Simultaneous Parallel Signal Amplification and Analyte-Ligand Capture Functions

Abstract

A device and system for conducting accurate diagnosis of states of disease or health, including cancer, with high sensitivity and specificity is described. The system employs a portable cassette-based testing system which is configured to detect pp analytes captured by hydrogel particles equipped with affinity bait(s) and an amplification reporter system. The affinity baits bind to a wide range of analytes, including: metabolites, proteins, nucleic acids, lipids, hormones, cytokines, growth factors, biomarkers, virus particles, exosomes, bacteria, fungi, drug compounds, synthetic organic compounds, volatile odorants, toxicants, and pollutants. The affinity baits bind with extremely high affinity, preserving the captured analyte. This system can increase the sensitivity of detection of said analytes up to ten-thousand-fold.

Claims

1. A method for detecting analyte-ligand binding, comprising: flowing an analyte with an antigen across a sample and conjugate pad; functionalized hydrogel particles capturing the antigen; the functionalized hydrogel particles continuing to flow after capturing the antigen; the antigen binding to a capture antibody when the functionalized hydrogel particles reach an electrode system; reactions occurring; free electrons generating a response; the functionalized hydrogel particles continuing to flow across the electrode system; and a control line on a counter electrode capturing the functionalized hydrogel particles.

2. The method of claim 1, wherein the reactions occurring are chosen from the group: D-Glucose+H.sub.2O+O.sub.2.fwdarw.D-Gluconic Acid+H.sub.2O.sub.2; H.sub.2O.sub.2+HRP.sub.red.fwdarw.H.sub.2O+HRP.sub.ox

3. The method of claim 1, wherein the functionalized hydrogel particles have an enzyme placed in an internal volume of the functionalized hydrogel particles.

4. The method of claim 3, wherein the enzyme is horseradish peroxidase.

5. The method of claim 1, wherein the functionalized hydrogel particles have an enzymatic amplification system.

6. A system for detecting analyte-affinity molecule binding, comprising: a particle; an affinity molecule, configured to bind to an analyte of interest, within said particle; and an amplification reporter system associated with said particle.

7. The system of claim 6, wherein said particle is a porous polymer particle.

8. The system of claim 7, wherein said amplification reporter system is excluded from a surface of said particle.

9. The system of claim 8, wherein said particle has substantial open void volume.

10. The system of claim 9, wherein said amplification reporter system is an unlinked chemical reporter system.

11. The system of claim 9, wherein said amplification reporter system is an electrochemical reporter system.

12. The system of claim 9, wherein said affinity molecule is immobilized within an internal structure of said particle.

13. The system of claim 9, wherein a pore size of said particle is configured to allow a secondary affinity molecule to reach into an internal structure of said particle and to recognize said analyte bound to said affinity molecule.

14. The system of claim 13, wherein said secondary affinity molecule is chosen from the group: antibodies; antibody fragments; peptides; proteins; lipids; carbohydrates; nucleic acids, synthetic molecular recognition compounds.

15. The system of claim 13, wherein said pore size of said particle is configured to allow a secondary affinity molecule to reach into an internal structure of said particle and to recognize said analyte bound to said affinity molecule only when said particle resides at a predetermined detection zone.

16. The system of claim 15, wherein said amplification reporter system is configured to be activated after said secondary affinity molecule has recognized said analyte of interest at said predetermined detection zone.

17. The system of claim 16, wherein said amplification reporter system is configured to generate a signal signifying said particle in said pre-determined detection zone only when said analyte is present in said particle.

18. The system of claim 17, wherein said analyte is chosen from the group: metabolites; proteins; nucleic acids; lipids; hormones; cytokines; growth factors; biomarkers; virus particles; exosomes; bacteria; fungi; drug compounds; synthetic organic compounds; volatile odorants; toxicants; pollutants.

19. The system of claim 16, wherein said pre-determined detection zone has a sensor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate the present invention and, together with the description, further serve to explain the principles of the invention and to enable a person skilled in the pertinent art to make and use the invention.

[0029] The present invention will be better understood with reference to the appended drawing sheets, wherein:

[0030] FIG. 1 shows an exploded view of the cassette test device of the present invention.

[0031] FIG. 2 depicts the current amperometric sensor of the present invention as a traditional 2-prong electrode.

[0032] FIG. 3 shows results of a test to determine if the hydrogel particles capture and arrest at the capture antibody line against the ESAT-6.

[0033] FIG. 4 depicts a result of adding the chromogenic substrate DAB to the reactive solution of G and GOx, producing a dark brown precipitate and amplifies the colorimetric intensity of the LFI.

[0034] FIG. 5 depicts a paired t-test with unequal variance, showing results of the test and device of the present invention.

[0035] FIG. 6 exhibits a paired t-test with unequal variance was performed, and using the charge threshold, there was significant differentiation between the positive and negative test cases

[0036] The features and advantages of the present invention will become more apparent from the detailed description set forth below when taken in conjunction with the drawings, in which like reference characters identify corresponding elements throughout. In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0037] The present specification discloses one or more embodiments that incorporate the features of the invention. The disclosed embodiment(s) merely exemplify the invention. The scope of the invention is not limited to the disclosed embodiment(s). The invention is defined by the claims appended hereto.

[0038] References in the specification to “one embodiment,” “an embodiment,” “an example embodiment,” etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment, Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

[0039] The present invention is a test and device for Simultaneous Parallel Signal Amplification and Analyte-Ligand Capture functions configured to provide sufficient signal while additionally detecting low abundance biomarkers. The present invention employs a test, which, for convenience, is housed within a biological cartridge of a cassette. The biological cartridge of the present invention remains separated from an electronics section of the cassette, and only accommodates a nitrocellulose strip. The cassette of the present invention was inspired by current pregnancy tests available on the market. The cassette system of the test of the present invention allows for the biological cartridge to be removable and discarded after use. A new biological cartridge is inserted into the electronics section to start the next test, making the device of the present invention reusable.

[0040] The cassette of the present invention is preferably printed with PLA material in its current state, which is very fast to prototype with. In the future, the model cassette is envisioned to be printed with a more resilient plastic such as ABS. It is also envisioned to be designed with a user-friendly design approachable for the end user. It should be understood that the current model depicted in FIG. 1 simply serves as a shell, and that future embodiments of the cassette may be more robust and/or have a differing form factor. The cassette, as shown disassembled/exploded in FIG. 1, includes an absorbent pad (10), an application site (20), an electrode site (30), a conjugate pad (40), a screen (50), a microcontroller (60), a battery (70), a test line (80), and a control line (90).

[0041] Capture Hydrogel Particles

[0042] Pertinent biomarkers found in blood and other bodily fluids are exceptionally low in concentration. Biomarkers are also subject to degeneration over time, especially during the processes of collecting and transporting samples.

[0043] Hydrogel particles are an open-mesh network of polymers with chemical affinity baits dyed to their surface, and can be used to overcome the obstacles discussed above. When combined with blood or bodily fluids, these hydrogel particles are able to segregate and eliminate significantly large amounts of undesirable proteins, enhance the target analytes, and amplify the sensitivity of immunoassays 100 fold. The chemical affinity baits have conductive elements within their structure, which can be exploited electrically. The manufacturing of the hydrogel particles is cost-effective, and the quality of this technology is able to maintain stability and complete solubility in bodily fluids over time. Additionally, these hydrogel particles can be functionalized with enzymes by affinity interactions. The hydrogel particles replace the traditional solid-core gold nanoparticles used in lateral flow immunoassays for the prototype of the present invention.

[0044] Immobilize and Detect: Amperometric Sensor

[0045] Enzymatic Amplification

[0046] Though the hydrogel articles contain ionic elements, it is imperative to amplify the signal in order to achieve sensitivity and performance standards. The method of amplification employed by the present invention produces both visual and electrical data. First, the hydrogel particles are functionalized with the enzyme horseradish peroxidase (HRP). HRP is an enzyme taken from the roots of horseradishes. Using enzymatic reactions to release electrons is a common and proven method of electrochemical sensing.

[0047] The system and device of the present invention combines the following chemicals and enzymes to increase the amperometric response: Glucose (G), Glucose Oxidase (GOx), Hydrogen Peroxide (H.sub.2O.sub.2), and HRP. GOx is an enzyme produced by fungi to readily oxidize G to produce H.sub.2O.sub.2. GOx is typically used to detect concentrations of G in medical diagnostics. In addition, it is an economical enzyme that has high durability.

[0048] The amplified signal will mainly be generated from the creation of H.sub.2O.sub.2 from G reducing GOx. After, HRP further reduces H.sub.2O.sub.2 to release electrons which interact with the sensor. The aforementioned reactions are simplified with the following equations:

D-Glucose+H.sub.2O.sub.2.fwdarw.D-Gluconic Acid+H.sub.2O  (1)

H.sub.2O.sub.2.fwdarw.HRP H2O+HRP  (2)

[0049] To ensure that the amplification of the reactive solution is occurring, the chromogenic substrate DAB (3, 3-diaminobenzidine) is added to it. In the presence of the reactive solution, DAB produces a dark brown precipitate. Thus, the two-part amplification using enzymatic reactions and hydrogel particles with the DAB substrate will produce enhanced visual results as well as released electrons for electrochemical sensing.

[0050] Amperometric Sensor

[0051] Amperometric sensors are the most common and well-known electrochemical sensing devices, and will work best in conjunction with the enzymatic reaction described above. Amperometric sensors work in a framework of a voltage divider circuit, in which there is a variable resistor. The amperometric sensor behaves as the variable resistor. By applying a constant voltage potential across two electrodes, the change in current is detected. This sensing technique works best with recognizing oxidation and reduction reactions. In addition, amperometric sensing has compatibility with nanomaterials and has shown a wide range of nanotechnology applications to improve diagnostic sensing.

[0052] The electrical data acquisition system of the present invention consists of an Arduino pro mini, which is a low-cost microcontroller board, and an amperometric sensor across the test lines. The current amperometric sensor is a traditional 2-prong electrode as shown in FIG. 2. The Arduino samples the current across the test line at a frequency of 10 Hz. Since the enzymatic reaction takes place within seconds, it only samples the current for the first 15 seconds.

[0053] In theory, the hydrogel particles functionalized with HRP are immobilized at the antibody line in the positive test case will produce a higher current response in comparison to the negative test case, when the particles do not immobilize at the antibody line, and flow to the absorbent pad.

[0054] To further differentiate between the positive and negative test cases, the current is integrated over the 15 seconds period. This allows for a threshold to be established, allowing for a binary screening diagnostic of “LTBI Negative” or “LTBI Positive.” This threshold was found by running 15 positive and 15 negative tests, then examining the data for the decision boundary.

[0055] The final system of the present invention preferably functions as follows: [0056] 1. Analyte with TB antigen flows across the sample and conjugate pad. [0057] 2. The HRP/GOx functionalized hydrogel particles captures the TB antigen and continue to flow. [0058] 3. When the hydrogel particles reach the electrode system, the TB antigen binds with the TB capture antibody. [0059] 4. The G/GOx/H.sub.2O.sub.2/HRP reactions mentioned in equations 1, 2 occurs. [0060] 5. The free electrons generates a current response. [0061] 6. The functionalized hydrogel particles continue flowing across the electrode and are captured at the control line on the counter electrode. [0062] 7. The system gives a diagnostic read out based on the change in current on the working electrode.
Overall, this approach combines the advantages of enzymatic reactions for amplifying a current response, the simplicity of lateral flow design, and the advanced protein capturing capabilities of the hydrogel particles for an optimal LTBI diagnostic test.

[0063] Dot Blots

[0064] To ensure that the reagents and antibodies used are compatible with each together, dot blot analyses were performed. A dot blot is a molecular biological assay that detects the presence of a target analyte. Since the hydrogel particles must be functionalized with HRP and they are targeting the LTBI antigen, ESAT-6, tests have been performed for the efficacy of the hydrogel particles being functionalized with HRP and capturing ESAT-6.

Efficacy of NP functionalization with HRP Enzyme

[0065] To test the efficacy of the hydrogel particles being functionalized with the HRP enzyme, washed hydrogel particles were incubated with HRP before proceeding with the standard dot blot procedure. FIG. 5 demonstrates that the particles can be functionalized with HRP when in PBS and urine. The RB-urine, AB-urine, and TB-urine spots are faint and do not show a dark signal like the initial solutions, functioning as the negative controls. The reactive black (RB) and alcian blue (AB) spots for urine and HRP were dark, similar to those of the initial solution, which indicate a positive result and thereby exhibit that the hydrogel particles were successfully functionalized with HRP.

[0066] Results

NP Visual Amplification at Antibody Capture Line

[0067] To demonstrate that the hydrogel particles are capturing the TB antigen, ESAT-6, and reactive solution is amplifying the visual response, a LFI was created using a conventional pregnancy test strip to determine if the hydrogel particles capture and arrest at the capture antibody line against the ESAT-6. Visual results are displayed in FIG. 3. The test strip with the positive test result demonstrates that the hydrogel particles have captured the TB biomarker, immobilized at the capture antibody line, and produced a dark brown precipitate. The negative test result demonstrates that the hydrogel particles produced no visually detectable line due to the absence of TB biomarkers.

[0068] Thus, it has been shown that the hydrogel particles effectively capture ESAT-6 and arrest at the capture antibody line. Additionally, adding the reactive solution with the chromogenic substrate DAB will increase the visual response of the system 1000 fold within seconds, as exhibited in FIG. 3 and FIG. 4. FIG. 4 depicts adding the chromogenic substrate DAB to the reactive solution of G and GOx produces a dark brown precipitate within seconds and amplifies the colorimetric intensity of the LFI by 1000 fold.

[0069] FIG. 5 shows visual results of T-Test. A paired t-test with unequal variance is depicted, where p«0.05, n=50 samples, 25 positive, and 25 negative. Image Software was used to quantify the visual intensity of the lateral flow lines produced. Positive samples produce dark lines with low gray scale intensity values, while negative samples have whiter lines creating higher gray scale intensity values. This test was performed for 50 trials with 25 TB positive samples and 25 TB negative samples. To quantify the visual result generated at the test line, gray scale intensity software was employed. TB positive samples produced darker visual results generating a lower gray scale intensity measurement while TB negative samples did not produce a visual result, thus creating a higher gray scale intensity value as shown in FIG. 5.

[0070] To analyze the sensitivity of this visual amplification, a standard confusion matrix was used. Of the 25 known positive samples, the system of the present invention correctly identified them as TB positive, and out of the 25 known negative samples, the system of the present invention identified them as TB negative. This demonstrates that the visual amplification of the present invention achieves 100% sensitivity and 100% specificity over the 50 trials performed.

[0071] Amperometric results indicate that it is evident that, across the test line, there is a current for both positive and negative samples, as shown in FIG. 5. However, the current across the test line in the positive case is significantly higher, as it can be seen that it stabilizes around 50 μA while the negative test case is about 12 μA. This is due to the enzymatic reaction generating free electrons, as well as the conductive hydrogel particles between two prongs of the amperometric sensor, meanwhile the control does not have either of these two components.

[0072] As shown in FIG. 6, a paired t-test with unequal variance was performed, and using the charge threshold, there was significant differentiation between the positive and negative test cases. Additionally, the average coulombs of charge transferred across the test line for the positive test case was averaged at 0.649 mC while the negative test case was averaged at 0.402 mC, demonstrating that the immobilization of the functionalized, copper-dyed particles produces a higher current at the test line.

[0073] Finally, a standard confusion matrix was used to assess the sensitivity and specificity of the electrical detection system. The electrical system correctly classified 24 of the 25 known positive test cases as TB positive, producing a 96% sensitivity. The system correctly classified 20 of the 25 known negative test cases at TB negative, producing an 80% specificity.

[0074] It should be understood that the purpose of the design process of the present invention was to develop a novel diagnostic that improved upon existing tools while also developing a quantifiable method of detection. This study has successfully demonstrated an improved method of capturing the ESAT-6 TB biomarker by utilizing hydrogel particles while also improving the sensitivity of a typical LFI. Combining the NP with enzymatic reactions and incorporating HRP within the volume of the hydrogel particles has enhanced the visual response of the LFI by 100%. Additionally, the enzymatic reactions have aided in the quantifiable detection of the hydrogel particles by using a biosensor. Amperometric methods of identifying free electrons has been successfully applied to this testing platform. Using simple sensor design, the system of the present invention has achieved 96% sensitivity and 80% specificity, thus providing a proof of concept that an amperiometric LFI using hydrogel particles for tuberculosis diagnosis (or other diagnoses) is feasible.

[0075] These results are valuable because the current TB diagnostics on the market fail to achieve the sensitivity and cost of the device of the present invention. Having a quantifiable LFI for TB, not only improves upon LFI sensitivity, but also produces data which could track the concentration of the target biomarker within a patient. Since the hydrogel particles can be configured for any biomarker, this methodology can be applied for a plethora of diseases and diagnostic applications. Additionally, having HRP within hydrogel particles is a completely new method of capturing and detecting the presence of low abundance biomarkers.

[0076] To improve this prototype, implementing AG/AgCl electrode designs, as well as incorporating a potentiostatic circuit design would greatly improve the variability seen in the electrical method of detection. Another suggestion for improvement could be incorporating new chromogenic substrates such as 3,3′, 5,5′-Tetramethylbenzidine or TMB. This substrate produces a blue color when exposed to a peroxidase reaction which would be beneficial to the test of the present invention since the hydrogel particle dye for tuberculosis is Reactive Blue 221, a blue color. Also, TMB has been known to produce better electrical effects, thus potentially enhancing the amperometric data.

[0077] The HRP incorporation inside the NP volume and attached to the dye bait can be modified. Currently, the system of the present invention has demonstrated that activated HRP retains its reactivity. However, this response could be greater if the addition of a spacer is added to provide space for the HPR to further catalyze or the HRP could be covalently bound to the hydrogel particles which would also promote better catalysis.

[0078] It is important to note that the visual and electrical systems can work independently, but in case the electrical components fail, the visual system functions on its own.

[0079] It should be understood that the hydrogel particles referenced are open porous hydrogel particles containing tunable pores and containing an immobilized novel high-affinity bait chemistry. The novel baits, which are derivatives of dyes, bind to proteins, nucleic acids, glycoproteins, glycolipids, and nucleic acids with extremely high affinity (10″2), preserving the captured analyte and effectively increasing the sensitivity of detection up to ten thousand fold. Hydrogel particles are commercialized, and have been applied to a wide variety of diagnostics and discovery projects by scientists worldwide. The bait chemistries of the present invention have provided a completely new approach to sequence the interface regions of interacting proteins. Such interfaces are the drug targets of the future. The system of the present invention engineers the bait of the hydrogel particles to create a slow release depot that can release therapeutic molecules in vivo.

[0080] Having illustrated the present invention, it should be understood that various adjustments and versions might be implemented without venturing away from the essence of the present invention. Further, it should be understood that the present invention is not solely limited to the invention as described in the embodiments above, but further comprises any and all embodiments within the scope of this application.

[0081] The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiment was chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated.