

In general terms, the present invention proposes determining the amount of interactions of the molecules of interest based on how much the transport rate of carriers through a microfluidic channel changes, when the carriers are embedded with the molecules of interest.

Specifically, a first aspect of the present invention is a method for analyzing interactions of molecules of interest, wherein the method uses a plurality of carriers, some of the plurality of carriers being embedded with the molecules of interest to form embedded carriers, and wherein the method comprises:

causing a first and second sample to flow separately (that is, successively, e.g. spaced apart by a certain time) through a microfluidic channel, whereby the first sample comprises the embedded carriers and the second sample comprises the remaining carriers;

for each of the first and second samples, detecting presence of the carriers at a plurality of positions along the microfluidic channel as the sample flows through the microfluidic channel, the plurality of positions defining one or more parts of the microfluidic channel;

determining, based on the detections, rates at which the embedded carriers and the remaining carriers travel through each of the one or more parts of the microfluidic channel during the flow of the first and second samples respectively; and

for each of the one or more parts of the microfluidic channel, obtaining data indicative of the amount of interactions of the molecules of interest during the flow of the first sample through the part based on a difference in the rates determined for the part.

A second aspect of the present invention is an apparatus for analyzing interactions of molecules of interest, wherein the apparatus uses a plurality of carriers, some of the plurality of carriers being embedded with the molecules of interest to form embedded carriers, and wherein the apparatus comprises:

a microfluidic device comprising:

a microfluidic channel, wherein the microfluidic device is configured to cause a first and second sample to flow separately through the microfluidic channel, whereby the first sample comprises the embedded carriers and the second sample comprises the remaining carriers; and

a plurality of detectors configured to detect presence of the carriers at respective positions along the microfluidic channel as each of the first and second samples flows through the microfluidic channel, said positions defining one or more parts of the microfluidic channel; and

an operations unit configured to:

determine, based on the detections, rates at which the embedded carriers and the remaining carriers travel through each of the one or more parts of the microfluidic channel during the flow of the first and second samples respectively, and

for each of the one or more parts of the microfluidic channel, obtain data indicative of the amount of interactions of the molecules of interest during the flow of the first sample through the part based on a difference in the rates determined for the part.

A third aspect of the present invention is a microfluidic device for determining transport dynamics of a plurality of carriers in a sample, wherein the plurality of carriers is capable of embedding molecules of interest and the transport dynamics are used for analyzing interactions of the molecules of interest, and wherein the microfluidic device comprises:

a microfluidic channel configured to allow flow of the sample through itself; and

a plurality of detectors configured to detect presence of the carriers at respective positions along the microfluidic channel as the sample flows through the microfluidic channel, wherein the respective positions define one or more parts of the microfluidic channel and the detection indicates the transport dynamics of the carriers through the one or more parts of the microfluidic channel.

The microfluidic channel may be lined with molecules of the same type as the molecules of interest and/or molecules of a different type but which are believed to interact with the molecules of interest.

Brief Description of the Figures

Embodiments of the invention will now be illustrated for the sake of example only with reference to the following drawings, in which:

Fig. 1 shows a typical structure of a cell in the human body, whereby the cell comprises different types of membrane protein in its cell membrane;

Fig. 2 shows a diagram illustrating different routes materials can take across the renal epithelium of a kidney;

Fig. 3 shows a diagram illustrating how a tight junction strand is formed between membranes of adjacent cells; Fig. 4 shows a micrograph illustrating transfected MDCK cells and the locations of fluorescent antibodies raised against Claudin-2 molecules of these cells;

Fig. 5 shows an apparatus for analyzing interactions of molecules of interest according to an embodiment of the present invention, wherein the apparatus uses a plurality of carriers, some of which embed the molecules of interest to form embedded carriers;

Fig. 6 shows an AutoCad rendering of 2-dimensional features of a microfluidic channel of the apparatus of Fig. 5;

Fig. 7 shows a PDMS cast formed during the process of forming the microfluidic channel;

Fig. 8 shows the microfluidic channel formed from the PDMS cast of Fig.

7;

Fig. 9 shows a structure of a sensor of the apparatus of Fig. 5;

Fig. 10 shows the structure of a known sensor;

Fig. 1 1 shows a template used to form the sensor of Fig. 9;

Fig. 12 shows the sensor formed from the template of Fig. 1 1 ;

Fig. 13 shows how the microfluidic channel is positioned with the sensor; Fig. 14 shows how the microfluidic channel is secured with the sensor; Fig. 15 shows a schematic diagram illustrating how the microfluidic channel is used with the sensor to determine a transport rate of carriers through the microfluidic channel;

Fig. 16 shows a graph illustrating a possible current waveform across the sensor when a sample comprising carriers is caused to flow through the microfluidic channel whereby this current waveform is used to determine the transport rate of the carriers through the microfluidic channel;

Fig. 17 shows a graph illustrating possible current waveforms across the sensor when samples respectively comprising the embedded carriers and the remaining carriers are separately caused to flow through the microfluidic channel, whereby these current waveforms are used to determine the transport rates of the carriers through the microfluidic channel; Fig. 18 shows results from a Western blot illustrating the success of preparing Claudin-2-embedded ABA vesicles;

Fig. 19 shows a graph plotting the current through the sensor when ABA vesicle suspensions of increasing concentrations are successively applied to the sensor;

Fig. 20 shows a graph plotting the current through the sensor when a droplet of water is caused to flow through the microfluidic channel;

Fig. 21 shows a graph plotting the current through the sensor when a sample comprising non-embedded ABA vesicles is caused to flow through the microfluidic channel;

Figs. 22(a) - (d) show images illustrating the success of immobilizing anti-Claudin-2 antibodies along walls of the microfluidic channel;

Figs. 23(a) - (c) show graphs plotting the current through the sensor when a sample comprising non-embedded ABA vesicles, a first aliquot of Claudin-2-embedded ABA vesicles and a second aliquot of Claudin-2- embedded ABA vesicles are separately caused to flow through the microfluidic channel with the anti-Claudin-2 antibodies immobilized along its walls;

Figs. 24(a) - (b) show graphs plotting the current through the sensor when a sample comprising non-embedded ABA vesicles and a sample comprising Claudin-2-embedded ABA vesicles are separately caused to flow through the microfluidic channel with Claudin-2-embedded ABA vesicles immobilized along its walls; and

Figs. 25(a) - (b) show graphs plotting the current through the sensor when a sample comprising non-embedded BD21 vesicles and a sample comprising Claudin-2-embedded BD21 vesicles are separately caused to flow through the microfluidic channel with Claudin-2-embedded BD21 vesicles immobilized along its walls.

Detailed Description of the Embodiments

Apparatus 500 Fig. 5 shows an apparatus 500 which is an embodiment of the present invention and which is capable of analyzing interactions of molecules of interest. The apparatus 500 uses a plurality of carriers, some of which embed the molecules of interest to form embedded carriers. The remaining carriers which do not embed the molecules of interest are referred to as non-embedded carriers.

As shown in Fig. 5, the apparatus 500 comprises a microfluidic device 502 which in turn comprises a microfluidic channel 502a and a detector unit 502b. The microfluidic channel 502a is secured with the detector unit 502b. The apparatus 500 further comprises a voltage unit 504 and an operations unit 506. The voltage unit 504 comprises a voltage source/picoammeter and is electrically coupled with the detector unit 502b via leads 510. The operations unit 506 comprises attendant equipment in the form of a desktop personal computer (PC). A link 512 serves to allow communication of data between the voltage unit 504 and the operations unit 506.

Apparatus 500 will now be described in more detail. Microfluidic device 502

As mentioned above, the microfluidic device 502 comprises a microfluidic channel 502a and a detector unit 502b. The microfluidic device 502 is capable of determining the transport dynamics of the carriers in a sample. These transport dynamics are used for analyzing the interactions of the molecules of interest and are indicated by detections made by the detector unit 502b.

Microfluidic channel 502a The microfluidic channel 502a is configured to allow flow of a sample through itself. Fig. 6 shows an AutoCAD rendering of the two-dimensional features of the microfluidic channel 502a. As shown in Fig. 6, the microfluidic channel 502a comprises a plurality of loops. Including loops as part of the structure of the microfluidic channel 502a is advantageous as a longer channel within a given area can be achieved. The microfluidic channel 502a also comprises inlet and outlet perforations 602 at its ends. Each of these perforations 602 may be used as either the inlet or the outlet of the microfluidic channel 502a. In particular, when one of the perforations 602 is used as the inlet, the other of the perforations 602 is used as the outlet. These perforations 602 are marked by the circles in Fig. 6. Furthermore, the microfluidic channel 502a has a rectilinear cross-section which is not shown in Fig. 6.

The microfluidic channel 502a is fabricated with poly (dimethylsiloxane) (in short, PDMS) using soft-lithography. In particular, a master mold for the microfluidic channel 502a is first made. This is followed by a PDMS micromolding process to fabricate the microfluidic channel 502a.

Master mold fabrication

The master mold is designed to yield a microfluidic channel 502a with the following dimensions: 100 im in width, 100 μιη in height and 10 cm in length. The master mold is fabricated as follows.

A 4-inch silicon wafer is first rinsed with isopropyl alcohol (IPA) and then blow- dried with gaseous nitrogen. The wafer is next treated with reactive oxygen plasma (plasma cleaner, Harrick, USA) at 100 W for 80 sees to remove dirt and organic contaminants, as well as to hydrophilize the surface of the wafer. SU-8 2050 (Microchem, USA) is then coated onto the wafer using a spin-coater with a final speed of 800 rpm for 80 sees to obtain a 100 jjm thick SU-8 2050 film. The thickness of this SU-8 2050 film defines the height of the microfluidic channel 502a. The SU-8 2050 film is subsequently soft-baked on a hotplate at 65 degrees Celsius (°C) for 10 minutes (mins) and then at 95°C for 30 mins to remove the solvents from the SU-8 2050 film. The SU-8 2050 film is then allowed to cool at 65°C for 1 min.

Next, a photo mask which defines the two-dimensional structure of the master- mold is used. This photo mask is formed by first drawing its design using AutoCAD software to produce an AutoCAD rendering (see Fig. 6), and then sending this AutoCAD rendering for emulsion printing on transparent material (Infineon Graphics, Singapore). To form the master mold, ultraviolet (UV) radiation at 240 - 260 mJ/cm² is shone through the photo mask onto the SU-8 2050 film for 37 sees. The resulting exposed SU-8 2050 film is then baked at 65°C for 5 mins and subsequently, at 95°C for 12 mins. After this post-exposure baking, the SU-8 2050 film is left to cool, initially at 65°C for 1 min and then, to room temperature. Next, the cooled SU-8 2050 film is developed by rinsing the film three times in a developer provided by the company "Microchem, USA" for 5 mins each time. This is followed by rinsing of the SU-8 2050 film with IPA for 1 min. Then, a final rinsing of the SU-8 2050 film with deionised water is performed, after which, the SU-8 2050 film is blow-dried with gaseous nitrogen. The resulting SU-8 2050 structure forms a SU-8 master mold.

PDMS micromolding

Next, a PDMS micromolding process is performed using the SU-8 master mold to fabricate the microfluidic channel 502a.

The SU-8 master mold is used as follows. 10 parts of PDMS base (Sylgard, USA) are first mixed thoroughly with 1 part of cross-linking agent (Sylgard, USA) to form a PDMS precursor. This PDMS precursor is then poured over the master mold, taking care that the volume of the PDMS precursor used will yield a final thickness of about 4 mm for easy handling. A vacuum dessicator is then used to de-gas the PDMS precursor mix in the master mold. After an hour of degassing, the PDMS precursor is incubated together with the master mold in a convection oven at 65°C for 2 hours (hrs) to form a PDMS cast. Fig. 7 shows the PDMS cast formed on the SU-8 master mold. Next, the PDMS cast is de-molded (i.e. removed from the SU-8 master mold) using absolute ethanol as a lubricant, and is then cut into an appropriate size and shape. Inlet and outlet perforations 602 are made on the PDMS cast. This is done using a Harrick Unicore sampler with a 1 mm inner diameter. The resulting PDMS cast comprising the inlet and outlet perforations 602 forms the microfluidic channel 502a. This microfluidic channel 502a is shown in Fig. 8.

Detector unit 502b

The detector unit 502b comprises a plurality of detectors configured to detect the presence of the carriers.

The plurality of detectors are integrated to form a single detector structure, such that the detectors can be moved simultaneously by moving the single detector structure. The single detector structure is configured to allow current flow through itself, with the plurality of detectors provided on the same current path of the current flow such that the current flow changes whenever one of the detectors detects the carriers. Preferably, the resistance across the single detector structure lies within the range of 200 kn - 500 kQ. . Integrating the detectors to form a single detector structure as opposed to using separately manipulable detectors is advantageous due to the following reasons. Firstly, this integration facilitates the fabrication of the detector unit 502b as less effort is required to form the single detector structure than to form a plurality of separately manipulable detectors. Secondly, it is easier to assemble the plurality of detectors with the microfluidic channel 502a when the detectors are integrated to form the single detector structure. Thirdly, using the single detector structure makes it easier to provide the detectors on the same current path. This in turn obviates the need for more than one voltage source/picoammeter.

More specifically, the single detector structure is in the form of a conjoined carbon nano-tube (CNT)-based sensor as shown in Fig. 9.

The conjoined CNT-based sensor comprises a plurality of detector regions 902 configured to serve as the plurality of detectors. These detector regions 902 are semiconductor regions capable of detecting the presence of the carriers.

The conjoined CNT-based sensor further comprises a plurality of metallic regions 904 which are arranged with the detector regions 902 to allow current flow through the conjoined CNT-based sensor. Each detector region 902 is flanked (bracketed) by two metallic regions 904 and the current flows through the sensor from one metallic region 904 to another across the detector regions 902.

The current flow through the sensor is voltage-driven by the voltage unit 504 and is configured to change whenever any one of the detector regions 902 contacts the carriers. Changes in the current flowing through the conjoined CNT-based sensor thus serve as a means for detecting the presence of the carriers.

The detector regions 902 are fabricated using low-density CNT's to form low density CNT regions whereas the metallic regions 904 are fabricated using high-density CNT's to form high-density CNT regions. Single-walied CNT's having a diameter of 1 - 2nm and a length of 5 - 50 m are used for the fabrication of these CNT regions. As an alternative to the conjoined CNT-based sensor, two CNT-based sensors, each with the structure as shown in Fig. 10, may be used. In particular, each CNT-based sensor comprises one detector region 1002 and two metallic regions 1004 with the detector region 1002 flanked by the metallic regions 1004. In the sensor of Fig. 9, two such sensors are conjoined to achieve the two detector regions 902 and three metallic regions 904 as shown in Fig. 9. The conjoined CNT-based sensor is fabricated by imprinting it on a sheet of transparent material as follows.

A template for the conjoined CNT-based sensor is first formed by casting and cutting a 1 mm thick layer of PDMS. Fig. 1 1 shows the template and its dimensions.

The template is then laid over a cut sheet of transparent material to serve as a mask for printing the conjoined CNT-based sensor onto the transparent surface. Next, the cut portions of the PDMS (i.e. the unshaded portions of the template shown in Fig. 1 1 ) are filled with a 500 g/mL (high density) suspension of single- walled CNT's (CheapTubes.com) in 1 % (w/v) sodium dodecyl benzene sulfate (SDBS). The suspension, together with the template and the transparent material, is then left to dry in a convection oven at 65°C for 30mins. After the suspension dries, the CNT's in the suspension are precipitated onto the transparent surface, forming CNT deposits on the transparent surface. The template is subsequently removed and the CNT deposits are rinsed gently with ultrapure water. The remaining CNT deposits form the metallic regions 904 of the conjoined CNT-based sensor. To form the detector regions 902 of the conjoined CNT-based sensor, 500 μί of a 5Mg/mL (low density) suspension of single-walled CNT's in 1 % (w/v) SDBS is applied to the areas on the transparent surface previously masked by the template. These areas are between the metallic regions 904 previously formed and correspond to the uncut portions of the PDMS (i.e. the shaded portions of the template shown in Fig. 1 1 ). Similarly, this suspension is left to dry in a convection oven at 65°C for 30 mins, after which, CNT's are precipitated onto the transparent surface. This forms CNT deposits on the transparent surface at the areas between the previously formed metallic regions 904. Another gentle rinsing with ultrapure water removes the residual SDBS from the CNT deposits. The remaining CNT deposits (at the areas between the previously formed metallic regions 904) form the detector regions 902 of the conjoined CNT-based sensor.

A cotton swab soaked with isopropanol is then used to carefully scrub away unintentional CNT stains or undesired portions of the conjoined CNT-based sensor on the transparent surface. Fig. 12 shows the resulting conjoined CNT- based sensor imprinted on the transparent material.

Voltage unit 504 The voltage unit 504 comprises a voltage source/picoammeter, more specifically, a Keithley 6487 voltage source/picoammeter.

The Keithley 6487 voltage source/picoammeter serves as both a DC voltage generator for generating voltage across the conjoined CNT-based sensor, as well as an ammeter for measuring the current flowing through the conjoined CNT-based sensor. The Keithley 6487 voltage source/picoammeter achieves its functions by using its leads which are shown as the leads 510 in Fig. 5. These leads 510 electrically couple the Keithley 6487 voltage source/picoammeter with the conjoined CNT-based sensor.

Operations unit 506

The operations unit 506 comprises attendant equipment in the form of a desktop personal computer (PC) and is configured to receive data from the voltage unit 504, and store, process and analyze these data. These data comprise information regarding the current flow through the conjoined CNT- based sensor. The operations unit 506 is further configured to control the voltage unit 504. In particular, the PC comprises Labw^'ew-compatible hardware and is configured to control the voltage unit 504 with a Labview-based program. For the operations unit 506 to perform the above, a wired link 512 (see Fig. 5) is established to allow communication of data between the operations unit 506 and the voltage unit 504.

Assembling the apparatus 500

The following steps are performed to assemble the apparatus 500.

The microfiuidic device 502 is first assembled. In particular, the microfiuidic channel 502a is sealed against the transparent material on which the conjoined CNT-based sensor is printed to integrate the conjoined CNT-based sensor with the microfiuidic channel 502a. This is done such that when a sample flows through the microfiuidic channel 502a, the detector regions 902 are able to contact the sample at different positions along the microfiuidic channel 502a. Care is taken to position two portions of the microfiuidic channel 502a directly over the detector regions 902 of the conjoined CNT-based sensor. Fig. 13 shows how the microfiuidic channel 502a is positioned with the conjoined CNT- based sensor.

As shown in Fig. 13 (and more clearly shown in Fig. 5), although the detector regions 902 are relatively near each other, the distance along the microfiuidic channel 502a between the positions corresponding to the detector regions 902 is still considerably long, especially with the loops of the channel 502a. This is advantageous as allowing the carriers to travel through a larger distance from one detector region 902 to the other allows changes in the current flow through the sensor due to the detection of the carriers to be more reliably detected, while having the detector regions 902 near each other allows easier assembly of the apparatus 500 and a shorter current flow distance between the detector regions 902, so the noise in the current measured may be reduced. Having the detector regions 902 near each other also facilitates the fabrication of the conjoined CNT-based sensor. As mentioned above, integrating the detectors to form such a single detector structure makes it easier to provide the detectors on the same current path and this in turn obviates the need for more than one voltage source/picoammeter.

The microfluidic channel 502a is then secured with the transparent material on which the conjoined CNT-based sensor is printed, using securing elements in the form of two binder clips as shown in Fig. 14.

The leads 510 of the Keithley 6487 voltage source/picoammeter are then clipped to the metallic regions 904 of the conjoined CNT-based sensor. Furthermore, the link 512 between the operations unit 506 and the voltage unit 504 is established.

Using the apparatus 500 to analyze interactions of molecules of interest

As mentioned above, the apparatus 500 is capable of analyzing interactions of molecules of interest and uses a plurality of carriers, with the plurality of carriers comprising embedded carriers and non-embedded carriers. To analyze the interactions of the molecules of interest, the apparatus 500 performs quantitative analysis of samples caused to flow through the microfluidic channel 502a.

In particular, to analyze interactions of the molecules of interest, the microfluidic device 502 is configured to cause a first and second sample to flow separately through the microfluidic channel 502a. The first and second samples respectively comprise the embedded carriers and the non-embedded carriers. Note that the first sample need not be caused to flow through the microfluidic channel 502a first. In other words, the second sample can be caused to flow through the microfluidic channel 502a before the first sample. During the flow of each sample through the microfluidic channel 502a, the carriers in the sample travel through the channel 502a with the flow of the sample and via diffusion. The plurality of detectors in the form of the two detector regions 902 are configured to detect presence of the carriers at respective positions along the microfluidic channel 502a as each of the first and second samples flows through the microfluidic channel 502a. These positions define a part of the microfluidic channel 502a. In particular, they serve as start and end points of a defined path along the microfluidic channel 502a. More specifically, when the carriers reach a position along the channel 502a corresponding to a detector region 902, contact of the detector region 902 with the carriers causes a change in the current through the conjoined CNT-based sensor. Based on changes in this current, the presence of the carriers at the points along the channel 502a corresponding to the detector regions 902 can be detected.

The operations unit 506 is configured to determine transport rates of the embedded carriers and the non-embedded carriers, more specifically, rates at which the embedded carriers and the non-embedded carriers travel through the part of the microfluidic channel 502a (defined by the positions corresponding to the detector regions 902) during the flow of the first and second samples respectively. This is done based on the detections made by the detector regions 902. The operations unit 506 is further configured to analyze the interactions of the molecules of interest based on these determined rates.

Figs. 15 - 17 illustrate how the rates are determined by the operations unit 506 and how these rates may be used to analyze the interactions of the molecules of interest:

In particular, Fig. 15 shows a schematic diagram of the microfluidic channel 502a together with the detector regions 902 shown as "Detector 1 " and "Detector 2". As shown in Fig. 15, a sample comprising a plurality of carriers 1502 (which may be the first sample or the second sample) is caused to flow through the microfluidic channel 502a. Note that Fig. 15 only serves to illustrate how the rates are determined and does not accurately show the structure of the microfluidic channel 502a or how this channel 502a is arranged with the detector regions 902.

To determine the rate the carriers 1502 travel through the part of the microfluidic channel 502a flanked by Detector 1 and Detector 2, the amount of time taken for the carriers to travel through this part is determined. This is done based on the detections made by Detector 1 and Detector 2 as explained below with reference to Fig. 16.

In particular, Fig. 16 shows a graph illustrating a possible current waveform through the conjoined CNT-based sensor when a sample comprising carriers is caused to flow through the microfluidic channel 502a. As shown in Fig. 16, at time = ti , a first spike in the current occurs and at time = t₂, a second spike in the current occurs. The first and second spikes in the current respectively represent signals from Detector 1 and Detector 2, more specifically, signals indicating that Detector 1 and Detector 2 have detected the presence of the carriers at their respective positions along the microfluidic channel 502a. By taking the difference between the time the first spike occurs and the time the second spike occurs (i.e. t₂ - ti), the amount of time taken by the carriers 1502 to travel through the part of the microfluidic channel 502a flanked by Detector 1 and Detector 2 can be determined. Again, note that Fig. 16 simply serves to illustrate how the rates are determined and the current waveform through the conjoined CNT-based sensor need not be identical to that shown in Fig. 16.

The rate at which the carriers 1502 travel through the part of the microfluidic channel 502a flanked by Detector 1 and Detector 2 can be simply expressed as the amount of time determined above (since the distance between Detector 1 and Detector 2 is fixed). Alternatively, the rate can be calculated by dividing the distance between Detector 1 and Detector 2 with the amount of time determined above. As mentioned above, to analyze the interactions of the molecules of interest, first and second samples respectively comprising the embedded carriers and the non-embedded carriers are caused to flow through the microfluidic channel 502a. Interactions between the molecules of interest in the embedded carriers tend to result in the transient binding of the embedded carriers to each other while they are entrained in the microfluidic flow of the first sample. The cumulative effect of the transient binding events between the plurality of embedded carriers retards the transport of the embedded carriers through the microfluidic channel 502a sufficiently, such that it results in a noticeable difference between the rates at which the embedded carriers and the non- embedded carriers travel through the part of the microfluidic channel flanked by Detector 1 and Detector 2. Based on this difference, the amount of interactions of the molecules of interest during the flow of the first sample through the part of the microfluidic channel 502a can be determined. Fig. 17 illustrates this.

Particularly, Fig. 17 shows two graphs illustrating possible current waveforms through the conjoined CNT-based sensor when the first and second samples are caused to flow through the microfluidic channel 502a. As shown in Fig. 7, the amount of time taken for the non-embedded carriers to travel through the part of the microfluidic channel 502a flanked by Detector 1 and Detector 2 is t₂ - ti . This is used as a reference. The amount of time taken for the embedded carriers to travel through the same part of the microfluidic channel 502a is t₃ - ti. The difference between these amounts of time is t3 - t₂ and indicates the amount of interactions of the embedded carriers as they travel through the part of the microfluidic channel 502a flanked by Detector 1 and Detector 2. Similar to Fig. 16, Fig. 17 simply serves to illustrate how interactions of the molecules of interest may be analyzed based on the rates. The current waveforms through the conjoined CNT-based sensor need not be in the form shown in Fig. 17.

Further molecules may be integrated with the microfluidic channel 502a prior to the flow of the first and second samples through the microfluidic channel 502a. For example, the further molecules may be immobilized along the walls of the microfluidic channel 502a. If the molecules of interest interact with the further molecules, more transient binding events will occur when the first sample flows through the microfluidic channel 502a. These additional transient binding events further retard the transport of the embedded carriers through the microfluidic channel 502a. Therefore, the amount of interactions between the molecules of interest and the further molecules can be determined based on the rate the embedded carriers travel through the microfluidic channel 502a integrated with the further molecules. The further molecules may be embedded with carriers of the same type as those in the first and second samples before they are integrated with the channel 502a.

