Method for detecting small molecule analytes using magnetoresistant sensors
Small molecule analytes (less than 1000 Daltons) in a fluid sample are detected using a competitive assay in a magnetic biosensor. The fluid sample is added to a biosensor detection chamber together with detection probes and magnetic tags which bind to the detection probes. The magnetic biosensor is functionalized with a capture probe that shares an epitope with the analytes, and the detection probe is capable of binding the epitope shared by the analytes and the capture probe, so that the presence of the analyte prevents detection probes (and magnetic tags) from binding to the biosensor. By measuring the binding of the magnetic tags to the magnetic biosensor, an amount of analytes in the solution is determined.
This application is a continuation of U.S. patent application Ser. No. 15/281793 filed Sep. 30, 2016, which claims priority from U.S. Provisional Patent Application No. 62/236708 filed Nov. 2, 2015, both of which are incorporated herein by reference.
FIELD OF THE INVENTIONThe invention relates generally to biosensing devices and techniques. More specifically, it relates to methods of detecting small molecule analytes in samples using magnetic sensors.
BACKGROUND OF THE INVENTIONThe need for methods to detect small molecule analytes in small quantities has motivated the development of a variety of biosensing techniques. For example, current high sensitivity immunoassay techniques use a competitive assay reaction scheme that requires washing between sample addition and analyte detection to remove background signal and provide required sensitivity. This washing adds additional steps and complexity to the technique, limiting its use. There remains a need for biosensing techniques with very high sensitivity that are simple enough to be suitable for point of care applications.
SUMMARY OF THE INVENTIONIn one aspect, the present invention provides a technique for detection of small molecule analytes without sacrificing sensitivity. To the best of our knowledge, this is the first demonstration of magnetic biosensors that are capable of detecting small molecules (i.e., molecules less than 1000 Daltons in molecular weight). A method for detecting small molecule analytes according to embodiments of the invention uses magnetic sensors and nanoparticle tags. The assay involves a capture probe, which shares an epitope with the sample analyte. The capture probe competes with the sample analyte for small molecule detection probes (such as antibodies, Fab fragments, single chain variable fragments, aptamers, or whole receptors). This capture probe is immobilized onto the surface of a sensor to capture detection probes in close proximity to the sensor. The presence of the analyte of interest in the sample interferes with the binding of the detection probes to the capture probes in a concentration dependent manner. After the detection probe binds the capture probe, the binding of magnetic tag to the detection probe brings the magnetic tag into close proximity to the sensor, which then detects the binding event. The binding of detection probes to the capture probe inversely relates to the degree to which the detection probe has been bound by sample analyte in solution. Thus, the amount of sample analyte in solution can be calculated.
In one aspect of the invention, a method is provided for detection of analytes in a fluid sample using competitive assay in a magnetic biosensor. The method includes adding the fluid sample containing the analytes, detection probes, and magnetic tags to the magnetic biosensor. The analytes being detected are small molecules, which is defined herein to mean that they have weights less than 1000 Daltons. The magnetic biosensor is functionalized with a capture probe that shares an epitope with the analytes, and the detection probe is capable of binding the epitope shared by the analytes and the capture probe. The method also includes measuring binding of the magnetic tags to the magnetic biosensor via the detection probes and the capture probes, and determining an amount of analytes in the solution from the measured binding of the magnetic tags to the magnetic biosensor.
In some instances of the method, two or more of the fluid sample, detection probes, and magnetic tags may be mixed together prior to adding them to the magnetic biosensor. For example, the detection probes and the magnetic tags may be conjugated prior to their addition to the magnetic biosensor. In other instances of the method, the fluid sample, detection probes, and magnetic tags may be added to the magnetic biosensor sequentially.
In the present context, adding a compound to the biosensor is understood to mean introducing it to a biosensor reaction chamber containing a biosensor element so that it can react with the biosensor element.
The magnetic biosensor preferably includes multiple magnetic biosensor elements. Some implementations include control biosensor elements that are not functionalized with capture probes that can bind to the analytes or to the detection molecules, in which case determining the amount of the analytes includes comparing the measurements of binding of the magnetic tags to the magnetic biosensor with measurements from the control biosensor elements. In some implementations, the magnetic biosensor elements are functionalized with different types of capture probes that share corresponding distinct types of epitopes with multiple corresponding distinct types of analytes, and the fluid sample contains the multiple distinct types of analytes; the method then includes adding to the magnetic biosensor multiple distinct types of detection probes that are capable of binding the multiple corresponding distinct types of epitopes. The method may also include measuring binding of the magnetic tags to the magnetic biosensor elements functionalized with different types of capture probes, and determining amounts of the distinct types of analytes in the solution from the measured binding of the magnetic tags to the magnetic biosensor elements.
