WO2010088219A2

WO2010088219A2 - Reusable biosensor platform

Info

Publication number: WO2010088219A2
Application number: PCT/US2010/022132
Authority: WO
Inventors: Junseok Chae; Seokheun Choi
Original assignee: Arizona Board Of Regents For And On Behalf Of Arizona State University
Priority date: 2009-01-27
Filing date: 2010-01-26
Publication date: 2010-08-05
Also published as: WO2010088219A3

Abstract

Techniques, apparatus and systems are described for generating reusable and reconfigurable biosensors. A microfluidic device includes a substrate layer and an electrode layer disposed above the substrate layer. A spacer layer is disposed above the electrode layers to form at least two channels. Another electrode layer is disposed above the spacer layer. Also, another substrate layer is disposed above the other electrode layer. The other substrate layer can include at least two inlets, each connected to one of the at least two channels, two outlets corresponding to the two inlets, and two electrical contacts filled with a conductive material to form a conductive feedthrough to the other electrode layer.

Description

REUSABLE BIOSENSOR PLATFORM

CLAIM OF PRIORITY

[0001 ] This application claims priority under 35 USC §119(e) to U.S. Patent Application Serial No. 61/147,728, filed on January 27, 2009, the entire contents of which are hereby incorporated by reference.

BACKGROUND

[0002] This application relates to biosensors. [0003] A diverse number of potentially interacting ligands exists with a broad range of chemical properlies, such as DNA, RNA, peptides, aptamer, proteins, lipids, and carbohydrates. The interactions of specific ligands involve formation of a strong and specific chemical bond, allowing biosensors in detecting target biomolecules. A specific bioreceptor immobilized on a sensing surface recognizes a specific target molecule and a sensor element transduces the change in the biomolecular interactions into an electrical signal. The biosensors can be implemented in various applications, such as medicine, biotechnology, environmental monitoring, food industry, and even military technology. For example, Micro-Electro-Mechanical- Systems (MEMS) technology offers miniaturization of bulky, heavy, and high power consumption macro-size system to improve performance and functionality. Several biosensors have utilized MEMS technology to detect proteins, DNA mismatch, and pathogens.

SUMMARY

[0004] This application discloses techniques, apparatus and systems for generating reusable and reconfigurable biosensors. Low-voltage electrochemical desorption can be used to remove self-assembled monolayers (SAMs) on a metal surface of a biosensor.

[0005] In one aspect, a microfluidic device includes a substrate layer and an electrode layer disposed above the substrate layer. A spacer layer is disposed above the electrode layers to form at least two channels. Another electrode layer is disposed above the spacer layer. Also, another substrate layer is disposed above the other electrode layer. The other substrate layer can include at least two inlets, each connected to one of the at least two channels, two outlets corresponding to the two inlets, and two electrical contacts filled with a conductive material to form a conductive feedthrough to the other electrode layer.

[0006] Implementations can optionally include one or more of the following features. The different la/ers can be bonded together by using oxygen plasma or adhesives. The substrate layer and the other substrate layer can include at least one of glass, silicon, ceramic, non-conductive composites and plastic substrate layers. The electrode layer and the other electrode layer can include one or more conducting films. The electrode layer and the other electrode layer can include at least one of chromium and gold. The electrical contacts can be filled with a conducting material. The conducting material can include silver paste. One of the two inlets can be connected to a reference channel to be used in reference measurements and the other inlet can be connected to a sample inlet to receive a sample protein and to be used in sample measurements. A surface plasmon resonance (SPR) analytical system can be connected to the microfluidic device to record SPR angle shift in real time. Recording SPR angle shift in real time can include the reference channel being configured to receive a reference solution comprising a self-assembled-monolayer (SAM) to modify a surface of the electrode layer in the reference channel; the sample channel being configured to receive a sample solution comprising the SAM to modify a surface of the electrode layer in the sample channel, and a target molecule that immobilizes on the SAM; the SPR analytical system being configured to apply a laser beam and measure a differential measurement of an angle shift of the applied laser beam incident on the modified surface of the electrode layer without the target molecule immobilized on the SAM in the reference channel and an angle shift of the applied laser beam incident on the modified surface of the electrode layer with the target molecule immobilized on the SAM in the sample channel. The electrode layer can include a cathode to receive a negative potential, and the other electrode layer can include an anode to receive a positive potential. The electrode layer and the other electrode layer can be connected to a potentiostat device to conduct ex-situ electrochemical measurement using linear sweep voltammetry. The microfluidic device can be configured to be reusable by desorbing the SAM and the target molecule from the surface of the electrode layer in the sample channel responsive to a reductive voltage applied to the electrode layer in the sample channel. The reductive voltage applied can be based on at least one of a length of an alkyl chain, a type of terminal group, or binding of proteins.

