MULTIPLEXABLE SUPER-RESOLUTION FORCE SPECTROSCOPY USING ULTRASOUND METHODS AND MAGNETIC DETECTION

Abstract

A method of measuring dissociation of the biomolecular bonds in one or multiple sample wells using super-resolution force spectroscopy (SURFS). SURFS utilizes precise ultrasound radiation to exert an acoustic radiation force on the biomolecular bonds labeled with magnetic particles. The force-induced dissociation of the protein bonds labeled with magnetic particles may be measured as a reduced magnetic signal by a magnetic sensor. The force resolution allows for differentiating biomolecular bonds with an extremely high level of precision. The biomolecular bonds include protein-protein, protein-nucleic acid, nucleic acid-nucleic acid, small molecule-protein, and small molecule-nucleic acid interactions.

Claims

1. Apparatus to measure the dissociation of molecular bonds, said apparatus comprising: at least one piezo plate; a compartment comprising liquid, wherein the compartment is positioned above the piezo plate; a sample-plate, positioned above said compartment; a magnetic sensor; and transport apparatus configured to move the sample-plate between a first location, and a second location, wherein said sensor is configured to measure changes in magnetism proportional to bond dissociation.

2. The apparatus of claim 1, further comprising a function generator.

3. The apparatus of claim 2, further comprising an amplifier, wherein the amplifier is an ac (alternating current) amplifier.

4. The apparatus of claim 1, wherein the first location is at the piezo plate.

5. The apparatus of claim 1, wherein the second location is at the magnetic sensor.

6. The apparatus of claim 1, wherein the transport apparatus comprises a motor or multiple motors.

7. The apparatus of claim 1, wherein the piezo plate is about 1 mm-100 mm in width, about 1 mm-100 mm in length, and about 0.3-10 mm in thickness.

8. The apparatus of claim 3, wherein the function generator, and an amplifier generate an amplified alternating current, wherein the amplified alternating current has a frequency of between 20 kHz and 5 MHz, and wherein the alternating current has an amplitude of between 0.01 V and 20 V.

9. The apparatus of claim 3, wherein the maximum power of the amplifier is 10-1000 watts.

10. The apparatus of claim 1, wherein the magnetic sensor is an atomic magnetometer, a superconducting quantum interference device, a Hall probe, or a magnetoresistive-based sensor.

11. The apparatus of claim 1, wherein said sample plate comprises 1 to 10,000 sample wells.

12. The apparatus of claim 1, wherein the piezo plates are in the range of 1-1000.

13. The apparatus of claim 1, wherein the first location and the second location overlap.

14. Apparatus for generating an acoustic force and measuring bond dissociation in multiple samples simultaneously, said apparatus comprising: at least one piezo plate, wherein the plate is driven by an amplified ultrasound radiation; a layer of a lubricant positioned on a first surface of said piezo plate; a sample plate comprising at least one sample well, wherein the piezo plate is configured to generate an acoustic force onto each said sample well; a magnetic sensor; and transport apparatus configured to move the sample plate between a first location, and a second location; wherein said sensor is configured to measure the changes in magnetism proportional to bond dissociation for the multiple samples.

15. The apparatus of claim 14, wherein the piezo plate is in the range of 1-1000.

16. The apparatus of claim 14, wherein the layer of lubricant is a vacuum grease positioned between the sample and the piezo plate, and wherein the thickness of the layer of vacuum grease is about 0.001-0.5 mm.

17. The apparatus of claim 14, wherein the layer of lubricant is liquid positioned between the sample and the piezo plate, wherein said layer has a thickness of about 0.001 mm-5 mm.

18. The apparatus of claim 14, wherein the first location and the second location overlap.

19. An apparatus configured for producing multiple acoustic forces within a single application of ultrasound, said application comprising: an array of piezo plates, wherein each piezo plate is coupled in series or parallel with a capacitor, wherein said capacitor adjusts voltage; a sample, positioned within a sample-plate at each said piezo plate; a motor; and a single magnetic sensor or an array of magnetic sensors for detection.

20. The apparatus of claim 19, wherein the said array of piezo plates are configured as a one dimensional array, and wherein the one dimensional array is a straight line or curve.

21. The apparatus of claim 19 wherein the array of piezo plates are configured as a two dimensional array, wherein the two dimensional array are multiple lines and patterns.

22. The apparatus of claim 19, wherein the motor is one of: a linear motor for a one-dimensional scan, or two orthogonal motors for two-dimensional scan.

23. A method of measuring bond dissociation with super resolution force spectroscopy (SURFS), said method comprising: (a) transporting a sample-plate comprising at least one sample to a first location, wherein said first location comprises a magnetic sensor, and measuring a first magnetic signal; (b) transporting said sample plate to a second location, wherein said second location comprises a piezo plate, and applying a first amplitude of ultrasound radiation; (c) transporting the sample plate to said first location and measuring a second magnetic signal; (d) transporting said sample plate to said second location, and applying a second amplitude of ultrasound radiation, wherein said second amplitude of ultrasound radiation is greater than said first amplitude of ultrasound radiation; (e) calculating the difference between the first magnetic signal, and said second magnetic signal; and (f) repeating steps a-e until a statistically significant difference is measured, wherein the difference between the first and the second magnetic signal is proportional to the force required to dissociate molecular bonds of said sample.

24. The method of claim 23, wherein said sample comprises biomolecules, wherein said biomolecules are labeled with magnetic particles, and wherein the biomolecules are nucleic acids, proteins, ribosome complexes, receptors on cell surfaces, or combinations thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] For a detailed description of the disclosed embodiments, reference will now be made to the accompanying drawings, wherein:

[0023] FIG. 1 depicts an embodiment of the SURFS technique described herein, where (a) depicts the precise control of ultrasound radiation to the sample; and (b) depicts a technical drawing of an embodiment of the apparatus setup; and (c) depicts a force spectrum of 12-bp DNA duplex, with a sequence of CCCAAT CGA CCC;

[0024] FIG. 2 depicts an embodiment of the multiplexed detection using a large piezo plate, wherein (a) depicts an embodiment of the setup, in which a 15-well sample was placed on the piezo plate and (b) depicts a sample prior to ultrasound application, while (c) is a depiction of the sample after sonication, wherein samples in rows 2 and 4 were visibly dissociated, while those in rows 1, 3, and 5 remained intact;

[0025] FIG. 3 depicts a graphic representation of an embodiment of a circuit to produce different forces at different piezo plates (the use of piezo elements and piezo plates in the context of this application are interchangeable) indicated as a circle by varying the capacitors C.sub.1-C.sub.4 as voltage dividers in accordance with principles described herein;

[0026] FIG. 4 depicts a graphical representation of resolving DNA-drug bonds from DNA duplexes, wherein (a) depicts resolving two close binding forces, 12-bp DNA and drug-bound 11-bp DNA. 11-bp DNA: ds(CCAAT CGA CCC); drug: daunomycin, and (b) depicts the calibration of force vs. the square of voltages, V.sup.2, in accordance with principles described herein;

[0027] FIG. 5 depicts an embodiment of SURFS used to probe molecular motion in ribosome translocation, wherein (a) provides a depiction of an embodiment of the probing scheme, in which a magnetically labelled DNA (bottom) is used to form duplexes with various ribosome-mRNA complexes, shown as Post(MFEK) and Pre(MFEKR), while panel (b) depicts a force spectra for the two DNA-ribosome structures depicted in a; and

[0028] FIG. 6 depicts an illustration of using SURFS to resolve multiple binding steps, wherein panel (a) depicts a schematic of different binding, and panel (b) depicts a simulated force spectrum; and

[0029] FIG. 7 depicts an further embodiment of the SURFS technique described herein, where (a) depicts the precise control of ultrasound radiation to the sample and indicates a first and second location for a magnetic senor; and (b) depicts a technical drawing of an embodiment of the apparatus setup and connectivity of an amplifier, and generator as described in embodiments herein,; and (c) further depicts a force spectrum of 12-bp DNA duplex, with a sequence of CCCAAT CGA CCC.

DETAILED DESCRIPTION OF DISCLOSED EXEMPLARY EMBODIMENTS

[0030] The following description is directed to various exemplary embodiments of the invention. However, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be exemplary of that embodiment, and that the scope of this disclosure, including the claims, is not limited to that embodiment.

[0031] Certain terms are used throughout the following description, and claims to refer to particular features or components. As one skilled in the art will appreciate, different persons may refer to the same feature or component by different names. This document does not intend to distinguish between components or features that differ in name but not function. The drawing figures are not necessarily to scale. Certain features and components herein may be shown exaggerated in scale or in somewhat schematic form, and some details of conventional elements may be omitted in interest of clarity and conciseness. In the following discussion and in the claims, the terms including and comprising are used in an open-ended fashion, and thus should be interpreted to mean including, but not limited to . . . .. As used herein, the term about, when used in conjunction with a percentage or other numerical amount, means plus or minus 10% of that percentage or other numerical amount. For example, the term about 80%, would encompass 80% plus or minus 8%. References cited herein are incorporated in their entirety. Also, the term couple or coupled is intended to mean either an indirect or direct electrical connection. Thus, if a first device is coupled to a second device, that connection may be through a direct electrical connection of the two devices, or through an indirect electrical connection, established via other intermediate devices and connections.

[0032] Embodiments disclosed herein are intended to overcome certain above mentioned limitations by using SURFS, which provides for a number of advancements over the prior art as disclosed herein and throughout the disclosure. One such advancement is to use precisely controlled ultrasound as the force source. By using a water reservoir as the medium between the piezo disk or plate and the sample, a precise force field may be obtained, and the thermal effect is significantly reduced. A second advancement is to automate the associated apparatus such that the sample can be alternately scanned for magnetic measurement and fixed for force application in many cycles (see FIG. 3b). The ultrasound power was gradually increased after each cycle wherein the automation substantially reduced operational errors. This is achieved by incrementing the amplitude of the output of a function generator prior to amplification.

EXAMPLES

[0033] FIG. 1a shows the scheme of an embodiment of such a precise ultrasound control. A piezo plate was placed on a piece of rubber. On top of the piezo, a water reservoir was constructed. The sample was mounted on a glass rod for scanning. The bottom of the sample, which contains the immobilized molecular system, makes contact with the water surface prior to ultrasound application. The water level was carefully monitored to be constant during experiment. FIG. 1 b shows the technical drawing of the sample, piezo, and water reservoir. A DNA duplex with 12-basepair (bp) was used to calculate the force resolution. The dissociation voltage was determined to be 0.204 V on the function generator, with 0.001 V resolution. The dissociation force of the same 12-bp DNA was also measured using FIRMS, which was revealed to be 45 pN. Because the acoustic radiation force is proportional to the ultrasound power, or V.sup.2, the force resolution can be calculated as

F=(2V/V)*F=(2*0.001/0.204)*45=0.5 pN (1)

[0034] Here, F is the bond dissociation force, and V is the dissociation voltage for ultrasound. F and V represent resolution of F and V, respectively.

[0035] Multiplexed SURFS using a single piezo plate: To accommodate multiple samples, a larger piezo plate is used. Depending on the size of each sample well and the total number of sample wells needed, the typical in-plane dimension of the piezo plate may be 5-100 mm in various shapes, and the thickness is typically 0.2-10 mm. Various large piezo plates are commercially available, for example, from American Piezo Company, and can be easily cut into customized sizes. Larger piezo plates have larger capacitances than the smaller ones, which lead to lower applied voltages. The equation governing the maximum voltage (Vmax) is:

Vmax=Imax/(2fC) (2)

[0036] Herein, Imax is the current rating of the amplifier, f is the frequency of sine ultrasound wave, and C is the capacitance. Because the acoustic force is proportional to the square of voltage, a high-current amplifier is required to drive a large piezo plate.

[0037] In addition to the scheme shown in FIG. 1a, another viable scheme is shown in FIG. 2a. Herein, in this embodiment a thin layer of grease may be applied on the piezo surface; then the sample plate containing multiple sample wells was placed on top of the piezo. Using a 4040 mm.sup.2 piezo (2 mm thick) as an example, the capacitance was measured to be 15.6 nF (FIG. 2b, bottom square). The resonance frequency is 1 MHz. In order to apply 8 V on the piezo (0.2 V prior to amplification), the current rating of the amplifier must to be greater than Imax, which will be in the current embodiment 0.78 A. The corresponding force will be approximately 45 pN based on the result in FIG. 1c. An amplifier 75A250A from AR (Souderton, Pa., USA) was used in this embodiment.

[0038] The sample plate contained fifteen sample wells (FIG. 2b, top plate). The first, third, and fifth rows were specifically bound magnetic particles via DNA duplexes; the second and fourth rows were non-specifically bound magnetic particles. Therefore, at a small force, particle dissociation would be expected for Rows 2 and 4, but not for Rows 1, 3, and 5. The results are shown in FIG. 2c, in which only Rows 2 and 4 contained dissociated particles as expected. Thus, this embodiment disclosed that it is possible to use a large piezo plate to apply force on multiple samples.

[0039] Gradient force field using piezo plates coupled with voltage dividers: Prior to the current disclosure, the time resolution of force spectroscopy has been poor because the data points were acquired one at a time. For example, to obtain a force spectrum with 15 data points, 15 different forces are needed and magnetic measurement will follow each force application. Each data point will take about 3 minutes. Hence, the time resolution is on the order of one hour. This may not be sufficient to study multiple-step protein binding events, such as the sequential binding of gp120 onto CD4 receptor and CXCR4 co-receptor for example.

[0040] The time resolution of such measurements is advanced by embodiments of SURFS as disclosed herein, and is thus achieved by generating a gradient force field. Herein, in some embodiments an array of individual piezo plates may be used instead of a single one. The plates are in parallel, but each is coupled with a different capacitor in series. The capacitances of the capacitors (C.sub.1-C.sub.4 in FIG. 3) may be calculated based on voltage dividers. For example, if only half of the voltage needs to be applied on a particular piezo, the capacitor should have the same capacitance as the piezo. The resulting force will be one quarter of the force without the capacitor, denoted as F.sub.0. In general, if the designated force is a factor of of F.sub.0 and C represents the piezo capacitance, then the capacitance of the coupling capacitor will be given by:

C.sub.i=(+1)C (3)

[0041] The gradient force field produced by adjusting the coupling capacitors may therefore improve the time resolution of SURFS. For example, in some embodiments the number of force applications may be reduced by 4 if four identical samples are used at different forces, which improves the force resolution by a factor of 4. By using more piezo elements in parallel, the whole force spectrum can be obtained within a single ultrasound application.

[0042] Applications of SURFS

[0043] With high force resolution, spatial resolution, and time resolution, SURFS may be utilized in biological research and biotechnology industrial branches, some such embodiments are disclosed herein.

[0044] Drug Molecules

[0045] Key questions in the design and development of drug molecules include the issue of how a drug interacts with their molecular target(s), including how effective is the drug for inhibiting the targeted ligand-receptor pair. Another is how the drug functions, i.e., whether it works through allosteric effect or directly blocks the ligand's binding pocket. Typically, binding affinity in concentration is used to characterize drug's efficacy. However, it does not provide information on the binding mechanism. Specifically, considering as an example CXCR4 and CCR5, both have been viable drug targets for HIV. A number of drug candidates have been developed, with one of them Maraviroc having been approved. X-ray structure showed that Maraviroc functions through allosteric effect that weakens the binding between gp120 and CCR5. However, it is unknown how much weaker the binding becomes and how effective it is on the cell surface.

[0046] SURFS, in some embodiments may be used to provide a precise differential binding force, F, to quantify the effect of drug molecules: wherein the presence of the drug altered the binding force of ligand-receptor bonds from F to F, so F=FF. [See U.S. Pat. No. 9,778,249 further incorporated herein in its entirety] Given that F in some will be different for allosteric vs. direct blocking: F will be smaller for allosteric effect than for direct blocking. Embodiments of the methods disclosed herein have showed that F accurately revealed the chiral selectivity of drug molecules and sequence specificity of DNA duplexes. The values of F were typically 5-16 pN. F may have a greater range in such an embodiment because drug molecules usually have higher affinity for proteins than for DNAs.

[0047] FIG. 4 shows the advantage of the high force resolution methods disclosed herein for resolving drug interactions. In FIG. 4a, the force spectra of 12-bp DNA and daunomycin bound 11-bp DNA are compared. The two dissociation voltages were well distinguished, with the former at 0.204 V and the latter at 0.226 V. However, previous techniques of the prior art cannot distinguish the two forces, which are 45.sup.2 and 48.sup.2 pN respectively. In other words, it can now be concluded using the methods disclosed herein that daunomycin-11-bp DNA is stronger than 12-bp DNA.

[0048] Another embodiment of the SURFS method herein disclosed was used to measure a series of DNA and DNA-drug complexes, and correlated the dissociation voltages with forces obtained by FIRMS. Linear fitting produces:

F=740V.sup.2+11 (4)

[0049] This equation confirms that the dissociation force linearly depends on the ultrasound power (represented by V.sup.2).

[0050] Molecular Motion Revealed by SURFS

[0051] Ribosomal translocation is a key step in protein synthesis. For each translocation step, the ribosome moves by three nucleotides from the pre-translocation state (Pre-complex) to the post-translocation state (Post-complex). Then, the ribosome will be in the pre-translocation state for the next translocation cycle. No technique of the prior art has revealed any difference between the Post-complex of the previous step and the Pre-complex of the next step. In other words, in light of the prior art it is unclear how the ribosome moves along the mRNA to decode genetic information.

[0052] However, embodiments are herein disclosed that comprise a probing scheme based on SURFS to precisely determine the position of the ribosome on the mRNA. Shown in FIG. 5a, a probe DNA labelled with magnetic particle to form duplexes with the exposed mRNA is disclosed. The number of base-pairs can be derived from the dissociation forces of the duplexes. Typically, one A-T nucleotide pairing will increase the force by 8-12 pN, and one C-G pairing will increase by 15-20 pN, after the minimum number of base pairing (9-10) to form a stable duplex.

[0053] The force spectra in FIG. 5b reveal that the force difference is very small but detectable. The dissociation voltages differ by 0.03 V. Compared to the results in FIG. 4, the force difference is on the order of about 3-5 pN, much less than a complete base-pair. This indicates there is a definite motion of the ribosome going from Post-complex step to the next Pre-complex step, but the motion is less than one nucleotide. Thus, such a step cannot be revealed by prior art methods, but is disclosed by the application of SURFS as described herein (because the space between neighbouring nucleotide is about 0.5 nm, the motion can be considered to be a fraction of this distance, or about 0.2 nm).

[0054] Resolving Multiple Binding Steps

[0055] Embodiments of the scheme of differential force field disclosed herein, enables the study of multiple-step binding, such as the infusion of the HIV infection. The most current prior art model for the infusion mechanism is that the viral envelope glycoprotein, gp120, first binds with the CD4 receptor on the cell surface and then with a co-receptor, CCR5 or CXCR4; fusion subsequently takes place between the virus and the cell. However, direct evidence for the molecular mechanism of this process remains elusive.

[0056] SURFS with time resolution as disclosed herein is well suited to address the resolution of this mechanistic question. It has been shown that gp120 binds with CD4 fairly rapidly on the substrate surface, within 2-3 min, whereas the complete fusion between the HIV virus and the cells takes approximately 60 min. Therefore, both force resolution and time resolution should be used to resolve the two steps. Shown in FIG. 6, there will be three types of bonds: weak nonspecific bonds (F.sub.1), strong gp120-CD4 bonds (F.sub.2), and stronger gp120-CD4-CCR5/CXCR4 (F.sub.3) bonds. At an earlier time (such as t1), the force spectrum should only show binding forces of F.sub.1 and F.sub.2, but not F.sub.3. Gradually (t>t1) F.sub.3 will appear in the force spectrum at longer reaction time, with amplitude designated as B.sub.3. The binding kinetics between gp120 and the CCR5/CXCR4 co-receptor will thus be obtained by plotting B.sup.3 vs. time t.

[0057] SURFS may also be employed in some embodiments to investigate drug molecules targeting the second binding step. The efficacy will be shown as two parameters, one is the differential binding force and the other is the second step binding amplitude, B.sup.3.

[0058] Thus, disclosed herein is a multiplexable SURFS technique that addresses the unmet need for sub-pN force resolution for the binding forces of intermolecular bonds, 0.2 nm spatial resolution on molecular motion along nucleic acids, and 2 min time resolution for multiple-step protein binding, capabilities of SURFS as disclosed herein may be extended to study protein binding on living cells.