The further molecules may be of the same type as the molecules of interest embedded in the carriers of the first sample. In this case, the number of binding events between the molecules of interest increases as the embedded carriers in the first sample travel through the microfluidic channel. This helps in the analysis of the interactions between the molecules of interest as the difference in the transport rates of the embedded carriers and the non-embedded carriers will be more significant.

Alternatively, the further molecules may be of a different type from the molecules of interest embedded in the carriers of the first sample. This allows the interactions between the molecules of interest and the further molecules to be analyzed.

The apparatus 500 can be used for drug or compounds screening. In particular, the microfluidic device 502 may be further configured to cause a third sample to flow through the microfluidic channel 502a. The third sample comprises another plurality of carriers (of the same type as those in the first and second samples) embedded with the molecules of interest, as well as, additives such as drugs or compounds which may potentially modify the interactions of the molecules of interest. The operations unit 506 can be configured to determine the rate at which this plurality of carriers in the third sample travel along the part of the microfiuidic channel 502a flanked by the detector regions 902 of the conjoined CNT-based sensor. Based on this rate and the rate at which the embedded carriers in the first sample travel through the same part of the microfiuidic channel 502a, the effects of the additives on the interactions of the molecules of interest can be determined.

More specifically, it can be determined if the additives modify the interactions of the molecules of interest. The additives may induce interactions of the molecules of interest, for example, they may increase the amount of interactions between these molecules or bring about interactions between nominally non- interacting partners. In this case, the transport rate of the carriers in the third sample will be lower than that of the carriers in the first sample, and hence, as compared to the carriers in the first sample, the carriers in the third sample will take a longer time to travel through the part of the microfiuidic channel 502a flanked by the detector regions 902. The additives may alternatively inhibit interactions of the molecules of interest. In this case, the transport rate of the carriers in the third sample will be higher than that of the carriers in the first sample, and hence, as compared to the carriers in the first sample, the carriers in the third sample will take a shorter time to travel through the part of the microfiuidic channel 502a flanked by the detector regions 902. If the additives have a negligible effect on the interactions of the molecules of interest, the transport rates of the carriers in the third sample and that of the carriers in the first sample should not have any significant difference.

Instead of including the additives in a third sample and subsequently causing the third sample to flow through the microfiuidic channel 502a, the additives may alternatively be immobilized (i.e. coated) along the walls of the microfiuidic channel 502a. In this case, the effect of the additives on the interactions of the molecules of interest can be determined based on the difference in the transport rates of the embedded carriers through the microfiuidic channel 502a with and without the additives-coated walls. Similarly, instead of immobilizing the above- mentioned further molecules to the walls of the channel 502a, these further molecules may be comprised together with embedded carriers in a sample to be caused to flow through the channel 502a. In general, any type of further molecule or additive can be immobilized along the walls of the microfluidic channel 502a as long as the immobilization does not damage the microfluidic channel 502a. The decision on whether to immobilize the further molecules and/or additives along the walls of the channel 502a, or to incorporate them together with embedded carriers in a sample to be caused to flow through the channel 502a depends on the context and aim of the experiment. For example, to analyze typical interactions between nominally free-moving molecules and the molecules of interest, it is not advisable to immobilize the nominally free-moving molecules to the walls of the microfluidic channel 502a. Instead, it is better to incorporate these nominally free-moving molecules together with embedded carriers in a sample to be caused to flow through the channel 502a. This is because immobilizing nominally free-moving molecules may change the behavior of these molecules and in turn, change the interactions of these molecules with the molecules of interest. On the other hand, if a researcher aims to study how immobilizing nominally free-moving molecules changes their interactions with the molecules of interest, then immobilizing these nominally free-moving molecules along the walls of the channel 502a can allow the researcher to achieve this aim. In cases whereby both decisions are equally effective in achieving the aim of the experiment, it may be preferable to adopt the decision which requires less effort.

The microfluidic device 502 can be further configured to cause a fourth sample comprising a further plurality of carriers to flow through the microfluidic channel 502a whereby the further plurality of carriers in this fourth sample are embedded with the molecules of interest and are geometrically different from the carriers in the first and second samples. Similarly, the operations unit 506 can be configured to determine the rate at which this further plurality of carriers travel through the part of the microfluidic channel 502a flanked by the detector regions 902. Based on this rate and the rate at which the embedded carriers in the first sample travel through the same part of the microfluidic channel 502a, the effects of using geometrically different carriers on the interaction of the molecules of interest can be determined.

The carriers in the first and second samples, or those in the third and fourth samples as mentioned above may comprise one or more of: cells, cell-mimics, viruses, artificial membranes, polymer vesicles, lipid vesicles. The molecules of interest, or the further molecules integrated with the channel 502a, may comprise one or more of: membrane associated proteins, cell adhesion proteins, synthetic proteins, peptides, lipids, carbohydrates, proteoglycans, metabolites, macromolecules, drugs. In one example, the molecules of interest are the Claudin-2 molecules discussed above. A more specific example of how the apparatus 500 may be used to analyze interactions between Claudin-2 molecules is described below.

Specific example of using the apparatus 500 to analyze interactions between Claudin-2 molecules

The following describes a specific example of using apparatus 500 for analyzing interactions between Claudin-2 molecules.

Preparation of carriers in the form of ABA vesicles

In this example, the carriers are in the form of ABA vesicles which are polymer vesicles. The ABA vesicles are prepared from poly(2-methyloxazoline)2o- poly(dimethylsiloxane)5₄-poly(2-methyloxazoline)₂o which is an ABA tri-block copolymer (Polymer Source, Canada) in the following manner. 5mg of the ABA tri-block copolymer is first dissolved in 200 L of analytical grade ethanol in a vessel. The resulting solution is then carefully blow-dried using nitrogen or argon gas until a thin polymer film is deposited against the vessel wall. The thin polymer film is dried further under a constant stream of nitrogen for 4hrs under a Schlenk line. One milliliter of ultrapure water is then added to the thin polymer film to form a mixture. Next, the mixture is stirred for about 18hrs at 150rpm, using a magnetic stirrer. The resulting suspension is then filtered 4 times through a 0.45 pm PVDF syringe filter, followed by 6 times through a 0.2 μιη PVDF syringe filter. This forms a vesicle suspension comprising ABA vesicles. This vesicle suspension is extruded using a Millipore filter of 100nm pore size and is dialyzed against water for 24hrs to remove any residual ethanol.

Preparation of Claudin-2-embedded ABA vesicles

Performing an in vitro synthesis of Claudin-2 molecules in the presence of the ABA vesicles allows the Claudin-2 molecules to embed into the ABA vesicles and assume their native conformations. In this way, the ABA vesicles can serve as scaffolds to provide membrane-mimic support for the Claudin-2 molecules.

In this example, Claudin-2-embedded ABA vesicles are prepared using a commercially-available cell-free in vitro synthesis kit as follows.

Complementary DNA (cDNA) encoding Claudin-2 is first inserted into a pTNT T7-promoter expression vector using standard molecular biology techniques. The resultant plasmid is amplified in DH10cc Escherichia coli culture, extracted and purified, again using standard molecular biology techniques.

In vitro synthesis of the Claudin-2 molecules is then carried out using a commercially-available cell-free in vitro synthesis kit in the form of a wheat germ extract cell-free expression system (Promega, USA). This is done together with the amplified plasmid, as well as the ABA vesicles prepared in the above manner. These ABA vesicles serve as carrier membrane architectures. The reaction mixtures for the in vitro synthesis of the Claudin-2 molecules are prepared according to the supplier's instructions and are incubated at 37°C for 90 mins. The wheat germ extract cell-free expression system produces the Claudin-2 proteins (i.e. the Claudin-2 molecules) and during this protein production, the Claudin-2 molecules insert into the ABA vesicles, forming a suspension comprising the Claudin-2-embedded ABA vesicles. Fig. 18 shows the results of performing a Western blot with anti-Claudin-2 antibodies on four samples (numbered 2 - 5) respectively comprising Claudin- 2-embedded ABA vesicles, Claudin-2 molecules not embedded in vesicles, ABA vesicles embedded with DRD2 molecules (i.e. DRD2-embedded ABA vesicles) and non-embedded ABA vesicles. The ABA vesicles and Claudin-2- embedded vesicles are formed using the methods as described above. DRD2 refers to an alternative membrane protein, more specifically, dopamine receptor D2, and is used here as a negative control. DRD2-embedded ABA vesicles are formed in the same manner as the Claudin-2-embedded ABA vesicles except that DRD2 molecules instead of Claudin-2 molecules are used.

To perform the Western blot, the four samples are electrophoresced using denaturing SDS-PAGE at 80V, 100 mA and are then transferred to a nitrocellulose membrane for immunoblotting using the iBIot® Dry Blotting system (Invitrogen). The nitrocellulose membrane is then stained with anti- Claudin-2 antibodies using the Invitrogen Western Breeze® kit. Two molecular weight markers i.e. the MagicMark™ XP Western Protein Standard and the SeeBlue^® Plus 2 Pre-stained Standard (respectively numbered 1 and 6 in Fig. 18) are used. In the Western blot results of Fig. 18, the black stains as referred to by the arrows indicate the presence of Claudin-2. These black stains are not present for sample 4 comprising the DRD2-embedded ABA vesicles or sample 5 comprising the non-embedded ABA vesicles, whereas they are present for samples 2 and 3 respectively comprising the Claudin-2-embedded ABA vesicles and the Claudin-2 molecules. Therefore, the results of Fig. 18 indicate that the Claudin-2 proteins in samples 2 and 3 are successfully formed. Furthermore, the black stain indicating the presence of Claudin-2 in lane 2 (corresponding to sample 2) is slightly higher than that in lane 3 (corresponding to sample 3). This is due to the fact that the Claudin-2 proteins in Jane 2 were traveling more slowly through the PAGE gel than the Claudin-2 proteins in lane 3. This indicates that the Claudin-2 molecules in sample 2 are associated with the ABA vesicles (most likely embedded into the ABA vesicles). In other words, the Claudin-2-embedded ABA vesicles are successfully formed using the method as described above.

Ability of the conjoined CNT-based sensor to detect ABA vesicles

The following is performed to determine if the conjoined CNT-based sensor is capable of detecting the presence of the ABA vesicles. The ABA vesicles used here are non-embedded ABA vesicles. With the leads from the Keithley 6487 picoammeter/voltage source clipped to the metallic regions 904 of the conjoined CNT-based sensor, a plurality of vesicle suspensions comprising increasing concentrations of the ABA vesicles in de-ionized water are successively added to the detector regions 902 of the conjoined CNT-based sensor.

Fig. 19 shows a graph plotting the current through the conjoined CNT-based sensor over the time period the plurality of vesicle suspensions are successively added to the detector regions 902. The graph in Fig. 19 reveals a dose- dependent change in the current through the sensor. In particular, the addition of each vesicle suspension causes a decrease in the current through the sensor. As a more highly concentrated vesicle suspension is added, the decrease in current caused by the addition becomes greater. This shows that the conjoined CNT-based sensor is capable of detecting the non-embedded ABA vesicles.

Note however that for the last two suspensions added, even though these suspensions comprise higher concentrations of the ABA vesicles than the remaining suspensions, the change in current for each of these suspensions is of a smaller magnitude than the change in current for the previously added suspensions. This is due to the saturation of the conjoined CNT-based sensor surface.

Ability to detect ABA vesicles perfused through the microfluidic channel 502a

After determining if the conjoined CNT-based sensor is capable of detecting the presence of the ABA vesicles, the following is performed to determine if the conjoined CNT-based sensor is capable of detecting the presence of the ABA vesicles as they are perfused through the microfluidic channel 502a. The ABA vesicles used here are also non-embedded ABA vesicles.

First, the transparent material on which the conjoined CNT-based sensor is imprinted (as shown in Fig. 12) is thoroughly rinsed with isopropanol, followed by water and finally ethanol. The transparent material is then left to dry in a convection oven at 65°C for 30mins. This serves to remove any ABA vesicles left on the transparent material from the above-mentioned process of determining if the sensor is capable of detecting the presence of the vesicles.

Next, the apparatus 500 is assembled in the manner as described above with reference to Fig. 14. In particular, the microfluidic channel 502a is sealed against the transparent sheet on which the conjoined CNT-based sensor is imprinted and the leads from the Keithley 6487 picoammeter/voltage source are then clipped to the metallic regions 904 of the conjoined CNT-based sensor. A poly(ether ether ketone) (in short, PEEK) tubing is then inserted into one of the perforations 602, in particular, the perforation 602 serving as the outlet of the microfluidic channel 502a. This is shown in Fig. 14. The PEEK tubing leads to a syringe installed into a syringe pump. The syringe pump is configured to withdraw fluid (which may be simply air) from the channel 502a when activated. If a sample is applied to the other perforation 602 serving as the inlet of the microfluidic channel 502a, the withdrawal by the syringe pump will cause the sample to be withdrawn into the channel 502a. The syringe pump is initially configured to withdraw at a rate of 10.5 μΙ_/ιηίη . In other words, the syringe pump is configured such that the sample applied at the inlet of the microfluidic channel 502a is caused to flow through the channel 502a at a flow rate of 10.5μΙ_/Ίηϊη . Acquisition of data indicating the current through the conjoined CNT-based sensor then commences, with a droplet of water comprising 100μί. of 0.22 pm - filtered ultrapure water applied to the inlet. The data is continuously acquired for 5mins or until the signal noise level of the data drops below 50pA. Thereupon, the syringe pump is activated, withdrawing the droplet of water applied to the inlet into the microfluidic channel 502a.

Fig. 20 shows a graph plotting the current through the conjoined CNT-based sensor during the above acquisition of data. As shown in Fig. 20, the current through the conjoined CNT-based sensor decreases at two time points (see arrows in Fig. 20). Since the contact of the droplet of water with each of the detector regions 902 is expected to trigger a change in the current through the conjoined CNT-based sensor, the results in Fig. 20 indicate that the droplet of water has successfully come into contact with the detector regions 902 and this contact is successfully detected. This in turn indicates that the apparatus 500 is properly assembled. The syringe pump is then de-activated and the PEEK tubing is removed from the outlet of the microfluidic channel 502a. Next, the syringe and PEEK tubing are completely filled with ultrapure water, taking care to remove all residual air. The microfluidic channel 502a is also completely filled with ultrapure water, with care taken to ensure no air bubbles are trapped therein.

Next, the PEEK tubing is reinserted into the outlet of the microfluidic channel 502a. The syringe pump is then activated to perfuse ultrapure water through the microfluidic channel 502a at a flow rate of 10.5μί-/ιηίη for a further 5mins, or until the baseline stabilizes and the noise level falls below 50pA.

The syringe pump is then reconfigured to withdraw at a reduced rate of Ο.δμϋΊηίη . An assay to detect if the conjoined CNT-based sensor is able to detect ABA vesicles under perfusion is then performed. This assay is done by first applying 2 μΙ_ of a vesicle suspension comprising non-embedded ABA vesicles in ultrapure water to the inlet of the microfluidic channel 502a with a micropipette, followed by a continuous application of ultrapure water to the inlet. The continuous application of ultrapure water is done immediately after the vesicle suspension has been completely consumed (i.e. immediately after the vesicle suspension has completely flowed through the microfluidic channel 502a).

The current flow across the conjoined CNT-based sensor is monitored throughout the above steps. Fig. 21 shows the current through the conjoined CNT-based sensor over the time period the assay is performed. As shown in Fig. 21 , the current shows significant changes at two time points (see arrows in Fig. 21 ). Since contact of the ABA vesicles with the detector regions 902 is expected to trigger changes in the current through the conjoined CNT-based sensor, the results in Fig. 21 indicate that the conjoined CNT-based sensor is able to successfully detect the presence of the ABA vesicles under perfusion. Furthermore, the arrows in Fig. 21 provide an indication of when the ABA vesicles contact each of the detector regions 902 of the conjoined CNT-based sensor. The amount of time taken for the ABA vesicles to travel through the part of the microfluidic channel 502a flanked by the detector regions 902 can be determined from the time span between the time points corresponding to the arrows in Fig. 21 . This amount of time may also be referred to as the elution time for the assay.

Five assays are performed consecutively in the manner as described above, yielding elution times differing with a relative standard deviation of 2.7%. This indicates that the apparatus 500 is able to produce relatively consistent results.

Coating the walls of the microfluidic channel 502a with Claudin-2-embedded ABA vesicles

Next, the walls of the microfluidic channel 502a are coated with Claudin-2- embedded ABA vesicles using first, an antibody immobilization protocol and subsequently, a vesicle immobilization protocol. The antibody immobilization protocol serves to immobilize a plurality of antibodies raised against Claudin-2 molecules (i.e. anti-Claudin-2 antibodies) along the walls of the microfluidic channel 502a whereas the vesicle immobilization protocol serves to immobilize the Claudin-2-embedded ABA vesicles to the antibodies.

The antibody immobilization protocol is as follows.