[0007] In another aspect, a method includes forming a self-assembly monolayer on a metal surface of a microfluidic device that comprises a reference channel, a sample channel, a cathode and an anode. Forming the self-assembly monolayer includes flowing a reference solution comprising a self-assembled-monolayer (SAM) through the reference channel and the sample channel to modify a metal surface of the cathode in the reference channel and the sample channel; and flowing a target molecule through the sample channel to be immobilized on the SAM. The method can also include detecting a presence of the SAM with the immobilized target molecule formed on the metal surface of the cathode; and applying a reductive potential to desorb the SAM along with the immobilized target molecule from the metal surface of the cathode in the sample channel. [0008] Implementations can optionally include one or more of the following features. Detecting the presence of the SAM with the immobilized target molecule formed on the metal surface of the cathode can include recording an angle shift of an applied laser beam incident on the metal surface of the cathode in the reference channel without the immobilized target molecule and the sample channel with the immobilized molecule in real time; and obtaining a differential measurement of the real time angle shift in the reference and sample channels. The process can include after desorbing the target molecule bound SAM, reusing the microfluidic device by flowing another solution that includes another SAM to reconfigure the surface of the cathode. Reusing the microfluidic device can include flowing another target molecule through the sample channel to bind with the other SAM. The other SAM can be desorbed as described above and another SAM can be formed as described above. Thus, the microfluidic device can be repeatedly reused. [0009] To detect the presence of the other SAM, contact angle measurement can be recorded on the other target molecule bound SAM. Also, the reductive potential can be applied to remove Ihe other target molecule-bound SAM to reconfigure the microfluidic device to be reused. The reductive potential applied can be based on at least one of a length of an alkyl chain, a type of terminal group, or binding of proteins. The target molecule can include streptavidin that immobilizes on the surface of the cathode. The cathode can include a conductive material, such as gold electrode. [0010] The techniques, systems and apparatus as described in this specification can optionally provide one or more of the following advantages. For example, low- voltage electrochemical desorption can be used to remove self-assembly monolayers on a metal surface. The self-assembling monolayers can be used to generate reusable and reconfigurable surfaces for sensor applications. A typical electrochemical desorption device requires high voltage which causes Hydrogen Evolution Reaction (HER) and corrodes metal electrodes that can damage the device. The low-voltage electrochemical desorption techniques as described in this specification can be implemented to mitigate HER and electrode corrosion. In addition, the techniques, systems and apparatus as described in this specification can be used to generate a device without the need for extreme pH, temperature, chemicals, etc.

[0011 ] These and other aspects and their exemplary implementations are described in detail in the attached drawings, the description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1A shows right-side and front-side views of an example microfluidic device to characterize a reusable and reconfigurable biosensor.

[0013] FIG. 1 B shows an example device configuration that includes three layers. [0014] FIG. 1 C shows a top-down view of a microfluidic device.

[0015] FIG. 1 D shows a top-down view of a microfluidic device on a surface plasmon resonance (SPR) window.

[0016] FIG. 1 E shows a cross-sectional view of a microfluidic device implemented as SPR protein sensors with proteins injected into a channel. [0017] FIG. 1 F shows an example SPR sensorgram.

[0018] FIG. 2A shows current density vs. potential profiles for reductive electrochemical desorption of ω-carboxylic acid alkanethiols (COOH(CH₂)_nSH) for n=2 and 10.

[0019] FIG. 2B shows current density vs. potential profiles for reductive electrochemical desorption of n-alkanethiols (CH₃(CH₂)_nSH) for n=3 and 1 1 (the longer the alkylchain, the more negative the reductive peak potential). [0020] FIGS. 2C & 2D show current density vs. potential profiles for reductive electrochemical desorptioπ of COOH(CH₂)_nSH modified by Streptavidin for n=2 and 10, respectively.

[0021 ] FIGS. 2E and 2F show current density vs. potential profiles for reductive electrochemical desorption of CH₃(CH₂)_nSH modified by fibrinogen for n=3 and 11 , respectively. [0022] FIG. 3A shows reductive peak potential of SH SAMs and protein-bound

SAMs COOH(CH₂)_n.

[0023] FIG. 3B shows reductive peak potential of SH SAMs and protein-bound SAMs for CH₃(CH₂)_nSH.

[0024] FIG. 3C shows a table of reductive peak potential of SAMs and protein- bound SAMs.

[0025] FIG. 4 shows example voltagrams (e.g., current density vs. potential profiles) for oxidative desorption of COOH(CH₂)_nSH for n=2 and CH₃(CH₂)_nSH for n=3. [0026] FIG. 5A shows SPR profiles with schematics of step-by-step procedure of surface modification.

[0027] FIG. 5B shows an enlarged SPR sensorgram of reductive desorption 1.

[0028] FIG. 5C shows an enlarged SPR sensorgram of reductive desorption 2.

[0029] FIG. 6 shows a table that summaries SPR angles shift and thickness. [0030] FIG. 7 shows reproducibility of SPR response from a protein modification with regeneration using SAM desorption.

[0031] FIG. 8 shows a table of SPR angle shift and thickness of FIG. 7.

[0032] FIGS. 9A, 9B and 9C are process flow diagrams of example processes for reconfiguring a microfluidic device to be reused

DETAILED DESCRIPTION

[0033] Biosensors can be categorized as qualitative, semi-quantitative and quantitative sensors. Qualitative sensors provide binary information, such as existence or absence of target analytes. An example of qualitative sensor includes the pregnancy test. Semi-quantitative sensors can detect and quantify the analytes in sub-threshold level. The DUI test is an example of a semi-quantitative sensor. Both techniques are useful for many areas of clinical specialty (i.e., glucose monitoring), food industry, and environment monitoring. In those cases, disposable biosensors can be implemented by taking advantage of low cost materials and batch fabrication.

[0034] Quantitative sensors can be used to cover a wide dynamic range. Quantitative sensors have high resolution and accuracy to produce large signal to noise ratio data. In particular, quantitative analysis that can elucidate the mechanisms that regulate the formation and activities of specific biomolecular complexes can be implemented for genomics and proteomics applications. For example, clinical diagnosis of cancer is supported with the elevation of cancer-specific biomarker. Quantitative techniques need detailed calibration of biosensors. Quantitation has been typically obtained using a series of measurements with disposable sensor units. However, variation coming from batch-to-batch irreproducibility and uncontrollable experimental condition often generates unalterable false-positive/negative responses. [0035] Dissociation of the target-bioreceptor complexes can be attempted as follows. Once target analytes bind to the specific bioreceptor and the recognition event is transduced, the target analytes are removed and the remaining bioreceptors are reused as a probe for detecting the same target molecules. However, such sensors need extreme pH, temperature, and chaotropic agents to detach the target from the bioreceptor. Also, as a consequence of the strong agents, a significant loss of biospecific activity occurs. Moreover, some biomolecules cannot be dissociated because of the high affinity of biomolecular interactions and their stability in extreme conditions.

[0036] While low frequency AC electric field may dissociate the C-reactive proteins from antibodies, consistent reproducibility of the technique may not be possible. Also, multiple use can lead to loss of affinity, and the low frequency AC electric field can cause reductive or oxidative desorption of linker molecules. Thus, these approaches impose limitations in dissociating the target-bioreceptor complexes and reduce the activity of biomolecular receptors.

[0037] Also, generation of reusable sensors can be attempted by removing the linker molecules with all bounded biomolecules. Generally, linker molecules are used to immobilize the bioreceptors on a solid surface to maintain permanent bonding. Self-assembled monolayer is a good candidate as a linker molecule because it is easy to be formed spontaneously and be used to control the orientation of the bioreceptors. To remove the linker molecules, existing techniques use acid, base or detergent. However, the extreme solutions damage the sensing sites and have a fatal impact on the next regeneration step. Electrochemical desorption of SAMs may be used. Electrochemical desorption of SAMs on metal surfaces occurs at negative potential or positive potential. At negative potential, a reductive desorption on a metal (Au) surface occurs according to the following reaction (1)

R - S - Au + e^" -» R - S^" + Au (1) where R and S represent an n-alkanethiols, i.e., alkanethiolates are desorbed from a gold surface by the one electron reduction process in an electrolyte. At voltammograms measured in 0.5 M KOH solutions (versus Ag/AgCI), reductive peak potential, E_p, ranges from -0.7 V to -1.2 V depending upon the thiol derivatives, length of the alkyl chain, the metal substrates and the electrolyte solutions. Also, an oxidative desorption occurs at positive potential (+0.8 V ~ +1.2 V) but this process has been studied less extensively. [0038] Desorption of SAMs can include electrical control for addressable immobilization of different bioreceptors on electrode arrays. However, when reductive potential is applied to the SAM-coated electrode, the opposite electrode suffers from corrosion, and hydrogen evolution reaction (HER) occurs at a similar potential to the potential of the SAM desorption. It is difficult to remove air bubbles by low flow rate of solution; typically several 10's of μl/min is used in microfluidic devices. The bubbles reduce the sensitivity, reliability, and accuracy of the biosensor. HER can be avoided by desorbing SAM in a strong alkaline electrolyte, but such physiological environments are impractical because most biosensors need to operate in a physiologically relevant medium such as Phosphate Buffered Saline (PBS) (pH=7.4). [0039] Techniques, systems and apparatus as described in this specification can be used to generate reusable and reconfigurable biosensors without the need for extreme pH, temperature, chemicals, etc. In addition, HER and electrode corrosion can be avoided. For example, reusable and reconfigurable microfluidic biosensors can be implemented in PBS. Metal electrode, such as gold (Au), corrosion and HER are attributed to high reductive potential for electrochemical desorption of SAMs, especially in a physiological solution. To minimize these limitations, the reduction potential is shifted less than -1.0 V (to the more positive direction, such as -0.9 V) using short chain SAMs (COOH(CH₂)₂SH and CH₃(CH₂)₃SH). SAMs desorb in sharp voltammetric peaks whose peak potentials shift in the negative potential direction as the hydrocarbon chain length of the alkanethiol molecule increases. Thus, as the chain length of the SAMs becomes shorter, the reduction potential becomes lower. Short-chain SAMs are often used as linker molecules to immobilize molecular probes including antibodies. Thus, the approach described in this specification can be used for many existing biosensors. [0040] Linear sweep voltammetry is used to characterize the desorption of SAMs as a function of the length of the alkyl chain, the type of terminal groups and the binding of proteins. Ellipsometry are used to evaluate the desorption of SAMs before and after the desorption. Surface plasmon resonance (SPR) insitu monitors successive experimental procedure of reconfigurable biosensor in real time, including SAM formation, protein modification, and SAM desorption in the microfluidic device. [0041] A reusable and reconfigurable biosensor platform in microfluidic systems can be generated using electrochemical desorption of SAMs. By applying a low DC voltage (e.g., 0.9 V) between two electrodes submerged in PBS, the sensing surface resets to be reusable and reconfigurable. Streptavidin-bound COOH-SAM completely desorbs and CH₃-terminated SAM forms on the sensing surface to capture the subsequent target molecule, fibrinogen, in a microfluidic device. The bio-molecular interactions are monitored by Surface Plasmon Resonance (SPR) in real time and ellipsometry and linear sweep voltammetry are used to evaluate the results. To avoid having the electrode peeling-off and electrolysis occurring at a similar potential to the potential of the SAM desorption, the reductive peak potential, E_p, for the SAM desorption is shifted to the positive direction using short chain alkanethiols in PBS. The peak potentials for both CH₃-and COOH-terminated SAMs shift by approximately 60 mV to the negative direction as proteins modify the surfaces. However, short chain SAMs shifts Ep to the positive direction by more than 100 mV. Linear sweep voltammetry measurements show the reductive potential of both SAMs modified by proteins to be approximately at -0.9 V and demonstrate that hydrogen evolution (~ -1.2 V) does not overlap the SAM desorption. Also, due to the low voltage, the electrode does not peel off during the desorption process. [0042] Microfluidic Device Fabrication [0043] FIG. 1 A shows an example microfluidic device 100 to characterize a reusable and reconfigurable biosensor. The microfluidic device includes multiple layers. The layers include a substrate layer 110 at the bottom of the device. The bottom substrate layer 110 can include a glass substrate, such as borosilicate glass,

Schott BK7. Electrode layers 112 and 114 are disposed on top of the bottom substrate layer. The electrode layers can 112 and 114 include Cr and Au materials deposited as a single or multiple layers. The bottom substrate layer 110 with the Cr/Au electrode layers 112 and 114 can be represented as the bottom layer. Above the electrode layers 112 and 1 14, a spacer layer 120 is disposed as the middle layer. The middle layer 120 can include a spacer material made of polydimethylsiloxane (PDMS), for example. PDMS is a silicon-based organic polymer, and is particularly known for its unusual rheological (or flow) properties. PDMS is optically clear, and is generally considered to be inert, non-toxic and non-flammable. PDMS is occasionally called dimethicone and is one of several types of silicone oil (polymerized siloxane). Above the spacer layer 120, a top layer 130 is disposed. The top layer 130 can include a substrate layer made of glass, for example. The top layer also includes additional electrode layers 132 and 134, such as the Cr/Au electrode layers. [0044] These layers are bonded together by using oxygen plasma, for example. Cr/Au of bottom layer (e.g., 2 nm/47 nm) is evaporated and patterned on a glass substrate (e.g., 150 um-thick) for SPR measurement. The top glass substrate can be made of various thickness (e.g., 1 mm-thick) and is mechanically drilled to have six holes including two inlets or ports 136 and 138, two outlets or ports 140 and 142, and two electrical contacts which are filled with silver paste or similar materials for feedthroughs. The PDMS layer is prepared to be 1 mm in thickness, for example and mechanically cut to have two channels including reference and sample channels. [0045] FIG. 1 B shows cross-sectional right-side 102 and front-side 104 views of the microfluidic device. In addition, the corresponding views 144, 146 and 148 of the top layer, middle layer and the bottom layers respectively are shown. [0046] FIG. 1C shows a top-down view 150 of a microfluidic device. The image shown in FIG. 1C represents applying a positive electrical potential between the top and bottom electrodes of a microfluidic device.

[0047] FIG. 1 D shows a top-down view 160 of a microfluidic device on a SPR window. The image shown in FIG. 1 D represents applying a negative potential to the bottom electrode with respect to the top electrode in a microfluidic device. [0048] FIG. 1 E shows a side cross-sectional view 170 of a microfluidic device implemented as SPR protein sensors with proteins injected into a channel. Proteins that include streptavidin and EDC/NHS are injected into the channel. The injected proteins adsorb on SAMs. When a laser beam is applied to the microfluidic device, the proteins adsorbed on the SAMs change the incident light angle of the applied laser

(^Δ θ).

[0049] FIG. 1 F shows an example SPR sensorgram 180. The y-axis represents the angle shift and the x-axis represents time. The straight linear line in FIG. 1 F represents the time dependent angle shift for a reference channel without proteins injected. For the reference channel, the measured angle shift does not change with time. The non-linear line represents the time dependent angle shift for a sample channel with proteins injected. Corresponding to the proteins changing the incident angle of the applied laser beam, the angle shift in the sample channel changes with time in a nonlinear manner. The difference between the reference and sample channels provides the Dθ. [0050] Methods and EEvaluation

[0051] The electrochemical desorption process of SAMs can be observed using many techniques including electrochemistry, Fourier Transform Infrared Spectroscopy (FTIR), electrochemical Quartz Crystal Microbalance (QCM), in-situ Scanning

Tunneling Microscope (STM), Second Harmonic Generation spectroscopy (SHG), Sum Frequency Generation (SFG), ellipsometry, Raman spectroscopy, and SPR. Among these techniques, electrochemical method may be the easiest way to characterize the electrochemical desorption process showing a current density (j) vs. potential (E) profile. Ellipsometry is used to evaluate the thickness of SAMs on a metal electrode. SPR is a real time, in-situ optical technique that monitors the incident light angle needed to excite surface plasmons at the interface of a thin film of metal and a dielectric layer, such as a SAM. As the thickness of the dielectric layer increases - for example, as proteins bind to a SAM - the resonance angle decreases. Thus the change in resonance angle shows the amount of protein adsorbing to a SAM. [0052] Linear Sweep Voltammetry

[0053] Ex-situ electrochemical measurement can be performed using linear sweep voltammetry to obtain the operating potential of SAM desorption in the microfluidic device and confirm that the reductive desorption would not be obscured by HER and the electrode corrosion. A set of custom-made electrodes are built. The electrodes are placed at the bottom hole of the cell with a silicone rubber O-ring and the top hole is tightly fitted with a silicon rubber stopper having a Pt wire counter and Ag/AgCI reference electrodes. Linear sweep voltammetry is conducted using a computer-controlled poteπtiostat (e.g., PGSTAT302N from Eco Chemie). [0054] SPR measurement & surface modification procedure [0055] Surface modification upon electrochemical desorption on the metal electrode can be monitored by SPR in situ in real time. SPR angle shift is produced by differential measurement using two (reference and sensing) channels in the microfluidic device. As shown in FIG. 1A-1 F above, the fabricated microfluidic chip is mounted on the SPR analytical system (Biosensing Instrument Inc.), and the angle shift is recorded in real time as each solution inducing surface modification flows through the microfluidic channels driven by an external syringe pump. Initially, PBS is circulated for 20 min until the angle shift stabilizes. Once the angle shift stabilizes, the Au electrode in sample channel is modified with COOH terminated SAM of 2 mM 3- Mercaptopropionic acid (MPA) solution in ethanol for 1 hour followed by thorough rinsing with PBS. The first target molecule, streptavidin, is immobilized on the SAM by activating the carboxylic acid groups of MPA with freshly prepared 400 mM EDC/100 mM NHS in water for 15 minutes. The NHS-esters produced react with amine functions present in the 1 uM streptavidine in 10 mM NaAc pH 5.3 (sodium acetate) solution. When the protein immobilization completes, PBS washes the electrode to remove excess weakly bound proteins. Then, a reductive potential is applied to desorb the immobilized molecules on the electrode. The DC potential applies to the Au bottom electrode (cathode) against the top electrode (anode) for 30 seconds and the electrode is immediately washed by PBS to avoid re-adsorption of streptavidin- bound SAM molecules. [0056] After complete desorption of the streptavidine-bound SAM, CH3- terminated SAM is formed at second cycle to demonstrate reconfigurability. 2 mM 1 - butanethiol (BT) solution in ethanol modifies the Au electrode in the sample channel for 1 hour. Following PBS washing, the second target molecule, fibrinogen, flows through the channel and forms hydrophobic bonding with the SAM. The contact angle measurement shows that the electrode covered by the SAM has strong hydrophobicity (e.g., 102^°). The reductive potential is applied to remove the fibrinogen-bound SAM. [0057] Evaluation using Ellipsometry

[0058] Ellipsometry can be performed to measure the thickness of the molecule layers on the electrode before and after the electrochemical desorption. Also, the thicknesses measured by ellipsometry are compared with those converted from SPR signals. Separately prepared Au-coated samples are used with the ex-situ ellipsometry setup.

[0059] Reductive desorption of SAMs on the cathode

[0060] The reusable and reconfigurable biosensor generated as described in this specification can overcome i) electrochemical reductive desorption of SAMs without HER and ii) reductive potential to avoid peeling of the metal electrode. HER may be the most challenging problem during the electrochemical desorption process. Electrolysis is a process to produce hydrogen at the cathode and oxygen at the anode when electrodes are in an aqueous solution and current runs through the solution. When reductive potential is applied on the cathode, reductive peak potential, E_p, can be difficult to measure because electrodesorption of SAMs and HER occur simultaneously. HER can be fatal in a microfluidic channel because the bubbles generated by HER tend to stay on the cathode and interfere with transduction signal. Also, even a tiny bubble in the channel can cause unreliable SPR measurements. All baseline data can be lost even after the bubble is removed by the buffer solution. [0061] Electrochemical desorption and HER can be monitored by voltammetry, which measures the current density vs. potential profile in the negative direction. The potential for the reductive desorption of thiol SAMs depends on the length of the alkyl chain, the type of terminal groups and the binding of proteins, for example. FIGS. 2A- 2F show linear sweep voltammograms of Au electrodes coated with SAMs in PBS solution, having different alkyl chain lengths and different terminal groups. The voltagrams shows current density vs. potential profiles for the reductive electrochemical desorption. For these voltagrams, the DC potential is swept from 0 V to -1.2 V at the rate of 20 mVs-1. The y-axis represents the measured current density (current per area). The x-axis represents the potential profile.

[0062] FIG. 2A shows current density vs. potential profiles 200 for reductive electrochemical desorption of ω-carboxylic acid alkanethiols (COOH(CH₂)_nSH) for n=2 (202) and 10 (204). E_p of the reductive desorption of adsorbed thiols appears at - 0.7680 V for n=2 and -0.8887 V for n=10 (see table 1 in FIG. 3C below). On the other hand, the E_p of the corresponding n-alkanethiols, CH₃(CH₂)_nSH, appears at -0.8047 V for n=3 and -0.9072 V for n=11 as shown in FIG. 2B and in table 1 of FIG. 3C. FIG. 2B shows current density vs. potential profiles 210 for reductive electrochemical desorption of n-alkanethiols (CH₃(CH₂)_nSH) for n=3 (212) and 11 (214). The longer the alkylchain, the more negative the reductive peak potential. [0063] Two reductive potential peaks are observed in short chain SAMs whereas long chain SAMs show only one peak in the voltammetry. The separated desorption peaks are due to the differences in the type of binding site and the subsequent strength of the interactions of the sulfur atom with gold surface. Among the two peaks, the final reductive desorption of the SAMs occurs at the second peak potential. The Ep of the reductive desorption varies depending on the length of alkyl chain and the type of terminal group of SAMs. The longer the alkylchain is, the more negative Ep becomes, reflecting the stronger van der Waals attractive interaction among alkylchains. Ep is also dependent on the terminal groups. Ep of the reductive desorption of COOH(CH2)nSH is more positive than that of CH3(CH2)nSH. This is due to the repulsive interaction between the negatively charged carboxylate groups in a COOH-SAM.

[0064] FIGS. 2C & 2D show current density vs. potential profiles for reductive electrochemical desorption of COOH(CH2)nSH modified by Streptavidin for n=2 (220) and 10 (23), respectively. The voltammograms in FIGS. 2C and 2D are of covalent bond of streptavidin to COOH(CH2)nSH by formation of an amide bond. In FIG. 2C, voltammogram 222 represents streptavidin-bound SAM and voltammogram 224 represents bare SAM. In FIG. 2D, voltammogram 232 represent streptavidin-bound SAM and voltammogram 234 represent bare SAM. Reductive desorption peak potentials are more negatively shifted by 128 mV for n=2 and by 58 mV for n=10 when the surface is modified by streptavidin.

[0065] FIGS. 2E and 2F show current density vs. potential profiles for reductive electrochemical desorption of CH3(CH2)nSH modified by fibrinogen for n=3 (240) and 1 1 (250), respectively. The voltagrams in FIGS. 2E and 2F are of adsorption of fibrinogen on CH3(CH2)nSH by hydrophobic attraction. In FIG. 2E, voltammogram 242 represent fibrinogen-bound SAM and voltammogram 244 represent bare SAM. In FIG. 2F, voltammogram 252 represent fibrinogen-bound SAM and voltammogram 254 represent bare SAM. The desorption peak potentials are also shifted to more negative values by 92 mV and 84 mV for n=2 and 11 , respectively, after the surface is modified by fibrinogen. Regardless of bonding mechanism of proteins, more potential is needed to desorb the protein bound SAMs than that of unmodified SAM. [0066] FIGS. 2D and 2F show that proteins-bound long chain SAMs need approximately -1.0 V to desorb the SAMs and such high potential seriously damages the opposite top electrode, especially when the electrode is a thin metal film. This is described further below. F^:or the different SAMs in FIGS. 2A-2F, HER occurs at approximately -1.2 V, which does not overlap the reductive desorption potential. Thus, short chain SAMs have a much less chance to encounter HER. [0067] FIG. 3A is a bar graph 300 showing reductive peak potential of SH SAMs and protein-bound SAMs COOH(CH2)n. FIG. 3B is a bar graph 310 showing reductive peak potential of SH SAMs and protein-bound SAMs for CH3(CH2)nSH. FIG. 3C shows a table 320 (Table 1) of reductive peak potential of SAMs and protein- bound SAMs. The first column represents various monolayers of SAMs and protein- bound SAMs. The right column represents the reductive peak potentials for the corresponding monolayers.

[0068] Oxidative desorption of SAMs on the anode

[0069] The biosensor device experiences both reductive and oxidative processes on the electrodes. Also, the biosensor device experiences reductive process on the cathode and oxidative process on the anode. FIG. 4 shows example voltagrams (current density vs. potential profiles) 400 for the oxidative desorption of

COOH(CH2)nSH for n=2 (402) and CH3(CH2)nSH for n=3 (404). SAM desorption of top electrode occurs at around +0.9V and Gold electrodes peel off the glass substrate at +1.0V. For the voltammograms of unmodified short chain SAMs, where the potential is swept between 0 V to +1.2 V. At +0.9 V, oxidative desorption of SAMs occurs and Au electrodes begin to corrode over +1.0 V. As the potential further increases, the gold film peels off from the glass substrate. To remove longer chain SAMs bound by proteins on the cathode (see, FIGS. 2A-2F), more than DC 1.0 V is needed, which induces corrosion of gold on the anode. For this reason, the short chain SAMs are used for the reusable/reconfigurable biosensor to minimize HER on the cathode and prevent the corrosion of a metal film.

[0070] Real time monitoring of SAM desorption in a Microfluidic device [0071 ] SPR and ellipsometry can be used in concert to verify assembly and removal of molecules in the microfluidic device. Electrochemical reduction is performed in-situ in a two electrode set-up and SPR monitors the molecular interactions in real-time. Ellipsometry is performed ex-situ to confirm the assembly of multiple layers on the bottom Au electrode surface. From the measured SPR angle shift, the thicknesses of the proteins and SAMs and their refractive indices are calculated according to the method of de-Feijter (see, J.A. De Feijter, J. Benjamins and F.A. Veer, Biopolymers, 1978, 17, 1759-1772), which is incorporated by reference, to allow the transformation of a film thickness parameter into an amount of adsorbed protein per unit area, r{ng » mm^~2) = d[(n_f - n_m)/(dn/dC)] (2) where d is the film thickness (nm), nf is the refractive index of the film, nm is the refractive index of the ambient, and dn/dC is the refractive index increment. This method is based on the assumption that the refractive index of a protein solution is a linear function of the protein concentration. The formula for the air/solid measurement is as follows, V(ng » mm^'2) = K» t (3) where K is the density of Ihe protein and SAMs and t is the thickness (nm). The density of protein and SAMs are 1.36 g/cm³ (proteins), 0.842 g/cm³ (CH₃(CH₂)₃SH), 0.845 g/cm³ (CH₃(CH₂)nSH), 1.218 g/cm³ (COOH(CH₂)₂SH), 0.792 g/cm³ (COOH(CH₂)ioSH), respectively. For the SPR instrument, an increase in the plasmon resonance angle of 120 mDeg corresponds to an average material layer growth of 1 ng/mm².

[0072] FIGS. 5A-5C show SPR profiles as a sequence of surface modification steps. The SPR profiles 500 of FIG. 5A include schematics of step-by-step procedure of surface modification including: © Injection of COOH-SAM solution; ©

COOH-SAM formation; © Activation of surface carboxylates; © Immobilization of

Streptavidin; © Reductive Desorption at -0.9V; © Injection of CH3-SAM solution; ®

CH3-SAM formation; ©Adsorption of Fibrinogen; and ® Reductive Desorption at -

0.9V. The y-axis represents the angle shift θ in milli-degrees (mDeg), and the x-axis represents the time in seconds (S). FIG. 5B shows an expanded view 510 of reductive desorption 1 as identified by reference (a) in FIG. 5A. FIG. 5B shows an expanded view 520 of reductive desorption 2 as identified by reference (b) in FIG. 5A. For both FIGS. 5B and 5C, the y-axis represents the angle shift θ in milli- degrees (mDeg), and the x-axis represents the time in seconds (S). [0073] FIG. 6 shows a table (Table 2) 600 that summaries the SPR angles from FIG. 5A, the thicknesses converted from SPR and ellipsometry results. The SPR angle shift and Thickness. The thickness measured by ellipsometry is in good agreement with the converted values from the SPR signals. [0074] First, COOH-Ierminated SAM is formed by flowing 40 ul of 2 mM MPA in ethanol at the rate of 10 ul/min. SPR angle shift increases by 1800 mDeg. When the value stabilizes, we stop the flowing for 1 hour to form SAMs. SAM formation is a two- step process, where thiol molecules first adsorb on a solid surface and then rearrange to become a packed monolayer. 40-50 % of the adsorption occurs very rapidly within - 10 s and over 90 % of formation is completed in 10-100 min. After 1 hour, PBS is reflowed to thoroughly wash unformed SAM residues. The thickness of the SAM is measured by ellipsometry to be 4.06 A, which is in good agreement with SPR. 58.2 mDeg shift corresponds to 3.98 A. Then EDC/NHS mixture activates carboxylic acid groups of MPA monolayer to effectively immobilize streptavidin. Streptavidin containing amine groups on the surface interact readily with the activated MPA intermediates and form covalent amide linkages, showing 11.95 A thick from SPR converted values. Reductive desorption of the immobilized proteins is performed by applying -0.9 V to the cathode against Au top electrode for 30 seconds. [0075] During the desorption process, PBS keeps flowing to avoid re-adsorption of the detached SAMs. The amplitude of the desorption potential is based on the data from linear sweep voltammetry measurement. By applying reductive desorption potential twice, almost complete removal of adlayers is achieved ((D in FIG. 5A and

Table 2 in FIG. 6). The cycle is repeated using CH₃-terminated short chain SAM (CH₃(CH₂)3SH) and capture the second target molecule, Fibrinogen, to demonstrate a reconfigurable biosensor. The bond between fibrinogen and CH₃-terminated SAM is formed by a strong hydrophobic attraction. These phenomena are attributed to the characteristic structure of proteins. Generally, proteins have hydrophobic residues buried within the core of the proteins and their hydrophilic residues facing outside. The adsorption of any protein onto a solid surface is a strong function of hydrophobic attraction. Hydrophilic residues form the orientation of the adsorbed protein and do not take part in the adsorption process itself. Therefore, proteins rapidly adsorb onto a hydrophobic surface, unfold and spread their hydrophobic core over the surface. The solid surface, CH₃- terminated SAM, employed here shows high hydrophobicity of 102° in contact angle measurement. As a result of fibrinogen adsorption, strong bonding occurs between the protein and the surface. FIG. 5A shows that even PBS flow does not dissociate the protein-surface binding. The Fibrinogen-bound SAM is also completely desorbed at -0.9 V (® in FIG. 5A and Table 2 in FIG. 6). This demonstrates that the sensing surface resets to be reusable/reconfigurable by undergoing electrochemical desorption of short chain SAMs while avoiding HER and electrode corrosion.

[0076] SAM formation and dissociation may occur on top Au electrode (anode) if high enough potential is applied. FIG. 4 shows that COOH(CH₂)₃SH has two oxidative peaks at +0.6756 V and + 0.913 V while a desorption peak of CH₃(CH₂)2SH is shifted to more positive values, +0.9502 V. Therefore, DC 0.9 V for the reductive desorption of SAMs on the cathode does not completely remove the SAMs formed on the top Au electrode. However, this imperfect desorption at the anode has no effect on the reductive desorption or adsorption of SAM on the cathode because SPR only measures biomolecular interactions within approximately 200 nm from the cathode. Moreover, 30 sec of DC is unlikely to re-adsorb newly detached alkyl molecules on both electrodes because the flow rate of 10 μl/min is fast enough to completely flow the dissociated residues out of the channel. [0077] Miniaturized biosensors can be designed to be disposable, taking advantage of low cost materials and batch fabrication. In contrast, macro-size biosensors tend to be designed as non-disposable units. Disposable biosensor chips are much favored for many applications where it is difficult to wash all residues in microfluidic channels and sensing surfaces. On the other hand, reusable and reconfigurable biosensors are useful where detailed calibration of sensors is required such as in biochemical science and analytical chemistry. The techniques, systems and apparatus as described in this specification can be used to generate a biosensor platform for reusable and reconfigurable sensing surfaces using electrochemical reductive desorption of linker molecules. HER in microfluidic channels and electrode corrosion are avoided by using short chain linker molecules. The linker molecules bound to proteins are desorbed by applying very small DC potential, which are characterized by ellipsometry, voltammetry, and SPR in real time. This technique can be comprehensively utilized in all communities where linker molecules are needed to immobilize biomolecular receptors. [0078] Reproducibility

[0079] Five measurements of the above procedure are performed in the same microfluidic device and monitored the change of the SPR signal (two cycles: COOH- SAM formation -> streptavidin immobilization -> regeneration -> CH₃-SAM formation -> fibrinogen adsorption -> regeneration). The reusability and reconfigurability of the microfluidic device are illustrated in FIG 7 and Table 3 in FIG. 8, which show absolute angle values of the SPR after each protein modification 700 and regeneration 800, respectively. The binding activity of streptavidin and fibrinogen retained over 95% of the original SPR change signal after 10 cycles of regeneration. The result indicated that the microfluidic device can be reused with good reproducibility with an RSD lower than 1.86%.

[0080] FIGS. 9A, 9B and 9C are process flow diagrams of example processes for reconfiguring a microfluidic device to be reused. The process 900 includes forming a self-assembly monolayer on a metal surface of a microfluidic device that comprises a reference channel, a sample channel, a cathode and an anode (910). Forming the self-assembly monolayer includes flowing a reference solution comprising a self- assembled-monolayer (SAM) through the reference channel and the sample channel to modify a metal surface of the cathode in the reference channel and the sample channel (912); and flowing a target molecule through the sample channel to be immobilized on the SAM (914). The process can also include detecting a presence of the SAM with the immobilized target molecule formed on the metal surface of the cathode (920); and applying a reductive potential to desorb the SAM along with the immobilized target molecule from the metal surface of the cathode in the sample channel (930). [0081] Detecting the presence of the SAM with the immobilized target molecule formed on the metal surface of the cathode can include recording an angle shift of an applied laser beam incident on the metal surface of the cathode in the reference channel without the immobilized target molecule and the sample channel with the immobilized molecule in real time (922); and obtaining a differential measurement of the real time angle shift in the reference and sample channels (924). The process can include after desorbing the target molecule bound SAM, reusing the microfluidic device by flowing another solution that includes another SAM to reconfigure the surface of the cathode (940). Reusing the microfluidic device can include flowing another target molecule through the sample channel to bind with the other SAM. The other SAM can be desorbed as described above and another SAM can be formed as described above. Thus, the microfluidic device can be repeatedly reused (950). [0082] To detect the presence of the other SAM, contact angle measurement can be recorded on the other target molecule bound SAM. Also, the reductive potential can be applied to remove the other target molecule-bound SAM to reconfigure the microti iridic device to be reused. The reductive potential applied can be based on at least one of a length of an alkyl chain, a type of terminal group, or binding of proteins. The target molecule can include streptavidin that immobilizes on the surface of the cathode. The cathode can include a conductive material, such as gold electrode. [0083] While this specification contains many specifics, these should not be construed as limitations on the scope of an invention or of what may be claimed, but rather as descriptions of features specific to particular embodiments of the invention. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or a variation of a subcombination.

[0084] Only a few implementations are disclosed. However, it is understood that variations and enhancements may be made.

Claims

CLAIMS What is claimed is:

1. A microfluidic device comprising: a substrate layer; an electrode layer disposed above the substrate layer; a spacer layer disposed above the electrode layers to form at least two channels; another electrode layer disposed above the spacer layer; and another substrate layer disposed above the other electrode layer, wherein the other substrate layer comprises at least two inlets, each connected to one of the at least two channels, two outlets corresponding to the two inlets, and two electrical contacts filled with a conductive material to form a conductive feedthrough to the other electrode layer.

2. The microfluidic device of claim 1 , wherein the different layers are bonded together by using oxygen plasma or adhesives.

3. The microfluidic device of claim 1 , wherein the substrate layer and the other substrate layer comprise at least one of glass, silicon and plastic substrate layers.

4. The microfluidic device of claim 1 , wherein the electrode layer and the other electrode layer comprise one or more conducting films.

5. The microfluidic device of claim 1 , wherein the electrode layer and the other electrode layer comprise at least one of chromium and gold.

6. The microfluidic device of claim 1 , wherein the electrical contacts are filled with a conducting material.

7. The microfluidic device of claim 6, wherein the conducting material comprises silver paste.

8. The microfluidic device of claim 1 , wherein one of the two inlets is connected to a reference channel to be used in reference measurements and the other inlet is connected to a sample inlet to receive a sample protein and to be used in sample measurements.

9. The microfluidic device of claim 8, wherein a surface plasmon resonance (SPR) analytical system is connected to the microfluidic device to record SPR angle shift in real time comprising: the reference channel to receive a reference solution comprising a self- assembled-monolayer (SAM) to modify a surface of the electrode layer in the reference channel; the sample channel to receive a sample solution comprising the SAM to modify a surface of the electrode layer in the sample channel, and a target molecule that immobilizes on the SAM; and the SPR analytical system to apply a laser beam and measure a differential measurement of an angle shift of the applied laser beam incident on the modified surface of the electrode layer in the reference channel without the target molecule immobilized on the SAM and an angle shift of the applied laser beam incident on the modified surface of the electrode layer in the sample channel with the target molecule immobilized on the SAM.

10. The microfluidic device of claim 9, wherein the microfluidic device is configured to be reusable by desorbing the SAM and the target molecule from the surface of the electrode layer in the sample channel responsive to a reductive voltage applied to the electrode layer in the sample channel.

1 1. The microfluidic device of claim 10, wherein the reductive voltage applied is based on at least one of a length of an alkyl chain, a type of terminal group, or binding of proteins.

12. The microfluidic device of claim 1 , wherein the electrode layer comprises a cathode to receive a positive potential; and the other electrode layer comprises an anode to receive a negative potential.

13. The microfluidic device of claim 1 , wherein the electrode layer and the other electrode layer are connected to a potentiostat device to conduct ex-situ electrochemical measurement using linear sweep voltammetry.

14. A method comprising: forming a self-assembly monolayer on a metal surface of a microfluidic device that comprises a reference channel, a sample channel, a cathode and an anode, wherein the forming comprises: flowing a reference solution comprising a self-assembled-monolayer

(SAM) through the reference channel and the sample channel to modify a metal surface of the cathode in the reference channel and the sample channel; flowing a target molecule through the sample channel to be immobilized on the SAM; detecting a presence of the SAM with the immobilized target molecule formed on the metal surface of the cathode; and applying a reductive potential to desorb the SAM along with the immobilized target molecule from the metal surface of the cathode in the sample channel.

15. The method of claim 14, comprising: after desorbing the target molecule bound SAM, reusing the microfluidic device by flowing another solution comprising another SAM to reconfigure the surface of the cathode.

16. The method of claim 15, comprising: flowing another target molecule through the sample channel to bind with the other SAM.

17. The method of claim 15, comprising: recording contact angle measurement on the other target molecule bound SAM; and applying the reductive potential to remove the other target molecule-bound SAM.

18. The method of claim 14, wherein the target molecule comprises streptavidin that immobilizes on the surface of the cathode.

19. The method of claim 14, wherein the cathode comprises gold electrode.

20. The method of claim 14, wherein the reductive potential applied is based on at least one of a length of an alkyl chain, a type of terminal group, or binding of proteins.

21. The method of claim 14, wherein detecting the presence of the SAM with the immobilized target molecule comprises: recording an angle shift of an applied laser beam incident on the metal surface of the cathode in the reference channel without the immobilized target molecule and the sample channel with the immobilized molecule in real time; and obtaining a differential measurement of the real time angle shift in the reference and sample channels.