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1. (WO2019005473) INTEGRATED OPTO NANO ELECTRONIC (IONE) SENSING
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WHAT IS CLAIMED IS:

5 1 . A method of electrochemical sensing of target analytes, the method comprising:

irradiating a complex comprising a nanoparticle and a target analyte, with a light source in proximity to an electrochemical sensor; and

1 0 conducting electrochemical sensing, by the electrochemical sensor, with respect to the irradiated complex to determine a presence or quantity of the target analyte.

2. The method of claim 1 , wherein the nanoparticle is conjugated to a c;

reporter enzyme.

3. The method of claim 2, wherein the reporter enzyme is horseradish peroxidase (HRP), alkaline phosphatase, or beta-galactosidase.

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4. The method of claim 1 , wherein the electrochemical sensing comprises a hydrogen evolution reaction (HER).

OR

5. The method of any one of claims 1 -4, wherein the target analyte comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, oncosomes, apoptotic bodies, and any combination thereof.

30 6. The method of any one of claims 1 -4, wherein the target analyte comprises a cell, a protein, a peptide, a lipid, nucleic acids, small molecules, microbes, food antigens, a toxin, opioids, or a metabolite.

35 7. The method of either one of claims 5-6, wherein the target analyte comprises at least one biomarker selected from the group consisting of EPCAM, EGFR, HER2, MUC1 , MUC2, MUC6, MUC5AC, GPC1 , WNT2, CEP, CD24, GPA33, CA125, CD44, CD44v6, CEA, Mesothelin, Trop2, Grp94, SSTR2, CD166, CD133,

1 MET, B7H3, CD63, CD9, CD81 , CD2, CD3, CD14, CD45, CD47, CD52, CD68, CD73 H LA-ABC, CXCL1 0, CXCL9, HVEM, FGFR3, NUMA, HSP70, IL-3, TSP2, PD- L1 , EGFRv3, EGFR T790M, IDH1 mutant, APC mutant, KRAS mutant, BRAF mutant, PIK3CA mutant, BR AC 1 /2 mutant, SMAD4 mutant, CDKN2 mutant, and

5 PTEN mutant biomarkers.

8. The method of any one of claims 1 -7, wherein the nanoparticle comprises at least one metal selected from the group consisting of gold, silver,

1 0 platinum, iron and copper.

9. The method of any one of claims 1 -8, wherein the nanoparticle ranges in size from about 1 nm to about 1000 nm.

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10. The method of any one of claims 1 -9, wherein a morphology of the nanoparticle comprises sphere, shell, cube, prism, pyramid, star, tube, popcorn, rod, cage, vesicle, or any combination thereof.

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1 1 . The method of any one of claims 1 -10, wherein the light source is a red light, a green light, a white light, a blue light, a yellow light, or any combinations thereof.

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12. The method of any one of claims 1 -1 1 , wherein the irradiating is for a time between about 5 seconds and about 5 minutes.

13. The method of any one of claims 1 -3, wherein the method further 30 comprises introducing an electron mediator into the electrochemical sensor.

14. The method of claim 13, wherein the electron mediator is 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o- 35 phenylenediamine dihydrochloride) (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), p- nitrophenyl phosphate (PNPP), o-nitrophenyl- -D-galactopyranoside (ONPG), Naphthol-AS-BI-beta-D-galactopyranosidase (Nap-Gal), or 4-Methyl-umbelliferyl- beta-D-galactopyranosidase (MUm-Gal).

15. The method of claim 14, wherein the reporter enzyme comprises horseradish peroxidase (HRP), and the electron mediator comprises 3,3',5,5'-tetramethylbenzidine (TMB).

16. The method of claim 1 or 4, wherein the method further comprises introducing an acid medium into the electrochemical sensor.

17. A method of detecting a target analyte in a sample, the method comprising:

providing a first binding moiety on a substrate to a sample comprising a target analyte, wherein the first binding moiety specifically binds to the target analyte;

allowing the target analyte in the sample to bind with the first binding moiety on the substrate, thereby forming a first complex comprising the target analyte and the first binding moiety on the substrate;

providing a nanoparticle comprising a second binding moiety that binds to the target analyte, wherein the nanoparticle is conjugated to a reporter enzyme;

allowing the first complex to bind with the second binding moiety of the nanoparticle, thereby forming a second complex comprising the first complex and the second binding moiety of the nanoparticle;

introducing an electron mediator into the electrochemical sensor;

applying the second complex to a sample detection region of a first electrode, wherein the first electrode is electrically coupled to a potentiostat;

irradiating the second complex in the sample detection region with a light source;

inducing an oxidation-reduction reaction between the electron mediator and the reporter enzyme; and

monitoring an output of the potentiostat to determine a presence or quantity of the target analyte in the sample.

18. The method of claim 17, wherein inducing the oxidation-reduction reaction comprises applying an electrical potential to a second electrode such that the oxidation-reduction reaction occurs, wherein the second electrode is electrically coupled to the potentiostat.

19. The method of claim 17 or 18, wherein

monitoring the output of the potentiostat comprises measuring a voltage or current from the first electrode, and

the voltage or current from the first electrode varies as a result of the oxidation-reduction reaction.

20. The method of any one of claims 17-19, wherein monitoring the output of the potentiostat comprises:

selecting the output of the potentiostat from a plurality of different potentiostats' outputs;

providing the selected output to a microcontroller unit; and

presenting the selected output on a display.

21 . The method of any one of claims 17-20, wherein the reporter enzyme is horseradish peroxidase (HRP), alkaline phosphatase, or beta-galactosidase.

22. The method of any one of claims 17-21 , wherein the electron mediator is 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride) (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), p-nitrophenyl phosphate (PNPP), o-nitrophenyl- -D-galactopyranoside (ONPG), Naphthol-AS-BI-beta-D-galactopyranosidase (Nap-Gal), 4-Methyl-umbelliferyl-beta-D-galactopyranosidase (MUm-Gal).

23. The method of any one of claims 17-22, wherein the reporter enzyme comprises horseradish peroxidase (HRP), and the electron mediator comprises 3,3',5,5'-tetramethylbenzidine (TMB).

24. The method of any one of claims 17-23, wherein the target analyte comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, oncosomes, apoptotic bodies, and any combination thereof.

25. The method of any one of claims 17-23, wherein the target analyte comprises a cell, a protein, a peptide, a lipid, nucleic acids, small molecules, microbes, food antigens, a toxin, opioids, or a metabolite.

26. The method of claim 24 or 25, wherein the target analyte comprises at least one biomarker selected from the group consisting of EPCAM, EGFR, HER2, MUC1 , MUC2, MUC6, MUC5AC, GPC1 , WNT2, CEP, CD24, GPA33, CA125, CD44, CD44v6, CEA, Mesothelin, Trop2, Grp94, SSTR2, CD166, CD1 33, MET, B7H3, CD63, CD9, CD81 , CD2, CD3, CD14, CD45, CD47, CD52, CD68, CD73 H LA-ABC, CXCL1 0, CXCL9, HVEM, FGFR3, NUMA, HSP70, IL-3, TSP2, PD-L1 , EGFRv3, EGFR T790M, IDH1 mutant, APC mutant, KRAS mutant, BRAF mutant, PIK3CA mutant, BRAC1 /2 mutant, SMAD4 mutant, CDKN2 mutant, and PTEN mutant biomarkers.

27. The method of any one of claims 17-26, wherein the nanoparticle comprises at least one metal selected from the group consisting of gold, silver, platinum, iron and copper.

28. The method of any one of claims 17-27, wherein the nanoparticle ranges in size from about 1 nm to about 1000 nm.

29. The method of any one of claims 1 7-28, wherein a morphology of the nanoparticle comprises sphere, shell, cube, prism, pyramid, star, tube, popcorn, rod, cage, vesicle, or any combination thereof.

30. The method of any one of claims 1 7-29, wherein the light source is a red light, a green light, a white light, a blue light, a yellow light or any combinations thereof.

31 . The method of any one of claims 17-30, wherein the irradiating is for a time between about 5 seconds and about 5 minutes.

32. The method of any one of claims 17-31 , wherein the substrate is a magnetic bead.

33. The method of claim 32, where applying the second complex to the sample detection region is by exposing the second complex to a magnetic field to retain the second complex next to the first electrode.

34. The method of any one of claim 17-33, wherein the substrate is a portion of the sample detection region of the first electrode.

35. A method of detecting a target analyte present in a fluid sample, the method comprising:

providing a plurality of magnetic beads to a first fluid sample, wherein the plurality of magnetic beads comprise first binding moieties that specifically bind to the target analyte;

allowing the first binding moieties of the plurality of magnetic beads to bind to the target analyte in the first fluid sample;

transferring the plurality of magnetic beads from the first fluid sample to a second fluid sample, wherein the second fluid sample comprises a plurality of nanoparticles, wherein the plurality of nanoparticles comprise second binding moieties that bind to the target analyte, and the plurality of nanoparticles are conjugated to a plurality of reporter enzymes;

allowing the second binding moieties of the plurality of nanoparticles to bind to the target analyte bound to the first binding moieties of the plurality of magnetic beads;

combining the second fluid sample comprising the plurality of magnetic beads and the plurality of nanoparticles with a solution comprising a plurality of electron mediators to obtain a third fluid sample;

providing the third fluid sample to a sample detection region of a first electrode, wherein the first electrode is electrically coupled to a potentiostat; irradiating the third fluid sample with a light source;

inducing an oxidation-reduction reaction between the plurality of electron mediators and the reporter enzymes in the third fluid sample; and

monitoring an output of the potentiostat to determine a presence or quantity of the target analyte in the third fluid sample.

36. The method of claim 35, wherein transferring the plurality of magnetic beads comprises:

immersing a sheath within the first fluid sample;

placing a magnet within the sheath such that the plurality of magnetic beads adhere to the sheath;

removing the sheath containing the magnet from the first fluid sample;

immersing the sheath containing the magnet in the second fluid sample; and removing the magnet from the sheath to release the plurality of magnetic beads to the second fluid sample.

37. The method of claim 35 or 36, wherein the third fluid sample is exposed to a magnetic field to retain the plurality of magnetic beads in the third fluid sample next to the first electrode.

38. The method of any one of claims 35-37, wherein inducing the oxidation-reduction reaction comprises applying an electrical potential to a second electrode such that the oxidation-reduction reaction occurs, wherein the second electrode is electrically coupled to the potentiostat.

39. The method of any one of claims 35-38, wherein monitoring the output of the potentiostat comprises measuring a voltage or current from the first electrode, wherein the voltage or current from the first electrode varies as a result of the oxidation-reduction reaction.

40. The method of any one of claims 35-39, wherein monitoring the output of the potentiostat comprises:

1 selecting the output of the potentiostat from a plurality of different potentiostats' outputs;

providing the selected output to a microcontroller unit; and

5 presenting the selected output on a display.

41 . The method of any one of claims 35-40, wherein the light source is a red light, a green light, a white light, a blue light, a yellow light, or any combinations thereof.

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42. The method of any one of claims 35-41 , wherein the irradiating is for a time between 5 seconds and 5 minutes.

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43. A system for detecting a target analyte, comprising:

an electrochemical sensor comprising a plurality of electrodes, the electrochemical sensor configured to perform an assay to detect a presence or quantity of a target analyte in a sample;

on

a potentiostat operatively coupled to the electrochemical sensor; and a light source configured to irritate a complex comprising the target analyte formed during the assay.

44. The system of claim 43, wherein the light source comprises a laser diode or a light emitting diode.

45. The system of claim 44, wherein the laser diode or light emitting diode 30 emits a light having a wavelength between 400 nm and 680 nm.

46. The system of claim 44 or 45, wherein a power of the laser diode or light emitting diode is 5 mW.

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47. The system of any one of claims 43-46, wherein the complex further comprises a nanoparticle conjugated to a reporter enzyme.

48. The system of claim 47, wherein the nanoparticle is a gold nanoparticle.

49. A method of detecting a target analyte in a sample, the method comprising:

providing a first binding moiety on a substrate to a sample comprising a target analyte, wherein the first binding moiety specifically binds to the target analyte;

allowing the target analyte in the sample to bind with the first binding moiety on the substrate, thereby forming a first complex comprising the target analyte and the first binding moiety on the substrate;

providing a nanoparticle comprising a second binding moiety that binds to the target analyte;

allowing the first complex to bind with the second binding moiety of the nanoparticle, thereby forming a second complex comprising the first complex and the second binding moiety of the nanoparticle;

introducing hydrogen ions into the electrochemical sensor;

applying the second complex to a sample detection region of a first electrode, wherein the first electrode is electrically coupled to a potentiostat;

irradiating the second complex in the sample detection region with a light source;

inducing an oxidation-reduction reaction involving the hydrogen ions and the nanoparticle; and

monitoring an output of the potentiostat to determine a presence or quantity of the target analyte in the sample.

50. The method of claim 49, wherein inducing the oxidation-reduction reaction comprises applying an electrical potential to a second electrode such that the oxidation-reduction reaction occurs, wherein the second electrode is electrically coupled to the potentiostat.

51 . The method of claim 49 or 50, wherein

monitoring the output of the potentiostat comprises measuring a voltage or current from the first electrode, and

the voltage or current from the first electrode varies as a result of the oxidation-reduction reaction.

52. The method of any one of claims 49-51 , wherein monitoring the output of the potentiostat comprises:

selecting the output of the potentiostat from a plurality of different potentiostats' outputs;

providing the selected output to a microcontroller unit; and

presenting the selected output on a display.

53. The method of any one of claims 49-52, wherein the hydrogen ions are in an acid medium.

54. The method of any one of claims 49-53, wherein the target analyte comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, oncosomes, apoptotic bodies, and any combination thereof.

55. The method of any one of claims 49-53, wherein the target analyte comprises a protein, a cell, a peptide, a protein, a lipid, a toxin, nucleic acids, microbes, food antigens, or a metabolite.

56. The method of claim 54 or 55, wherein the target analyte comprises at least one biomarker selected from the group consisting of EPCAM, EGFR, HER2, MUC1 , MUC2, MUC6, MUC5AC, GPC1 , WNT2, CEP, CD24, GPA33, CA125, CD44, CD44v6, CEA, Mesothelin, Trop2, Grp94, SSTR2, CD166, CD133, MET, B7H3, CD63, CD9, CD81 , CD2, CD3, CD14, CD45, CD47, CD52, CD68, CD73 H LA-ABC, CXCL1 0, CXCL9, HVEM, FGFR3, NUMA, HSP70, IL-3, TSP2, PD-L1 , EGFRv3, EGFR T790M, IDH1 mutant, APC mutant, KRAS mutant, BRAF mutant, PIK3CA mutant, BRAC1 /2 mutant, SMAD4 mutant, CDKN2 mutant, and PTEN mutant biomarkers.

57. The method of any one of claims 49-56, wherein the nanoparticle comprises at least one metal selected from the group consisting of gold, silver, platinum, iron and copper.

58. The method of any one of claims 49-57, wherein the nanoparticle ranges in size from about 1 nm to about 1000 nm.

59. The method of any one of claims 49-58, wherein a morphology of the nanoparticle comprises sphere, shell, cube, prism, pyramid, star, tube, popcorn, rod, cage, vesicle, or any combination thereof.

60. The method of any one of claims 49-59, wherein the light source is a red light, a green light, a white light, a blue light, a yellow light or any combinations thereof.

61 . The method of any one of claims 49-60, wherein the irradiating is for a time between about 5 seconds and about 5 minutes.

62. The method of any one of claims 49-61 , wherein the substrate is a magnetic bead.

63. The method of claim 62, where applying the second complex to the sample detection region is by exposing the second complex to a magnetic field to retain the second complex next to the first electrode.

64. The method of any one of claim 49-63, wherein the substrate is a portion of the sample detection region of the first electrode.

65. The method of any one of claim 49-64, wherein the oxidation-reduction reaction comprises a hydrogen evolution reaction (HER).

66. A method of detecting a target analyte present in a fluid sample, the method comprising:

providing a plurality of magnetic beads to a first fluid sample, wherein the plurality of magnetic beads comprises first binding moieties that specifically bind to the target analyte;

allowing the first binding moieties of the plurality of magnetic beads to bind to the target analyte in the first fluid sample;

transferring the plurality of magnetic beads from the first fluid sample to a second fluid sample, wherein the second fluid sample comprises a plurality of nanoparticles, wherein the plurality of nanoparticles comprise second binding moieties that bind to the target analyte;

allowing the second binding moieties of the plurality of nanoparticles to bind to the target analyte bound to the first binding moieties of the plurality of magnetic beads;

combining the second fluid sample comprising the plurality of magnetic beads and the plurality of nanoparticles with an acid medium to obtain a third fluid sample; providing the third fluid sample to a sample detection region of a first electrode, wherein the first electrode is electrically coupled to a potentiostat;

irradiating the third fluid sample with a light source;

inducing an oxidation-reduction reaction involving hydrogen ions and the nanoparticles in the third fluid sample; and

monitoring an output of the potentiostat to determine a presence or quantity of the target analyte in the third fluid sample.

67. The method of claim 66, wherein transferring the plurality of magnetic beads comprises:

immersing a sheath within the first fluid sample;

placing a magnet within the sheath such that the plurality of magnetic beads adhere to the sheath;

removing the sheath containing the magnet from the first fluid sample;

immersing the sheath containing the magnet in the second fluid sample; and removing the magnet from the sheath to release the plurality of magnetic beads to the second fluid sample.

68. The method of claim 66 or 67, wherein the third fluid sample is exposed to a magnetic field to retain the plurality of magnetic beads in the third fluid sample next to the first electrode.

69. The method of any one of claims 66-68, wherein inducing the oxidation-reduction reaction comprises applying an electrical potential to a second electrode such that the oxidation-reduction reaction occurs, wherein the second electrode is electrically coupled to the potentiostat.

70. The method of any one of claims 66-69, wherein monitoring the output of the potentiostat comprises measuring a voltage or current from the first electrode, wherein the voltage or current from the first electrode varies as a result of the oxidation-reduction reaction.

71 . The method of any one of claims 66-70, wherein monitoring the output of the potentiostat comprises:

selecting the output of the potentiostat from a plurality of different potentiostats' outputs;

providing the selected output to a microcontroller unit; and

presenting the selected output on a display.

72. The method of any one of claims 66-71 , wherein the light source is a red light, a green light, a white light, a blue light, a yellow light, or any combinations thereof.

73. The method of any one of claims 66-72, wherein the irradiating is for a time between 5 seconds and 5 minutes.

74. The method of any one of claim 66-73, wherein the oxidation-reduction reaction comprises a hydrogen evolution reaction (HER).