The microfluidic channel 502a is first treated with oxygen plasma at 100W for 80 sees and is then temporarily sealed against a flat PDMS substrate. Protein A (from Staphylococcus aureus, Sigma-Aldrich) dissolved at 25 pg/mL in phosphate-buffered saline with 0.05% (w/v) sodium azide (PBS-SA) is then perfused into the microfluidic channel 502a at a rate of 1 pL/min for 60mins. The protein A is subsequently left to incubate in the microfluidic channel 502a for a further 60mins. This adsorbs the protein A into the walls of the microfluidic channel 502a.

Next, PBS-SA is perfused onto the walls of the microfluidic channel 502a at a rate of l O LAnin for 120mins. An antibody mixture comprising anti-Claudin-2 antibodies is then perfused through the microfluidic channel 502a at a rate of 1 pL/min for 30mins. More specifically, the antibody mixture comprises two types of antibodies, namely, type (i) antibodies: IgG (rabbit, anti-Claudin-2, polyclonal, ZYMED® Laboratories) and type (ii) antibodies: goat, anti-mouse fluorescein- conjugated IgG, with each of type (i) and type (ii) at a concentration of 0.05mg/mL After the perfusion of the antibody mixture, the antibody mixture eluent is collected and reperfused at a rate of 1 pLAnin for 30mins. A final perfusion of ultrapure water through the microfluidic channel 502a at a rate of Ι Ομ-Jmin for 60 mins completes the antibody immobilization protocol.

The protein A is capable of binding the anti-Claudin-2 antibodies in the type (i) antibodies. Therefore, by inserting the anti-Claudin-2 antibodies into the microfluidic channel 502a via the above perfusions, the protein A absorbed into the walls of the channel 502a can bind the anti-Claudin-2 antibodies, thereby immobilizing these antibodies to the walls of the channel 502a.

The protein A is also able to bind the type (ii) antibodies to immobilize these antibodies to the walls of the channel 502a. The type (ii) antibodies serve to indicate how successful the antibody immobilization protocol is. In particular, the type (ii) antibodies are configured to emit green light (with an emission wavelength of 520nm) under blue light excitation (i.e. excitation wavelength of 490nm). Therefore, the success of the antibody immobilization protocol may be evaluated by illuminating the channel 502a under blue light and acquiring an image of the channel 502a under this illumination. If green areas are present in the acquired image, this indicates that the type (ii) antibodies are successfully immobilized to the walls of the channel 502a. This generally implies that the type (i) antibodies are also successfully immobilized to the walls of the channel 502a.

Figs. 22(a) - (d) show the results of the antibody immobilization protocol. In particular, Figs. 22(a) and (c) show images of a part of the microfluidic channel 502a before the antibody immobilization protocol, whereas Figs. 22(b) and (d) show images of the same part of the microfluidic channel 502a after the antibody immobilization protocol. Specifically, the part shown in Figs. 22(a) - (d) is a part of a loop of the channel 502a. Figs. 22(a) and (b) are bright field images i.e. images of the part of the channel 502a illuminated under white light, whereas Figs. 22(c) and (d) are fluorescent images i.e. images of the part of the channel 502a illuminated under blue light. Note that since Figs. 22(c) and (d) are in grayscale, any green area in these images will appear gray. As shown in Fig. 22(c), the fluorescent image obtained before the antibody immobilization protocol does not comprise any green area. On the other hand, as shown in Fig. 22(d), the fluorescent image obtained after the antibody immobilization protocol comprises green areas in the part of the channel 502a shown in this image. This indicates that the type (ii) antibodies are successfully immobilized to the walls of the microfluidic channel 502a. This in turn implies that the type (i) antibodies are also successfully immobilized to the walls of the channel 502a. In other words, the antibody immobilization protocol is successful.

Next, a vesicle immobilization protocol is performed as follows.

In particular, a first aliquot of Claudin-2-embedded ABA vesicles is perfused through the microfluidic channel 502a (with the walls previously immobilized with the anti-Claudin-2 antibodies). This is done at a rate of 0.5μΙ_Ληίη for 45 mins. Via this perfusion, some of the Ciaudin-2-embedded ABA vesicles are captured by the anti-Claudin-2 antibodies immobilized along the walls of the microfluidic channel 502a. Next, a second aliquot of Claudin-2-embedded ABA vesicles is perfused through the microfluidic channel 502a. Similarly, some of the Claudin-2-embedded ABA vesicles in the second aliquot are captured by the anti-Claudin-2 antibodies, in particular, those that have not already captured the Claudin-2-embedded ABA vesicles from the first aliquot. The Claudin-2- embedded ABA vesicles are thus immobilized along the walls of the channel 502a. A final perfusion of ultrapure water through the microfluidic channel 502a at a rate of 0.5μΙ_/ιηίη for 60 mins completes the vesicle immobilization protocol.

The current through the conjoined CNT-based sensor is measured during the above-mentioned perfusions of the first and second aliquots of Claudin-2- embedded ABA vesicles. For comparison, a sample comprising non-embedded ABA vesicles is also perfused through the microfluidic channel 502a (with the antibodies immobilized along its walls) in a manner similar to that for the Claudin-2-embedded ABA vesicles as described above. The current through the conjoined CNT-based sensor is also measured during this perfusion.

Figs. 23(a) - (c) respectively show the currents through the conjoined CNT- based sensor during the perfusions of the non-embedded ABA vesicles, the first aliquot of Claudin-2-embedded ABA vesicles and the second aliquot of Claudin- 2-embedded ABA vesicles. The arrows in each of Figs. 23(a) - (c) indicate the points on the current waveform showing significant changes. The time points at which these significant changes occur are the time points at which one of the detector regions 902 of the conjoined CNT-based sensor detects the ABA vesicles.

As shown in Figs. 23(a) - (c), compared to the non-embedded ABA vesicles, a longer time is taken by the Claudin-2-embedded ABA vesicles to travel the part of the channel 502a flanked by ^"the detector regions 902. In particular, the time taken for the non-embedded ABA vesicles to travel this part of the channel 502a is 17.5mins whereas the time taken for the Claudin-2-embeddecl ABA vesicles to travel this part of the channel 502a is 20mins (for both the first and second aliquots). This indicates that the transport of the Claudin-2-embedded ABA vesicles is retarded and this is probably due to the interactions of these vesicles with the immobilized anti-Claudin-2 antibodies and with each other.

Comparing the transport rates of Claudin-2-embedded ABA vesicles and non- embedded ABA vesicles through the microfluidic channel 502a To determine if the Claudin-2 molecules synthesized and inserted into the ABA vesicles are able to interact with each other, non-embedded ABA vesicles are used as a reference and Claudin-2-embedded ABA vesicles are used as test samples. Furthermore, the microfluidic channel 502a with its walls immobilized with the Claudin-2-embedded ABA vesicles is used.

In particular, a series of assays are performed with each assay carried out as follows. Two samples, one comprising the Claudin-2-embedded ABA vesicles and the other comprising the non-embedded ABA vesicles are perfused separately through the microfluidic channel 502a at the same flow rate. As before, the time taken for the vesicles in each sample to traverse the distance along the channel 502a flanked by the detector regions 902 (i.e. the elution time for each sample) is taken to be the time between the perturbations of the two detector regions 902 as indicated by significant changes in the current through the conjoined CNT-based sensor.

Figs. 24(a) and (b) illustrate the results for one of the assays. In particular, Fig. 24(a) shows the current through the conjoined CNT-based sensor when the sample comprising the non-embedded ABA vesicles is perfused through the microfluidic channel 502a whereas Fig. 24(b) shows the current through the sensor when the sample comprising the Claudin-2-embedded ABA vesicles is perfused through the channel 502a. As shown in Figs. 24(a) and (b), the elution time for the sample comprising the Claudin-2-embedded ABA vesicles is longer than that for the sample comprising the non-embedded ABA vesicles. More specifically, the assay yielded an elution time of 18.7mins for the sample comprising the non-embedded ABA vesicies, and 22.25mins for the sample comprising the Claudin-2-embedded ABA vesicles. In other words, in this assay, the Claudin-2-embedded ABA vesicles take 3.55mins longer than the non- embedded ABA vesicles to cross the distance along the channel 502a between the detector regions 902. This shows that the Claudin-2-embedded ABA vesicles are interacting with each other and apparatus 500 is able to detect the interactions.

Further assays for analyzing interactions between Claudin-2 molecules

Further assays using alternative anti-Claudin-2 antibodies and carriers in the form of polybutadiene vesicles are also performed. In particular, fresh samples of vesicles are prepared using an alternative polymer, poly (butadiene)-d-poly (ethylene oxide) (in short, BD21 ). The BD21 vesicles are used for the in vitro synthesis of Claudin-2. Furthermore, the walls of a fresh microfluidic channel 502a are coated with monoclonal anti-Claudin-2 antibodies (Abnova) and then immobilized with Claudin-2-embedded BD21 vesicles.

For each further assay, two samples respectively comprising non-embedded BD21 vesicles and Claudin-2-embedded BD21 vesicles are separately perfused through the microfluidic channel 502a (with its walls immobilized with Claudin-2- embedded BD21 vesicles). The interactions of the Claudin-2 molecules are then analyzed using the apparatus 500 in the same manner as described above.

Figs. 25(a) and (b) illustrate the results for one of these further assays. In particular, Fig. 25(a) shows the current through the conjoined CNT-based sensor when the sample comprising the non-embedded BD21 vesicles is perfused through the microfluidic channel 502a whereas Fig. 25(b) shows the current through the sensor when the sample comprising the Claudin-2- embedded BD21 vesicles is perfused through the channel 502a. As shown in Figs. 25(a) and (b), the elution time for the sample comprising the Claudin-2- embedded BD21 vesicles is longer than that for the sample comprising the non- embedded BD21 vesicles. More specifically, in this assay, the Claudin-2- embedded BD21 vesicles take 0.65mins longer than the non-embedded BD21 vesicles to cross the distance along the channel 502a between the two detector regions 902. This shows that the Claudin-2-embedded BD21 vesicles are interacting with each other and apparatus 500 is able to detect the interactions. In summary, all the assays, including those using the ABA vesicles and those using the BD21 vesicles, show that the in vitro synthesized Claudin-2 molecules are capable of claudin-claudin interaction and that this interaction noticeably increases the time taken for the vesicles to traverse the distance along the channel 502a between the two detector regions 902 of the conjoined CNT- based sensor.

Other alternative assays may also be performed to analyze the interactions between the Claudin-2 molecules.

In these alternative assays, the Claudin-2-embedded vesicles may be formed using cell- or virus-based in vitro synthesis protocols instead of the cell-free in vitro synthesis kit as described above.

Furthermore, in these alternative assays, the Claudin-2-embedded vesicles may be immobilized along the walls of the microfluidic channel 502a using chemical and/or physical means other than the one described above. The same applies for the immobilization of the anti-Claudin-2 antibodies along the walls of the channel 502a if these antibodies are used. Other proteins instead of Protein A capable of binding the anti-Claudin-2 antibodies may also^'1' be used and these proteins may be coated or immobilized onto the walls of the channel 502a instead of adsorbed into these walls.

Variations to apparatus 500 Although only a single embodiment of the invention has been described in detail above, it is to be understood that many variations are possible within the scope of the invention, as defined by the claims. A few examples of such variations are given below.

The microfluidic channel 502a may be of a different structure than that shown in Fig. 6 or it may be of the same structure but with different dimensions. The cross-section of the microfluidic channel 502a need not be rectilinear and may be of a different shape.

Although the detector unit 502b comprises only two detectors, more than two detectors may be used and may be configured to detect the presence of the carriers at more than two positions along the microfluidic channel 502a. These positions thus define more than one part of the microfluidic channel 502a which can be used for analyzing the interactions of the molecules of interest. Different parts of the microfluidic channel 502a may have walls coated with different types of further molecules and/or additives, so that the interactions between the molecules of interest and the different types of further molecules, and/or the effects of different types of additives may be analyzed using a single microfluidic channel 502a.

In addition, the conjoined CNT-based sensor need not be of the same structure as that shown in Fig. 9 and may comprise a different number of detector regions and metallic regions arranged differently. Alternatively, the detector unit 502b may comprise detectors which can be moved independently of each other. This allows the distance between the detectors to be varied (as opposed to the fixed distance between the detectors of the conjoined CNT-based sensor). The detector unit 502b may also comprise any type of detectors as long as they are capable of detecting the presence of the carriers. For example, since the electrical properties of parts of a sample comprising the carriers differ from the electrical properties of other parts of the sample, the detectors may be any detectors which are sensitive to the electrical properties of the part of the sample interacting with them. Alternatively, the carriers may comprise fluorescent probes. This may be done by modifying the carriers with the fluorescent probes, either by incorporation into the carrier membranes or encapsulation within the carriers. In this case, each detector may comprise a light source and a receptor. The light source may be configured to activate the fluorescent probes to generate fluorescent signals as the carriers travel through the position along the microfluidic channel 502a corresponding to the detector, whereas the receptor may be configured to receive the fluorescent signals to detect the presence of the carriers at the corresponding position.

Depending on the type of detectors used, the detectors of the detector unit 502b also need not be in contact with the sample flowing through the microfluidic channel 502a. However, it is preferable if the detectors are at least in close proximity with the channel 502a to achieve more accurate results.

The detectors of the detector unit 502b also need not be arranged with the microfluidic channel 502a in the manner shown in Figs. 5 and 12. But it is preferable if one or more distances between the detectors are minimized while one or more distances along the microfluidic channel 502a between the positions corresponding to the detectors are maximized. Furthermore, to secure the detector unit 502b with the microfluidic channel 502a, securing elements other than binder clips may be used.

Also, the voltage unit 504 need not comprise the Keithley 6487 picoam meter/voltage source. Instead, it may comprise an ammeter and a voltage source as two separate devices. The operations unit 506 also need not comprise a PC and can comprise any other device suitable for controlling the voltage unit 504 and for receiving, storing and analyzing data from the voltage unit 504. As hardware design improves over time, the operations unit 506 may be reduced in size to a portable hand-held unit for easy handling and use. The operations unit 506 also need not use a Labview-based program even though this is preferable for easier manipulation of the apparatus 500.

In addition, the link 512 for communication between the operations unit 506 and the voltage unit 504 need not be wired and may instead be wireless. Also, the quantitative analysis of samples caused to flow through the microfluidic channel 502a may be performed in a high-throughput format. More specifically, massively parallel sample analyses may be performed. Furthermore, the microfluidic device 502 may be provided separately from the rest of the apparatus 500. This allows the replacement of just the microfluidic device 502. It also allows different microfluidic devices 502 with differently configured microfluidic channels 502a and detector units 502b to be used with the same voltage unit 504 and operations unit 506. These different microfluidic devices 502 may have their walls coated with different types of molecules and/or additives, and may comprise different types and/or number of detectors.

Moreover, the components of the apparatus 500 may be fabricated using different materials and in different ways from those described above.

Commercial applications of the embodiments of the present invention

The embodiments of the present invention may be used for studying interactions between compounds and/or molecules, both within the same class of compounds and/or molecules as well as between different classes of compounds and/or molecules. These compounds and/or molecules may comprise one or more of proteins, lipids, carbohydrates, proteoglycans, metabolites, macromoiecules, drugs etc.

In one specific example, the embodiments of the present invention are used for studying protein-protein interactions. This may comprise the following:

i. Studying protein-protein interactions between membrane proteins which may be in vitro synthesized; ii. Studying protein-protein interactions between non-membrane proteins which may be in vitro synthesized;

iii. Studying protein-protein interactions between native systems such as cells, bacteria and viruses;

iv. Studying weak protein-protein interactions effectively;

v. Measuring the affinity constant of interacting protein pairs;

vi. Understanding how certain pathological conditions are linked to altered tight junction functions;

vii. Understanding how certain pathological conditions are linked to other protein-protein interactions;

viii. Identifying drugs or compounds modifying protein-protein interactions (for example, inhibiting the interactions or inducing interactions between nominally non-interacting protein or peptide partners). Studies similar to (i) - (viii) above may also be performed for other classes of compounds and/or molecules.

Therefore, the embodiments of the present invention may be used for many commercial applications. As the apparatus 500 is suitable to be used for both research and diagnostic purposes, it can be used by both research laboratories as well as medical facilities.

For example, the embodiments may be used by pharmaceutical companies to (i) identify drugs that can be used to treat tight junction-related diseases; (ii) identify compounds that potentially modify tight junction functions; and (iii) identify drugs that can be used to treat pathological diseases arising from other protein-protein interactions.

The embodiments of the present invention may also be used by medical professionals for (i) point-of-care identification of putative pathogens in patient samples and (ii) planning of treatment regimes for pathological conditions. A commercial form of the apparatus 500 may comprise the microfluidic device 502 in the form of a microfluidic chip with the operations unit 506 comprising attendant sample detection hardware. As mentioned above, the microfluidic device 502 may be provided separately from the rest of apparatus 500. In other words, replacement microfluidic chips or microfluidic chips suitable for different commercial purposes may be sold separately from the rest of apparatus 500.

Advantages of the embodiments of the present invention The following describes some advantages of the embodiments of the present invention.

Protein-protein interactions have typically been studied using static molecular and immunoiogical techniques. However, such techniques are ineffective in probing weak protein-protein binding, such as those between claudins. On the other hand, embodiments of the present invention are able to detect weak and transient molecular interactions as they detect the effect of these molecular interactions rather than the interactions themselves. More specifically, instead of studying barely perceptible individual molecular interactions, embodiments of the present invention probe the cumulative effect of the molecular interactions. This is done based on the transport of the molecules of interest through a microfluidic channel.

While protein-protein interactions have been studied in microfluidic channels, these studies are largely miniaturizations of conventional static protein binding assays. In contrast, the embodiments of the present invention are configured to analyze the interactions between molecules in a microfluidic flow-through format by studying the flow dynamics of samples through a microfluidic channel. Furthermore, the embodiments of the present invention use a plurality of carriers which serve as membrane-mimic supports for insertion and integration of the molecules of interest. This is achieved by synthesizing the molecules of interest in the presence of the carriers. Embedding the molecules of interest, for example proteins, into the carriers help to preserve the structure (i.e. conformation), orientation, organization and function of these molecules of interest. In particular, membrane proteins are notoriously difficult to synthesize owing to the need for a scaffold for folding and stabilizing them. Embodiments of the present invention use the carriers as scaffolds for insertion and folding of the membrane proteins, thus allowing the proteins to be synthesized without the use of cells (for example, a commercially-available cell-free in vitro synthesis kit may be used). While this technology has previously been described using lipid vesicles, and lipid and polymer membranes (see PCT applications PCT/EP2006/008318 and PCT/SG2010/000159), it has not been shown that polymer vesicles can be used for measuring protein-protein interactions.

Using the carriers has a further advantage. In particular, the embodiments of the present invention analyze the transport dynamics of the embedded carriers with the molecules of interest instead of the molecules of interest alone. Since each embedded carrier has a relatively greater mass as compared to each molecule of interest, it is easier to detect an embedded carrier than a molecule of interest. It is therefore easier to determine the rate at which embedded carriers travel through a part of the microfluidic channel than to determine the rate at which molecules of interest without the carriers travel through the same part of the channel.

Moreover, the components used in the embodiments of the present invention do not interfere with the binding events between molecules and thus, additives in the samples that interfere with these events can be identified. Hence, the embodiments of the present invention can be used for identifying drugs or compounds affecting tight junction functions. This can help in the development of therapeutic drug derivatives.

In addition, some embodiments of the present invention such as apparatus 500 as described above employs Labwew-compatible hardware. This, together with the straightforward means of sample application (achieved by use of a microfluidic channel), allows the embodiments to be used without the need for complex instructions. Thus, the embodiments can be used by researchers of various backgrounds. Furthermore, the embodiments can be readily developed into a functional prototype.

Claims

1 . A method for analyzing interactions of molecules of interest, wherein the method uses a plurality of carriers, some of the plurality of carriers being embedded with the molecules of interest to form embedded carriers, and wherein the method comprises:

causing a first and second sample to flow separately through a microfluidic channel, whereby the first sample comprises the embedded carriers and the second sample comprises the remaining carriers;

for each of the one or more parts of the microfluidic channel, obtaining data indicative of the amount of interactions of the molecules of interest during the flow of the first sample through the part, based on a difference in the rates determined for the part.

2. A method according to claim 1 , further comprising integrating further molecules with the microfluidic channel prior to causing the first and second samples to flow through the microfluidic channel.

3. A method according to claim 2, wherein integrating further molecules with the microfluidic channel comprises immobilizing the further molecules along walls of the microfluidic channel.

4. A method according to claim 3, wherein the further molecules comprise protein molecules and immobilizing the further molecules along walls of the microfluidic channel comprises:

immobilizing antibodies, raised against the protein molecules, along the walls of the microfluidic channel; and

inserting the protein molecules into the microfluidic channel to allow the antibodies to capture the protein molecules, thereby immobilizing the protein molecules along the walls of the microfluidic channel.

5. A method according to claim 4, wherein immobilizing the antibodies along the walls of the microfluidic channel comprises:

adsorbing further protein molecules capable of binding the antibodies into the walls of the microfluidic channel; and

inserting the antibodies into the microfluidic channel to allow the further protein molecules to bind the antibodies, thereby immobilizing the antibodies along the walls of the microfluidic channel.

6. A method according to any one of claims 2 - 5, wherein the further molecules comprise molecules of the same type as the molecules of interest.

7. A method according to any one of claims 2 - 5, wherein the further molecules comprise molecules of a different type from the molecules of interest.

8. A method according to any one of the preceding claims, wherein the embedded carriers are formed by synthesizing the molecules of interest in the presence of said some of the plurality of carriers such that the molecules of interest embed into said some of the plurality of carriers.

9. A method according to any one of the preceding claims, wherein the method further comprises: causing a third sample to flow through the microfluidic channel, whereby the third sample comprises additives and another plurality of carriers embedded with the molecules of interest;

determining the rate at which the another plurality of carriers travel through each of the one or more parts of the microfluidic channel during the flow of the third sample; and

analyzing, based on the determined rates, effects of the additives on the interactions of the molecules of interest.

10. A method according to any one of the preceding claims, wherein the method further comprises:

causing a fourth sample comprising a further plurality of carriers to flow through the microfluidic channel, whereby the further plurality of carriers are embedded with the molecules of interest and are geometrically different from the plurality of carriers of the first and second samples;

determining the rate at which the further plurality of carriers travel through each of the one or more parts of the microfluidic channel during the flow of the fourth sample; and

analyzing, based on the determined rates, effects of using geometrically different carriers on the interactions of the molecules of interest.

1 1. A method according to any one of the preceding claims, wherein the plurality of carriers comprise one or more of: cells, cell-mimics, viruses, artificial membranes, polymer vesicles, lipid vesicles.

12. A method according to any one of the preceding claims, wherein the molecules of interest comprise one or more of: membrane-associated proteins, cell adhesion proteins, synthetic proteins, peptides, lipids, carbohydrates, proteoglycans, metabolites, macromolecules, drugs.

13. A method according to claim 12, wherein the molecules of interest comprise Claudin-2 molecules.

14. An apparatus for analyzing interactions of molecules of interest, wherein the apparatus uses a plurality of carriers, some of the plurality of carriers being embedded with, the molecules of interest to form embedded carriers, and wherein the apparatus comprises:

a microfluidic device comprising:

an operations unit configured to:

for each of the one or more parts of the microfluidic channel, obtain data indicative of the amount of interactions of the molecules of interest during the flow of the first sample through the part, based on a difference in the rates determined for the part.

15. An apparatus according to claim 14, wherein the microfluidic channel comprises a plurality of loops.

16. An apparatus according to claim 14 or 15, wherein the detectors are arranged with the microfluidic channel to minimize one or more distances between the detectors, while maximizing one or more distances along the microfluidic channel between the positions corresponding to the detectors.

17. An apparatus according to any one of claims 14 - 16, wherein the detectors are arranged with the microfluidic channel such that the detectors are able to contact each of the first and second samples during the flow of the sample through the microfluidic channel.

18. An apparatus according to any one of claims 14 - 17, wherein for each of the first and second samples, electrical properties of parts of the sample comprising the carriers differ from electrical properties of other parts of the sample; and

wherein each detector is sensitive to the electrical properties of the part of the sample at the position along the microfluidic channel corresponding to the detector, so that the detector can detect the presence of the carriers at the corresponding position.

19. An apparatus according to any one of claims 14 - 17, wherein the plurality of carriers comprise fluorescent probes and each detector comprises: a light source configured to activate the fluorescent probes to generate fluorescent signals as the carriers travel through the position along the microfluidic channel corresponding to the detector; and

a receptor configured to receive the fluorescent signals to detect the presence of the carriers at the corresponding position.

20. An apparatus according to any one of claims 14 - 17, wherein the plurality of detectors are integrated to form a single detector structure configured to allow current flow through itself, the plurality of detectors being provided on the same current path of the current flow such that the current flow changes whenever the carriers reach a position along the microfluidic channel corresponding to one of the detectors.

21 . An apparatus according to claim 20, wherein resistance across the single detector structure is between 200Ι<Ω to 500kQ .

22. An apparatus according to claim 20 or 21 , wherein the single detector structure comprises:

a plurality of detector regions configured to serve as the plurality of detectors; and

a plurality of metallic regions arranged with the plurality of detector regions with each detector region flanked by two metallic regions to allow the current flow through the single detector structure.

23. An apparatus according to claim 22, wherein the detector regions are fabricated using low-density carbon nanotubes and the metallic regions are fabricated using high-density carbon nanotubes.

24. An apparatus according to claim 23, wherein the carbon nanotubes comprise single-walled carbon nanotubes.

25. An apparatus according to any one of claims 14 - 24, wherein the operations unit comprises a -a/bi Zew-compatible hardware.

26. A microfluidic device for determining transport dynamics of a plurality of carriers in a sample, wherein the plurality of carriers is capable of embedding molecules of interest and the transport dynamics are used for analyzing interactions of the molecules of interest, and wherein the microfluidic device comprises: