Patent Application
20160178649
These teachings relate generally to methods and systems for detecting a target molecule in the target fluid.
Molecular recognition is fundamental to a number of biological mechanisms. Sensors for molecular recognition are referred to herein as biosensors, although that name should not be considered limiting.
A biosensor typically has two components, a recognizing element that interacts with the target molecule and a transducing element that converts the interaction into a quantifiable effect. Some common recognizing elements are based on antibody, enzymatic, cellular or bio receptor interactions. Typical transducing elements are electrochemical optical and dielectric elements.
Although a number of biosensors configured as described above have been used, there are some basic disadvantages-poor chemical, physical and long-term stability, batch to batch variability and high cost. There is a need for biosensors that will overcome these disadvantages.
In order to provide a concrete example, the detection of oxytocin levels is described herein below.
Oxytocin is a peptide hormone widely known for its role in reproduction and child birth. In fact, the word “oxytocin” was coined from the Greek words meaning “quick birth” after its uterine-contracting properties were discovered by Dale. More recently, the role of oxytocin as a neuromodulator in the central nervous system of humans has been recognized. It is now known that oxytocin indeed plays a very important role in a variety of complex social behavior. For instance, high peripheral oxytocin levels have been associated with better relationship quality. Oxytocin may also be capable of modulating inflammation and promoting wound repair. It is also realized that the levels of oxytocin can affect human stress behaviors, interpersonal relations, and even wound healing.
While the importance of oxytocin has stimulated a major interest in monitoring oxytocin levels to better understand its role in human and animal behavior, there are some technical issues with regards to the current state-of-the art capability for measuring oxytocin levels.
Specificity Issue: Recent studies have shown that the regulation of oxytocin is a complex process involving two forms of oxytocin. Initially a 12-amino acid hormone is produced. Subsequently, it may be cleaved to a 9-amino acid hormone. This shortened form is the active neuropeptide credited with oxytocin's behavior-altering effects. While the biological role, if any, of the 12-amino acid pre-hormone is unknown, it has been associated with atypical social behaviors in autism and possibly related to obesity. Hence the measurement method of oxytocin level must have the specificity to distinguish between the 9- and 12-aminoacid forms of oxytocin. Current immunoassays fail to differentiate the neuroactive form from the pre-hormone version. In immunoassays, the specific recognition ability of antibodies relies on a short variable sequence of amino acids at the tips of the Y-structure, which is called the paratope and specific for one particular moiety of the analyte. In the scenario of oxytocin detection, 9- and 12- amino acid forms of oxytocin both bind to the paratope of antibody with a similar affinity because both the 9- and the 12-amino acid versions consist of an identical amino acid tip segment. Consequently, immunoassay cannot discriminate between the 9- and 12-amino acid forms.
Sensitivity issue: Basal blood levels of oxytocin are in the pg/mL range. This low biological level makes accurate measurements of oxytocin difficult. For instance, under normal physiological conditions, oxytocin levels in blood are ˜5 pg/ml and the corresponding salivary concentrations would be 0.25-0.50 pg/ml, which is undetectable by current immunoassay technologies.
Hence, the current immunoassay-based methods do not have either the specificity or sensitivity needed. Oxytocin assays with improved sensitivity and specificity would be the necessary tools to understand the function of this important neurohormone.
Biosensors and sensing methods that overcome the disadvantages—poor chemical, physical and long-term stability, batch to batch variability and high cost, are disclosed herein below. Oxytocin assays and oxytocin sensing methods with improved sensitivity and specificity are also disclosed herein below.
In one or more embodiments, the sensor of these teachings for detecting and recognizing target molecules includes a capture and release component and a sensing component. The capture release component includes a structure having one of molecularly imprinted polymer layer or molecularly imprinted polymer nanoparticles disposed on the structure, the structure being configured to receive a target fluid having the target molecules, the target molecules being captured by the molecularly imprinted polymer nanoparticles, and a source of a release solvent configured to release the target molecules captured by the molecularly imprinted polymer nanoparticles, the release solvent and released target molecules constituting a release solution. The sensing component includes a sensor surface having a layer of molecular imprinted polymer disposed on the sensor surface; the layer of molecularly imprinted polymer disposed to receive the release solution, the target molecules binding to the molecularly imprinted polymer, and a sensing circuit configured to detect impedance changes in the layer of molecularly imprinted polymer caused by the binding of the target molecules to the molecularly imprinted polymer.
In one or more embodiments, the method of these teachings includes disposing molecularly imprinted polymer nanoparticles on a surface of a structure, receiving, at the surface, a target fluid having the target molecules, capturing the target molecules in the molecularly imprinted polymer nanoparticles, releasing, after capture, the target molecules from the molecularly imprinted polymer nanoparticles, the target molecules being released into a release solution, providing the release solution to a sensor surface having a layer of molecular imprinted polymer disposed on the sensor surface, the target molecules binding to the layer of molecularly imprinted polymer, and detecting impedance changes in the layer of molecularly imprinted polymer caused by the binding of the target molecules to the molecularly imprinted polymer, the target molecules being detected by the impedance changes.
A number of other embodiments are also disclosed.
For a better understanding of the present teachings, together with other and further objects thereof, reference is made to the accompanying drawings and detailed description and its scope will be pointed out in the appended claims.
The following detailed description presents the currently contemplated modes of carrying out the invention. The description is not to be taken in a limiting sense, but is made merely for the purpose of illustrating the general principles of the invention, since the scope of the invention is best defined by the appended claims.
As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.
Except where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and the claims are to be understood as being modified in all instances by the term “about.” Further, any quantity modified by the term “about” or the like should be understood as encompassing a range of ±10% of that quantity unless otherwise specified.
Biosensors and sensing methods that overcome the disadvantages, poor chemical, physical and long-term stability, batch to batch variability and high cost, are disclosed herein below.
In one or more embodiments, the sensor of these teachings for detecting and recognizing target molecules includes a capture and release component and a sensing component. The capture release component includes a structure having ne of molecularly imprinted polymer layer or molecularly imprinted polymer nanoparticles disposed on the structure, the structure being configured to receive a target fluid having the target molecules, the target molecules being captured by the molecularly imprinted polymer nanoparticles, and a source of a release solvent configured to release the target molecules captured by the molecularly imprinted polymer nanoparticles, the release solvent and released target molecules constituting a release solution. The sensing component includes a sensor surface having a layer of molecular imprinted polymer disposed on the sensor surface; the layer of molecularly imprinted polymer disposed to receive the release solution; the target molecules binding to the molecularly imprinted polymer and a sensing circuit configured to detect impedance changes in the layer of molecularly imprinted polymer caused by the binding of the target molecules to the molecularly imprinted polymer. The capture and release components are operatively connected to receive from a fluid source, the fluid being the target fluid or the release solvent. The sensing component is operatively connected to the capture and release components in order to receive the release solution.
In one embodiment, the sensor of these teachings is designed to work in a two-stage scenario. The first stage is “Capture” in which the target molecule is captured from sample solution by a molecularly-imprinted polymer (MIP). A release solution is then introduced to induce changes in charge and conformation of the captured oxytocin to facilitate its release. The release solution subsequently delivers the released target to the “Detection” stage, which consists of another version of molecularly-imprinted polymer.
A principle of the above embodiment is based on the use of different forms of “molecular imprinting technology”. Molecular imprinting, which enables creation of stable and selective “artificial receptors,” is a method for preparing polymers of predetermined selectivity for the separation and analysis of a vast variety of biologically active molecules. This method has also been the focus of attention for peptide and protein extraction and purification. The technique involves the formation of complexes between a print molecule (template) and a functional monomer based on relatively weak, non-covalent interactions. These complexes appear spontaneously in the liquid phase and are then fixed sterically by polymerization with a high degree of cross-linking. After extracting the print molecules from the synthesized polymer, empty recognition sites remain in the polymer matrix and these sites can recognize the original template molecules during subsequent exposure. Molecularly-imprinted materials have been called “antibody mimics” because these systems attempt to mimic the interactions of their natural counterparts and have achieved affinity and selectivity that approach those of natural recognitions.
An embodiment of the sensor of these teachings that works in two stages as illustrated in
The circumstance that the conformations and charges of the target molecule (such as a peptide) can be tuned by experimental conditions such as pH and ion concentration forms a principle of the present teachings. Researchers have taken advantage of this property to control nanocrystal growth by tuning peptide conformation (Banerjee, I. A. et al, “Cu nanocrystal growth on peptide nanotubes by biomineralization: Size control of Cu nanocrystals by tuning peptide conformation,” PNAS 2003; 100: 14678-14682, which is incorporated by reference herein in its entirety and for all purposes). By tailoring the properties of polymers (by varying charge distribution or hydrophobicity or hydrophilicity or pore size), the formed specific binding site in molecularly imprinted polymer (MIP) matrix can record the conformation and charge state of the target peptide (see
Molecularly-imprinted polymer (MIP) particles for capturing the target molecules were developed. The relevant fabrication involves the use of “emulsion polymerization” approach to synthesize MIP nanoparticles (Zeng, Z., et al. “Synthetic polymer nanoparticles with antibody-like affinity for a hydrophilic peptide.” ACS Nano 2010, 4 (1), pp 199-204, which is incorporated by reference herein in its entirety and for all purposes). An important aspect of emulsion polymerization is that it involves an aqueous solution of monomers dispersed in droplets in an immiscible organic solvent (e.g. toluene and hexane). The droplets are stabilized by surfactants. If a hydrophilic peptide is to be used as an imprint molecule, the peptide will be restricted to the water domain and as a consequence, no accessible binding sites will be formed. Therefore, the position occupied by the peptides at the interface of the water and oil domains during polymerization is very important to create accessible binding sites. To overcome this challenge, the target molecule (also referred to as a peptide) was first modified with fatty acid chains by amide coupling. In this scenario, the modified peptides function as surfactant molecules, with the hydrophobic tail in the oil domain and the hydrophilic segment (peptide) at the surface of the aqueous domain which contains the monomers (see
The microcolumn array was modified with peptide-imprinted polymers using the target molecule as the template molecule. In one instance, fabrication was done at the physiological pH (7.4).
One challenge in forming the molecularly-imprinted polymer (MIP) coating layer is the optimizing the charge distribution, hydrophobicity and cross-link density to yield the highest purification efficiency for the target peptide. The molded PDMS channel was modified to introduce surface-bound acrylamide groups that covalently link the MIP to the channel wall.
A channel that can efficiently and specifically capture the target molecule was developed. A microfluidic channel consisting of micro-column arrays (
Various monomers (see
In one or more embodiments, the method of these teachings includes disposing molecularly imprinted polymer nanoparticles on a surface of a structure, receiving, at the surface, a target fluid having the target molecules, capturing the target molecules in the molecularly imprinted polymer nanoparticles, releasing, after capture, the target molecules from the molecularly imprinted polymer nanoparticles, the target molecules being released into a release solution, providing the release solution to a sensor surface having a layer of molecular imprinted polymer disposed on the sensor surface, the target molecules binding to the layer of molecularly imprinted polymer, and detecting impedance changes in the layer of molecularly imprinted polymer caused by the binding of the target molecules to the molecularly imprinted polymer, the target molecules being detected by the impedance changes.
In order to better elucidate these teachings, the exemplary embodiment of detection of oxytocin is disclosed herein below. It should be noted that these teachings are not limited only to the exemplary embodiment.
Utilization of molecular imprinting for distinguishing oxytocin variants. The circumstance that the conformations and charges of peptide can be tuned by experimental conditions such as pH and ion concentration forms the key principle of our proposed approach. Researchers have taken advantage of this property to control nanocrystal growth by tuning peptide conformation. The principle is also verified by the large difference in isoelectric point (pI) between the OXT-9 (pI, 6.96) and OXT-12 (pI, 8.62). By tailoring the properties of polymers (by varying charge distribution or hydrophobicity or hydrophilicity or pore size), the formed specific binding site in molecularly imprinted polymer (MIP) matrix can record the conformation and charge state of the target peptide (
Ensuring the necessary sensitivity for oxytocin detection. Oxytocin levels in human body are in the pg/mL range, which requires a sensitive measurement method. In this work, sensitive detection of oxytocin was proposed to combine surface imprinting with electrochemical measurement. Imprinting a matrix with binding sites situated at the surface has been proven to have several unique advantages (e.g. easily accessible binding sites, rapid mass transfer and binding kinetics). Meanwhile, methods of electrochemistry impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) were considered owing to their sensitive reliable properties and easy to miniaturization. The technical details about these two technologies are shown in
A passive electrical system comprises both resistor and capacitor elements. Given the non-conductive nature of most biomolecules, the increase in the resistance occurs with increasing surface loading. Before oxytocin binding, the resistance is low because of the existence of highly conductive pathways from the solution to the gold conductive substrate. Once the targeted molecules bind to the cavities and block the conductive pathways, the resistance increases. Based the above hypothesis, both methods are suitable for detection of oxytocin.
Capture Stage
The molecularly imprinted polymer (MIP) particles were modified for capturing oxytocin. The relevant fabrication involves the use of “emulsion polymerization” approach to synthesize MIP nanoparticles. An important aspect of emulsion polymerization is that it involves an aqueous solution of monomers dispersed in droplets in an immiscible organic solvent (e.g. toluene and hexane). The droplets are stabilized by surfactants. If a hydrophilic peptide is to be used as an imprint molecule, the peptide will be restricted to the water domain and as a consequence, no accessible binding sites will be formed. Therefore, the position occupied by the peptides at the interface of the water and oil domains during polymerization is very important to create accessible binding sites. To overcome this challenge, oxytocin was first modified with fatty acid chains by amide coupling. In this scenario, the modified peptides function as surfactant molecules, with the hydrophobic tail in the oil domain and the hydrophilic segment (peptide) at the surface of the aqueous domain which contains the monomers (see
In order to determine the “Capture efficiency” of MIPs, UV-Visible spectroscopy studies were conducted. MIP particles were immobilized within a syringe filter. 100 μL of oxytocin solution (containing either 0.5 mg/ml of OXT-9 or OXT-12) was then carefully injected through immobilized MIP particles, followed by a thorough rinsing with Phosphate Buffered Saline (PBS) buffer (900 μL). The eluting PBS buffer was combined with the post-capture oxytocin solution for UV measurement (
Release Efficiency for both OXT-9 and OXT-12 Forms after Capture
In the “capture and detection” scenario, the captured oxytocin shall be efficiently released and delivered for the detection. Therefore, an effective procedure to release most of captured oxytocin is critical. To do so, five capturing columns were prepared via immobilizing certain amount of cleaned and dialyzed MIP particles within syringe filters for each form of the oxytocin peptide. Meanwhile, five PBS solutions with different pH values were made for releasing the captured peptide. The typical procedure is described as followed: First, 100 μL of oxytocin solution (containing either 0.5 mg/ml of OXT-9 or OXT-12) was injected through a column, and then was thoroughly rinsed with PBS buffer (pH of 7.4) to remove any physically attached peptide. A releasing solution (1 mL) was then carefully injected through the above column and collected for UV characterization (dashed curves in
Specificity of Capture
Current immunoassays fail to differentiate the neuroactive 9-amino acid version (OXT-9) from the pre-hormone 12-aminoacid version (OXT-12). The reason is due to the nature of antibody: the specific recognition ability of antibodies relies on a short variable sequence of amino acids at the tips of the Y-structure [6], which is called the paratope and specific for one particular moiety of the analyte. In the scenario of oxytocin, OXT-9 and OXT-12 both can bind to the paratope of antibody with a similar affinity because both consist of an identical amino acid tip segment. Consequently, immunoassay does not have the specificity to discriminate between the OXT-9 and OXT-12 forms, In other words, they do not have the specificity required by DARPA. To ascertain that the capture stages described in the previous sections do have the specificity needed, two separate columns were prepared with particles imprinted for OXT-9 and OXT-12 (
Specificity from Mass Spectrometry Studies
While the previous section showed that the capture specificity for both OXT-9 and OXT-12 forms, these were based on the UV absorption studies. The capture process was optimized and the specificity determined by a more accurate method—mass spectrometry. For this purpose, a sample solution containing both OXT-9 (0.5 mg/mL) and OXT-12 versions (0.5 mg/mL) in PBS (pH 7.4) was prepared along with two capturing columns (one with OXT-9 imprinted particles and the other with OXT-12 imprinted). 100 μL of test oxytocin sample solution was then carefully injected through a capture column, followed by the thorough rinsing with use of 0.9 ml of PBS buffer (pH 7.4). The rinsing PBS buffer was collected and combined with the post-capture sample solution for mass spectroscopy measurement. For comparison, 100 μL of oxytocin sample solution was directly diluted into 1 mL and characterized with mass spectroscopy (
The ultra-high pressure liquid chromatography coupled with time of flight high-resolution mass spectroscopy (UPLC-QtoF HRMS) was applied to characterize the above solutions. The stationary phase was a C-18 column and the mobile phase was a gradient of water and acetonitrile. Mass spectroscopic studies clearly show that OXT-12-imprinted particles preferentially capture OXT-12 version while OXT-9-imprinted particles preferentially capture OXT-9 version
Sensitive Detectors
To ensure the necessary sensitivity, the critical challenge here was to form an ultra-thin and uniform molecularly-imprinted polymer (MIP), which is capable of sensitively transducing binding events into a detectable electronic signal. The ideal polymer material for coating on the sensor electrode should have the following properties: (1) It should be insulating and (2) It should form a thin and uniform layer. Polyphenol (PPn) yrs MIP for Sensitive detectors was used.
The electrochemical coating of PPn on the flat gold surface is expected to be highly uniform and ultra-thin, due to the self-limiting nature of the deposition. This technical approach has been experimentally demonstrated on a more challenging surface (CNT arrayed architecture) (see, for example, Dong, C. et al, “A molecular-imprint nanosensor for ultrasensitive detection of proteins.” NATURE NANOTECHNOLOGY 2010; 5: 597-601, which is incorporated by reference herein in its entirety and for all purposes). A typical procedure is schematically shown in
The oxytocin detectors were constructed via electrochemically depositing a layer of oxytocin-imprinted polyphenol (PPn) on a flat gold surface. The experimentally demonstrated technical approach (Dong, C. et al. “A molecular-imprint nanosensor for ultrasensitive detection of proteins.” NATURE NANOTECHNOLOGY 2010; 5: 597-601) was applied, which is briefly described below: In a three-electrode electrochemical system, oxytocin peptide was first attracted onto gold surface. Cyclic voltammetry was then applied in presence of phenol monomer at a scanning rate of +30 mV s−1 between 0.0 to 0.9 V versus the reference electrode (Ag wire). The deposited PPn layer has shown to be highly uniform and ultra-thin, due to the self-limiting nature of the electrochemical polymerization. The resultant detector was rinsed and incubated overnight in deionized water to remove the imprinted peptides. The resultant detector has a layer of molecular imprinted polymer 70 disposed on a conductive surface 75, a flat gold surface in the embodiment shown.
Sensitivity: The detection of oxytocin binding to its imprint site on the detector surface was evaluated using differential pulse voltammetry (DPV). A three-electrode electrochemical system was configured by connecting the sensor (gold substrate with polyphenol (PPn) coating) as the working electrode, using silver (Ag) as the reference electrode and platinum (Pt) wire as the counter electrode.
Dynamic range: Based on a literature survey, oxytocin levels in plasma or saliva appear to range from a few pg/mL several to approximately three hundred pg/ml. Such a broad variation indicates the importance of the sensing dynamic range for practical applications. Similarly, differential pulse voltammetry (DPV) was used to determine the sensing dynamic range within a three-electrode configuration.
The relative peak current changes were calculated and plotted with the oxytocin levels.
Integration Design of Capture and Detection Stages into a Common Platform.
The integrated device will include three major modules—a mechanical platform for fluid handling, and a cartridge containing capture column and electrochemical detector, and a data acquisition module containing an electrochemical workstation, a laptop, and a data acquisition card (
The integration approach, in one instance, involves several steps: i) Conversion of the first version of the cartridge to a PDMS-based microfluidic cartridge, which will render the process amenable for manufacturing, ii) Automatic control of the flow of fluids in the mechanical platform, and iii) development of the hardware and the software necessary to enable a fully automatic data collection.
Although these teachings have been described with respect to various embodiments, it should be realized these teachings are also capable of a wide variety of further and other embodiments within the spirit and scope of the appended claims.
This application claims priority of U.S. Provisional Application No. 62/064,681, filed Oct. 16, 2014, entitled METHOD AND SYSTEM FOR SENSING AND DETECTING A TARGET MOLECULE, which is incorporated by reference herein in its entirety for all purposes.
This invention was made partially with U.S. Government support from the U.S. Army Contracting Command Redstone under Contract No. W31P4Q-14-C-0004, The federal government may have certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62064681 | Oct 2014 | US |