The amount of analytes in the solution may be determined from the measured binding of the magnetic tags to the magnetic biosensor by measuring electrical signals from magnetic biosensor elements (e.g., giant magnetoresistive sensors) to detect changes in electromagnetic properties of the biosensor elements due to binding of the magnetic tags.
In another aspect, the invention provides a method for detection of analytes in a fluid sample using direct binding in a magnetic biosensor. The method includes adding the fluid sample containing the analytes and magnetic tags to the magnetic biosensor. The analytes are small molecules that have weights less than 1000 Daltons. The magnetic biosensor is functionalized with a capture probe that changes conformation upon binding to the analytes, and the conformation changes enable the capture probe to bind to the magnetic tags. The method also includes measuring binding of the magnetic tags bound to the capture probes of the magnetic biosensor, and determining an amount of analytes in the solution from the measured binding of the magnetic tags to the capture probes.
A biosensing technique according to one embodiment of the invention is illustrated in
Following the principles above, a method of detecting at least one analyte according to an embodiment of the invention uses the following competitive assay scheme:
1) Fabricate an array of magnetic sensors and functionalize the surfaces such that a capture probe can be bound to the sensor surface.
2) Spot the capture probe that shares an epitope with the analyte of interest.
3) Add sample containing the analyte of interest.
4) Add detection probe capable of binding the epitope shared by the analyte of interest and capture probe.
5) Add magnetic tags.
6) Monitor the binding of the magnetic tags to the detection probe bound to the capture probe, and determine the amount of sample analyte in the solution.
As an example of the technique adapted for detecting tetrahydrocannabinol (THC) analytes (314 Daltons) in a saliva sample, the biosensor elements may be giant magnetoresistive (GMR) sensor elements functionalized with bovine serum albumin (BSA) conjugated with THC molecules. The detection probe is anti-THC bound to a magnetic nanoparticle complex. Other sensor elements may be functionalized with BSA or BSA-Biotin, which serve as biological negative and positive controls, respectively. The technique may be implemented using a system including an 10×8 array of giant magnetoresistive sensors. Saliva samples containing unknown amounts of THC are mixed with biotin labeled antibodies for THC and are added to the reaction chamber with the sensors. Binding of anti-THC antibodies to the BSA-THC coated sensors is monitored with the addition of streptavidin labeled magnetic nanoparticles. The amount of binding is concentration dependent and inversely related to the amount of THC in saliva. As more THC is added to the saliva sample, the signal from the GMR sensor decreases.
The technique may be implemented as a hand-held point-of-care device that enables rapid and precise detection of small amounts of THC without the need for washing. Until now, GMR biosensors have only been used to measure large proteins, with a focus on cancer diagnosis. The combination of GMR biosensor technology with a competitive immunoassay assay scheme provides surprising improvement in biosensor sensitivity, with the ability to detect low nanomolar concentrations of very small molecules.
For illustrative purposes, an example of such a device for implementing the techniques of the present invention are now described in detail.
The disposable cartridge 138 and magnified view of the GMR biosensor chip 140 are shown in detail in
The array of GMR biosensors may be fabricated on a 10×12 mm piece of silicon wafer. An individual GMR biosensor has a spin valve structure of IrMn (8)/CoFe (2)/Ru (0.8)/CoFe (2)/Cu (2.3)/CoFe (4.5) (all thicknesses in nm) on seed layer, and has an active sensing area of 100×100 Electrical pads are connected to the biosensors via a network grid type of electrodes to allow external access to individual biosensors. 30 nm and 300 nm of oxide layers are deposited on the active sensing area and the rest of the chip, respectively, to passivate the electrodes. After washed with acetone, methanol, and isopropanol, the chip is glued on a customized PCB, and wire-bonded to the electrical pads. Then, the chip is treated with 10% (3-Aminopropyl)triethoxysilane (APTES, Sigma-Aldrich, USA) in acetone for 30 min. After washing with acetone and distilled water sequentially, a reaction chamber is installed on top of the chip to accommodate samples and reagents. THC-BSA (Fitzgerald, USA), BSA (Sigma-Aldrich, USA), and biotinylated BSA (Sigma-Aldrich, USA) are then spotted on different sensors with replicates using a non-contact arrayer (Scienion, USA). The cartridge is stored overnight in a humid chamber at 4° C. The chip is washed with rinsing buffer (PBS pH 7.4 with 0.1% BSA and 0.05% Tween-20), and blocked with 1% BSA (Sigma-Aldrich, USA) for 1 hour before use.
The measurement reader is composed of two stacked PCBs and a cartridge integrated with GMR biosensor chip. The two PCBs are connected via two connectors (16 pins and 20 pins). The top board is equipped with a receptacle for the cartridge and a toroid magnetic core coil with 470 μF ceramic capacitor for LC resonance. The LC tank is driven by two power amplifiers in a bridge-tied load configuration on the top board as shown in
To reduce the effect by flicker noise of GMR sensor, double modulation scheme is implemented as shown in
The bottom PCB provides a sinusoidal voltage to the GMR sensors while the magnetic field generated by the toroid core coil magnetically excites the GMR biosensors. The electrical current from GMR biosensors is amplified by carrier tone subtraction circuit. Then the 20-bit ADC chip (Linear Technology, LTC2378) samples the amplified signal and transfers the digitized signal to the microcontroller (ATSAM3X8EAAU, Atmel Corporation) for further analysis with Fast Fourier Transform. Temperature and magnetoresistive ratio (MR) correction algorithm is used to remove changes in signal due to temperature fluctuation and variation in sensor fabrication. The microcontroller transferred the processed data to the Bluetooth module (HC-06) on the bottom board using Serial Peripheral Interface (SPI). Then, a smartphone connected via Bluetooth receives and displays the result on the screen using a custom app.
Step 2: 50 μL of the mixture is added to the chip reaction chamber containing a functionalized biosensor chip. The chip has bovine serum albumin (BSA) 174 and THC-BSA 176 immobilized on different sensors 178, 180, respectively. In addition, some sensors are functionalized with biotinylated BSA (Biotin-BSA). The solution is incubated for unoccupied antibodies 170 to bind to THC-BSA 176 on the sensors for an additional 15 min.
Step 3: Unbound antibodies are washed, the disposable chip is inserted into the measurement reader, and 40 μL of streptavidin-coated MNPs 182 are then added to the chip reaction chamber, where they bind to antibodies 170. The stray field from the bound MNPs disturbs the magnetization of biosensors underneath, which changes the resistance of the biosensor. The changes in resistance, monitored as GMR biosensor signals (ΔMR/MR0), are proportional to the number of bound MNPs and have an inverse relationship with the concentration of THC in the sample due to the nature of competitive assays.
After the cartridge is inserted into the measurement reader, a custom app based on the Android operating system (Google, USA) controls the measurement procedure. First, the app starts to measure the resistances of the biosensors to ensure whether the cartridge is inserted correctly and exclude defective biosensors of the chip from the measurement. By applying two different magnitudes of magnetic field to the sensors, the app calibrates the individual biosensors and normalizes the signals. Then, the app asks the user to add MNPs (Miltenyi Biotec, USA) to the chip and monitors signals from biotin-BSA-coated sensors. Signals from the THC-BSA, BSA, and biotinylated BSA (Biotin-BSA) sensors are monitored. The app automatically aborts the measurement if the biotin-BSA signals remain below 100 ppm until 1 min, which is a good indication that the user did not add the MNPs. Otherwise, the app continues to monitor all signals from different sensors up to 10 min. The raw data can be sent via email.
Saliva samples can be easily collected in the field using various techniques. For example, a simple sample collection strategy uses a cotton swab 200, syringe 202, and filter unit 204, as shown in
For THC detection, values ranging from 2 to 25 ng/mL are of interest. To achieve better sensitivity around this range, the preferred concentration of anti-THC biotinylated antibodies is 1 μg/mL. To arrive at this value, the inventors tested three different concentrations of antibodies (5, 1, and 0.5 μg/mL) with THC analyte concentrations at 0, 5 and 20 ng/ mL, as shown in
To determine a preferred time frame for incubation of a sample mixture with the chip, three different incubation times (5, 10, and 15 min) for the chip incubation were tested with 15 min of preincubation (
Furthermore, preincubation and chip-incubation times were reduced to 5 and 10 min, respectively, and the GMR sensor signals were taken at 5 min after adding MNPs instead of 10 min to carry out the entire measurement within 20 min.
The array of sensors may be split into two groups and compartmentalized with separate reaction chambers. One of the compartments is used to measure a sample with known concentrations of analytes or without any of them, and the other compartment is used to measure unknown sample or sample of interest. The difference in signals between the sensors with the same capture probe in different compartments can be used to determine the concentrations of analytes in the unknown sample or sample of interest.
Since the binding of the antibodies to THC is a thermodynamic process, the temperature affects the assay results and there are day-to-day variations in measurement signals due to temperature fluctuations, chip-to-chip variations, or incubation time variations. Thus, to increase accuracy of the assay and minimize the measurement variation, a two-compartment cartridge 400 where two reaction chambers 402, 404 with a gasket made of polydimethylsiloxane (PDMS) are used with a GMR biosensor chip 406 to measure both the sample of interest and a reference sample simultaneously with the same chip 406 (
Although the examples discussed above for illustrative purposes focus on THC, the techniques of the present invention are not limited to THC, but are generally applicable to other small molecules. For example, the technique was demonstrated with morphine (285.3 Da) by replacing THC with morphine. The sensors were coated with morphine-BSA in lieu of THC-BSA, and antimorphine antibodies at 0.1 μg/mL were used. The signals from zero analyte, morphine at 10 and 100 ng/mL showed statistically significant differences.
Since the competitive assays are applicable to detection of any type of small molecules, the platform could be used to detect different drugs such as heroin and cocaine in addition to THC and morphine as well as to detect therapeutic small molecule inhibitors in cancer treatments.
In some embodiments of the invention, different capture probes for detecting different small molecules may be functionalized on different sensors in the array of sensors to perform multiplex assays. Then, the corresponding detection probes that can bind their target analytes specifically are added to the biosensor. For example, with the multiplexing capability of the GMR biosensor chip, the technique can be used to simultaneously detect multiple analytes (e.g., THC—COOH and THC, or THC and its metabolites in blood and urine).
An example of a multiplexing magnetic biosensor array 500 is shown in
It will be appreciated that the principles of the invention are not limited to the specific techniques or devices described above for illustrative purposes, but may be altered in various ways. For example,
The reaction scheme may have variations, such as:
a) The detection probe and magnetic tags may be pre-conjugated, then added to the sensor array after addition of sample.
b) The detection probe and magnetic tags may be pre-conjugated, pre-mixed with sample, and then added to the sensor array as one whole mixture.
c) The detection probe, magnetic tags, and sample may be simultaneously pre-mixed prior to addition to the sensor array.
d) The detection probe, magnetic tags, or a conjugate of the two may be lyophilized on or in proximity to the array of sensors.
The reagents may be delivered via microfluidics. The reagents may be delivered via paper-based or gel-based platform to the sensor or via lateral flow method.
Mechanical stimulation such as shaking, vibrating, or stirring may be used to mix reagents.
The capture probe may be spotted on each sensor locally by surface tension of the solution, structural confinement, chemical treatment (e.g., hydrophobic/philic treatment), or delivery via an imprinting method.
The magnetic sensors may be magnetic tunnel junction sensors, Hall effect sensors, or magnetoresistive sensors such as anisotropic magnetoresistive sensors and giant magnetoresistive sensors.
The magnetic tags may be magnetic beads, magnetic nanoparticles, magnetic disks, and magnetic rods. They can bind to the detection probes via protein-protein interactions, DNA-DNA interaction, or chemical reactions.
An aptamer may be used instead of an antibody for the detection probe. In some cases, this could yield improved sensitivity, specificity, and provide a more thermally stable alternative to an antibody based approach.
In addition, the technique is not limited to saliva, but is capable of detecting small analytes in blood, because GMR biosensors are matrix-insensitive.
In another aspect, the invention provides a method for detection of small molecule analytes in a fluid sample using direct binding in a magnetic biosensor, as illustrated in
Claims
1. A method for detection of analytes in a fluid sample using direct binding in a magnetic biosensor, the method comprising:
- adding the fluid sample containing the analytes and magnetic tags to the magnetic biosensor, wherein the analytes have weights less than 1000 Daltons;
- wherein the magnetic biosensor is functionalized with a capture probe that changes conformation upon binding to the analytes;
- wherein the conformation changes enable the capture probe to bind to the magnetic tags;
- measuring binding of the magnetic tags bound to the capture probes of the magnetic biosensor;
- determining an amount of analytes in the solution from the measured binding of the magnetic tags to the capture probes.
Type: Application
Filed: Feb 27, 2019
Publication Date: Jun 27, 2019
Inventors: Tyler O'Brien Shultz (Los Gatos, CA), Shan X. Wang (Portola Valley, CA), Jung-Rok Lee (Seoul)
Application Number: 16/287,000