阅读说明：本技术 产生循环分析物概况的方法和实施该方法的装置 (Method for generating a circulating analyte profile and apparatus for performing the method ) 是由 王善祥 庄之颖 余珩 迈克尔·J.·贝格斯 路易斯·卡波纳 于 2020-03-06 设计创作，主要内容包括：本公开的方面包括产生受试者的循环分析物概况的方法。所述方法包括使来自受试者的血液样本与用于特异性结合分析物的探针组接触,和检测分析物与探针组的探针的结合是否存在。还提供了包括捕获探针组并且可用于例如实施本公开的方法的传感器装置。(Aspects of the disclosure include methods of generating a circulating analyte profile of a subject. The method includes contacting a blood sample from the subject with a probe set for specifically binding the analyte, and detecting the presence or absence of binding of the analyte to the probes of the probe set. Sensor devices that include a set of capture probes and that can be used, for example, to perform the methods of the present disclosure are also provided.)

1. A method of generating a circulating analyte profile of a subject, comprising:

contacting a blood sample from a subject with a set of probes for specifically binding an analyte comprising:

two or more of carcinoembryonic antigen (CEA), C-X-C motif chemokine ligand 4(CXCL4), C-X-C motif chemokine ligand 7(CXCL7), and C-X-C motif chemokine ligand 10(CXCL 10); and

one or more of Epidermal Growth Factor Receptor (EGFR), pro-surface active protein B (pro-SFTPB), and tissue inhibitor of metalloproteinase 1 (TIMP 1); and

detecting the presence or absence of binding of said analyte to the probes of said set of probes,

to generate a circulating analyte profile of the subject.

2. The method of claim 1, wherein the blood sample is contacted with a set of probes for specifically binding analytes comprising three or each of CEA, CXCL4, CXCL7 and CXCL 10.

3. The method of claim 1, wherein the blood sample is contacted with a set of probes for specifically binding analytes comprising CEA, CXCL4, CXCL7, and CXCL 10.

4. The method of any one of claims 1 to 3, wherein the blood sample is contacted with a probe set for specific binding of an analyte comprising two or each of EGFR, pro-SFTPB and TIMP 1.

5. The method of any one of claims 1 to 3, wherein the blood sample is contacted with a set of probes for specifically binding analytes comprising EGFR, pro-SFTPB and TIMP 1.

6. The method of any one of claims 1 to 5, wherein the panel of probes further comprises one or more probes that specifically bind to one or any combination of additional analytes selected from the group consisting of:

anti-angiopoietin-like protein 3 antibody (anti-ANGPTL 3), anti-14-3-3 protein theta antibody (anti-ywaq), anti-laminin alpha 1 antibody (anti-LAMR 1), human epididymin 4(HE4), prodrag 2(AGR2), chromogranin a (chga), leucine rich alpha-2-glycoprotein 1(LRG1), anti-annexin 1 antibody (anti-ANXA 1), anti-ubiquitin 1 antibody (anti-UBQLN 24), interleukin 6(IL6), interleukin 8(IL8), C-X-C motif chemokine ligand 2(CXCL2), C-X-C motif chemokine ligand 12(CXCL12), C-X-C motif chemokine ligand 14(CXCL14), defensin, beta 1(DEFB1), fibroblast growth factor 2(FGF2), clusterin 97(CD97), proplatelet basic protein (PPBP), Procalcitonin (PCT) antigen, Receptor for advanced glycation end products (RAGE), S100 calcium binding protein A4(S100A4), S100 calcium binding protein A8(S100A8), and Osteopontin (OPN),

wherein the method further comprises detecting the presence or absence of binding of the one or any combination of the additional analytes to the probes of the probe set to generate a circulating analyte profile of the subject.

7. The method of any one of claims 1 to 6, wherein the set of probes comprises probes for binding 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, or 25 or more analytes.

8. The method of any one of claims 1 to 7, wherein the set of probes comprises probes for specifically binding 200 or fewer analytes, 150 or fewer analytes, 125 or fewer analytes, 100 or fewer analytes, 75 or fewer analytes, 50 or fewer analytes, 40 or fewer analytes, 30 or fewer analytes, or 25 or fewer analytes.

9. The method of any one of claims 1 to 8, wherein detecting the presence or absence of the analyte binding comprises quantifying the analyte detected.

10. The method of any one of claims 1-9, wherein the panel of probes further comprises a probe for binding to a circulating tumor cell, wherein the method further comprises detecting the presence or absence of binding of the circulating tumor cell to the probe of the panel to generate a circulating analyte profile of the subject.

11. The method of claim 10, wherein detecting the presence or absence of binding of the circulating tumor cell comprises quantifying the detected circulating tumor cell.

12. The method of any one of claims 1-11, wherein the panel of probes further comprises probes for binding tumor DNA, wherein the method further comprises detecting the presence or absence of binding of tumor DNA to the probes of the panel of probes to generate a circulating analyte profile of the subject.

13. The method of claim 12, wherein detecting the presence or absence of tumor DNA binding comprises quantifying the detected tumor DNA.

14. The method of any one of claims 1 to 13, wherein the subject is from a population with a high risk of lung cancer.

15. The method of claim 14, wherein the subject is a previous or current smoker.

16. A method according to claim 14 or claim 15, wherein the previous or current smoker has lung nodules.

17. The method of claim 16, wherein the previous or current smoker has lung nodules detected by Low Dose Computed Tomography (LDCT).

18. The method of claim 16 or claim 17, further comprising assessing the risk of malignancy of the lung nodule based on a circulating analyte profile of the subject.

19. The method of claim 18, wherein the assessment is further based on one or any combination of clinical parameters of the subject selected from the group consisting of: subject age, nodule size, subject gender, nodule edge (whether or not there is a burr), nodule location, subject cancer history, subject cancer family history, and smoking history (including smoking intensity).

20. The method of claim 18 or claim 19, comprising assessing the risk of the lung nodule being non-small cell lung cancer (NSCLC).

21. The method of any one of claims 1 to 20, wherein the blood sample is a whole blood sample, a plasma sample, or a serum sample.

22. The method of any one of claims 1 to 21, wherein the set of probes is a set of capture probes provided as an addressable probe array.

23. The method of claim 22, wherein the addressable probe array resides on a magnetic sensor chip of a magnetic sensor device.

24. The method of claim 23, wherein the magnetic sensor chip comprises two or more magnetic sensors having capture probes attached to surfaces thereof.

25. The method of claim 24, wherein each of the two or more magnetic sensors having capture probes attached to its surface comprises a capture probe for binding the same analyte.

26. A method according to claim 24 or claim 25, wherein each magnetic sensor comprises a magnetoresistive element.

27. The method of claim 26, wherein the magnetoresistive element is a spin valve magnetoresistive element or a Magnetic Tunnel Junction (MTJ) magnetoresistive element.

28. The method of any one of claims 23 to 27, wherein detecting the presence of binding of the analyte to the probes of the probe set comprises detecting a magnetically labeled detection reagent bound to the captured analyte.

29. The method of claim 28, wherein the magnetically labeled detection reagent indirectly binds to the captured analyte.

30. The method of claim 29, wherein the magnetically labeled detection reagent is part of a complex comprising a capture probe, the analyte, a primary detection reagent that specifically binds to the analyte, and a magnetically labeled detection reagent that binds to the primary detection reagent.

31. The method of any one of claims 23 to 30, wherein detecting the presence of binding of the analyte to the probes of the probe set comprises detecting a change in resistance in the magnetoresistive element induced by the magnetically-labeled detection reagent.

32. A sensor device, comprising:

a set of capture probes provided as an addressable probe array, wherein the set comprises probes for specifically binding an analyte comprising:

two or more of carcinoembryonic antigen (CEA), C-X-C motif chemokine ligand 4(CXCL4), C-X-C motif chemokine ligand 7(CXCL7), and C-X-C motif chemokine ligand 10(CXCL 10); and

one or more of Epidermal Growth Factor Receptor (EGFR), pro-surface active protein B (pro-SFTPB), and tissue inhibitor of metalloproteinase 1 (TIMP 1).

33. The sensor device of claim 32, wherein the set comprises probes for specifically binding analytes comprising three or each of CEA, CXCL4, CXCL7 and CXCL 10.

34. The sensor device of claim 32, wherein the set comprises probes for specifically binding analytes comprising CEA, CXCL4, CXCL7 and CXCL 10.

35. The sensor device of any one of claims 32 to 34, wherein the set comprises probes for specifically binding an analyte comprising two or each of EGFR, pro-SFTPB and TIMP 1.

36. The sensor device of any one of claims 32 to 34, wherein the set comprises probes for specifically binding analytes comprising EGFR, pro-SFTPB and TIMP 1.

37. The sensor device of any one of claims 32 to 36, wherein the set further comprises one or more probes for specifically binding to one or any combination of additional analytes selected from the group consisting of:

anti-angiopoietin-like protein 3 antibody (anti-ANGPTL 3), anti-14-3-3 protein theta antibody (anti-ywaq), anti-laminin alpha 1 antibody (anti-LAMR 1), human epididymin 4(HE4), prodrag 2(AGR2), chromogranin a (chga), leucine rich alpha-2-glycoprotein 1(LRG1), anti-annexin 1 antibody (anti-ANXA 1), anti-ubiquitin 1 antibody (anti-UBQLN 24), interleukin 6(IL6), interleukin 8(IL8), C-X-C motif chemokine ligand 2(CXCL2), C-X-C motif chemokine ligand 12(CXCL12), C-X-C motif chemokine ligand 14(CXCL14), defensin, beta 1(DEFB1), fibroblast growth factor 2(FGF2), clusterin 97(CD97), proplatelet basic protein (PPBP), Procalcitonin (PCT) antigen, Advanced glycation end product Receptor (RAGE), S100 calcium binding protein A4(S100A4), S100 calcium binding protein A8(S100A8), and Osteopontin (OPN).

38. The sensor device of any one of claims 32 to 37, wherein the set of probes comprises probes for binding 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, or 25 or more analytes.

39. The sensor device of any one of claims 32 to 38, wherein the set of probes comprises probes for specifically binding 200 or fewer analytes, 150 or fewer analytes, 125 or fewer analytes, 100 or fewer analytes, 75 or fewer analytes, 50 or fewer analytes, 40 or fewer analytes, 30 or fewer analytes, or 25 or fewer analytes.

40. The sensor device of any one of claims 32 to 39, wherein the set of probes comprises probes for binding circulating tumor cells.

41. The sensor device of any one of claims 32 to 40, wherein the set of probes comprises probes for binding tumor DNA.

42. The sensor device of any one of claims 32 to 41, wherein the sensor device is a magnetic sensor device.

43. The sensor device of claim 42, wherein the magnetic sensor device comprises a magnetic sensor chip comprising the set of capture probes.

44. The sensor device of claim 43, wherein the magnetic sensor chip comprises two or more magnetic sensors having capture probes attached to surfaces thereof.

45. The sensor device of claim 44, wherein the two or more magnetic sensors having capture probes attached to their surfaces comprise capture probes for binding the same analyte.

46. The sensor device of claim 44 or claim 45, wherein each magnetic sensor comprises a magnetoresistive element.

47. The sensor device of claim 46, wherein the magnetoresistive element is a spin valve magnetoresistive element or a Magnetic Tunnel Junction (MTJ) magnetoresistive element.

48. A kit, comprising:

a set of probes for specifically binding an analyte, the analyte comprising:

two or more of carcinoembryonic antigen (CEA), C-X-C motif chemokine ligand 4(CXCL4), C-X-C motif chemokine ligand 7(CXCL7), and C-X-C motif chemokine ligand 10(CXCL 10); and

one or more of Epidermal Growth Factor Receptor (EGFR), pro-surface active protein B (pro-SFTPB), and tissue inhibitor of metalloproteinase 1 (TIMP 1); and

contacting a blood sample from a subject with the probe set to generate an indication of a circulating analyte profile of the subject.

49. The kit of claim 48, wherein the set comprises probes for specifically binding analytes comprising three or each of CEA, CXCL4, CXCL7 and CXCL 10.

50. The kit of claim 48, wherein the set comprises probes for specifically binding analytes comprising CEA, CXCL4, CXCL7 and CXCL 10.

51. The kit of any one of claims 48 to 50, wherein the panel comprises probes for specifically binding analytes comprising two or each of EGFR, pro-SFTPB and TIMP 1.

52. The kit of any one of claims 48 to 50, wherein the panel comprises probes for specifically binding analytes comprising EGFR, pro-SFTPB and TIMP 1.

53. The kit of any one of claims 48 to 52, wherein said set further comprises one or more probes for specifically binding to one or any combination of additional analytes selected from the group consisting of:

anti-angiopoietin-like protein 3 antibody (anti-ANGPTL 3), anti-14-3-3 protein theta antibody (anti-ywaq), anti-laminin alpha 1 antibody (anti-LAMR 1), human epididymin 4(HE4), prodrag 2(AGR2), chromogranin a (chga), leucine rich alpha-2-glycoprotein 1(LRG1), anti-annexin 1 antibody (anti-ANXA 1), anti-ubiquitin 1 antibody (anti-UBQLN 24), interleukin 6(IL6), interleukin 8(IL8), C-X-C motif chemokine ligand 2(CXCL2), C-X-C motif chemokine ligand 12(CXCL12), C-X-C motif chemokine ligand 14(CXCL14), defensin, beta 1(DEFB1), fibroblast growth factor 2(FGF2), clusterin 97(CD97), proplatelet basic protein (PPBP), Procalcitonin (PCT) antigen, Advanced glycation end product Receptor (RAGE), S100 calcium binding protein A4(S100A4), S100 calcium binding protein A8(S100A8), and Osteopontin (OPN).

54. The kit of any one of claims 48 to 53, wherein the set of probes comprises probes for binding 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, or 25 or more analytes.

55. The kit of any one of claims 48 to 54, wherein the set of probes comprises probes for specifically binding 200 or fewer analytes, 150 or fewer analytes, 125 or fewer analytes, 100 or fewer analytes, 75 or fewer analytes, 50 or fewer analytes, 40 or fewer analytes, 30 or fewer analytes, or 25 or fewer analytes.

56. The kit of any one of claims 48 to 55, wherein the set of probes comprises probes for binding to circulating tumor cells.

57. The kit of any one of claims 48 to 56, wherein said set of probes comprises probes for binding to tumor DNA.

58. The kit of any one of claims 48 to 57, wherein the instructions comprise instructions for contacting a blood sample from a subject from a population at high risk of lung cancer with the set of probes to generate a circulating analyte profile of the subject.

59. The kit of claim 58, wherein the instructions comprise instructions for contacting a blood sample from a subject that is a previous or current smoker with the panel of probes to generate a circulating analyte profile of the subject.

60. The kit of claim 58 or claim 59, wherein the instructions comprise instructions for contacting a blood sample from a subject having a lung nodule with the panel of probes to generate a circulating analyte profile of the subject.

61. The kit of claim 60, wherein the instructions further comprise instructions for assessing the risk of malignancy of the lung nodule based on the circulating analyte profile of the subject.

Background

In the united states, lung cancer remains the most lethal and second most prevalent cancer, with a five-year survival rate of stage IV advanced non-small cell lung cancer (NSCLC) of 5-15%. NSCLC is the most common type of lung cancer, and when it is found early while still in stage I, the five-year survival rate is not much worse than 80%. Therefore, detection of early stage disease has been the focus of intensive research and public health programs aimed at identifying populations at risk for lung cancer are ongoing. The first risk of lung cancer remains smoking, and up to 90% of lung cancer deaths are reported by the CDC to be associated with smoking. The united states preventive services working group (USPSTF) has proposed the following: annual lung cancer screening using Low Dose Computed Tomography (LDCT) was performed on adults aged 55 to 80 years with a 30-pack year smoking history, whether they are current smokers or previous smokers who have quitted smoking in the last 15 years. Thus, an increasing number of individuals are receiving annual LDCT screening for evidence of lung nodules as a first indicator of lung cancer. The presence of nodules in LDCT scans is not evidence of lung cancer, as non-cancerous nodules also occur in the lung and occur at a much higher frequency than cancerous nodules. Of the lung nodules found in LDCT scans, up to 94% are due to benign disease. Such a high rate of false positive results subjects hundreds of thousands of individuals to unnecessary interventions and invasive procedures that not only cause significant injury, but also place a significant burden on the already overburdened healthcare system. There is a need for an effective non-invasive method to assess whether lung nodules detected by LDCT are cancerous or benign lesions.

Disclosure of Invention

Aspects of the disclosure include methods of generating a circulating analyte profile of a subject. The method includes contacting a blood sample from the subject with a probe set for specifically binding the analyte, and detecting the presence or absence of binding of the analyte to the probes of the probe set. In some embodiments, the set of probes comprises probes for specifically binding analytes comprising two, three, or each of carcinoembryonic antigen (CEA), C-X-C motif chemokine ligand 4(CXCL4), C-X-C motif chemokine ligand 7(CXCL7), and C-X-C motif chemokine ligand 10(CXCL 10). In some embodiments, such a probe set further comprises probes for specifically binding to two or each of Epidermal Growth Factor Receptor (EGFR), pro-surface active protein B (pro-SFTPB), and tissue inhibitor of metalloproteinase 1 (TIMP 1). Sensor devices (e.g., magnetic sensor devices) that include a set of capture probes and that can be used, for example, to implement the methods of the present disclosure are also provided.

Drawings

Figure 1 distribution of biomarkers in 405 samples stratified by smoking history.

Figure 2 ROC curves for model 217_3092 trained with a subgroup of previous smokers 1/3 and tested on a subgroup 2/3, compared to a Mayo model ROC curve.

Figure 3 ROC curves for model 217_3092 trained with a subset of previous smokers 1/3 and tested on a Mayo model Intermediate Risk (IR) subject in the subset 2/3, compared to a Mayo model ROC curve.

Figure 4 ROC curves for model 217_3092 trained with a current subgroup of smokers 2/3 and tested on the 1/3 subgroup, compared to the Mayo model ROC curve.

Figure 5 ROC curves for model 217_3092 trained with a subset of current smokers 2/3 and tested on the Mayo model Intermediate Risk (IR) subjects in the 1/3 subset, compared to the Mayo model ROC curves.

Detailed Description

Aspects of the disclosure include methods of generating a circulating analyte profile of a subject. The method includes contacting a blood sample from the subject with a probe set for specifically binding the analyte, and detecting the presence or absence of binding of the analyte to the probes of the probe set. Sensor devices that include a set of capture probes and that can be used, for example, to perform the methods of the present disclosure are also provided.

Before the methods, devices, and kits of the present disclosure are described in greater detail, it is to be understood that the methods, devices, and kits are not limited to the specific embodiments described, as such methods, devices, and kits may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods, devices, and kits will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the method, apparatus and kit. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the methods, apparatus, and kits, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods, devices, and kits.

Certain ranges are given herein wherein a numerical value is preceded by the term "about". The term "about" is used herein to provide literal support for the exact number following it, as well as a number that is close or approximate to the number following the term. In determining whether a number is near or approximate a specifically recited number, a near or approximate non-recited number may be a number that provides substantial equivalents of the specifically recited number in the context in which it is presented.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods, devices, and kits belong. Representative illustrative methods, devices, and kits are now described, although any methods, devices, and kits similar or equivalent to those described herein can also be used to carry out or test the methods, devices, and kits.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and were incorporated by reference herein to disclose and describe the materials and/or methods in connection with which the publications were cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the methods, devices, and kits are not entitled to antedate such publication by virtue of its inclusion in a publication, as it may differ from an actual publication date by virtue of independent confirmation.

It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is also noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

It is to be understood that certain features of the methods, devices, and kits, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods, devices, and kits that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments are specifically encompassed by the present disclosure and are disclosed herein to the extent that each combination is individually and specifically disclosed, such combination is intended to comprise an operable method and/or composition. Moreover, all sub-combinations listed in the examples describing such variables are also specifically embraced by the present methods, devices and kits and are disclosed herein as if each such sub-combination were individually and explicitly disclosed herein.

As will be understood by those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

Method

Aspects of the disclosure include methods of generating a circulating analyte profile of a subject. The method includes contacting a blood sample from the subject with a probe set for specifically binding the analyte, and detecting the presence or absence of binding of the analyte to the probes of the probe set. In certain aspects, detecting comprises quantifying the analyte detected.

The probes in the probe set may be any molecule that specifically binds to the target analyte. Target analytes include, but are not limited to, proteins (including non-antibody proteins, etc.), nucleic acids (e.g., tumor DNA or RNA), and cells, such as circulating tumor cells. The probes of the probe set may be selected according to the nature of the analyte to be detected. For example, if one of the two or more analytes is a protein (e.g., a non-antibody protein or an antibody protein), an antibody, ligand, or the like that specifically binds the protein can be used as a probe in a probe set. If one of the two or more analytes is an antibody, the corresponding antigen of the antibody may be used as a probe in a probe set, or an antibody that binds to the antibody may be used. For example, if one of the two or more analytes is a nucleic acid, a nucleic acid that is sufficiently complementary to a unique region of that nucleic acid to achieve specific binding under the desired contact conditions can be used as a probe in a probe set. Proteins (e.g., nucleic acid binding proteins, antibodies, etc.) can also be used to bind to nucleic acid analytes.

The term "binding" refers to direct binding between two molecules due to, for example, covalent, electrostatic, hydrophobic, ionic, and/or hydrogen bonding interactions. The probes of the probe set specifically bind to their corresponding analytes. Non-specific binding (NSB) generally refers to something other than an antibody and its cognate antigen (such as various other antibodies in a sample)Pro). Under certain assay conditions, NSB means a signal of less than about 10^-7Affinity binding of M, e.g. at 10^-6M、10^-5M、10^-4M, etc.

"specific binding" or "specific binding" refers to a probe that is, for example, greater than or equal to about 10⁵M^-1Or Ka (i.e., the equilibrium binding constant for a particular binding interaction in units of 1/M) binds to its corresponding analyte. In certain embodiments, the extracellular binding domain is greater than or equal to about 10⁶M^-1、10⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1、10¹¹M^-1、10¹²M^-1Or 10¹³M^-1The Ka of (1) binds to an antigen. By "high affinity" binding is meant binding at least 10⁷M^-1At least 10⁸M^-1At least 10⁹M^-1At least 10¹⁰M^-1At least 10¹¹M^-1At least 10¹²M^-1At least 10¹³M^-1Or higher Ka binding antigen. Alternatively, affinity can be defined as the equilibrium dissociation constant (KD) for a particular binding interaction in M (e.g., 10)^-5M to 10^-13M, or less). In some embodiments, specific binding is to the extracellular binding domain of less than or equal to about 10^-5M, less than or equal to about 10^-6M, less than or equal to about 10^-7M, less than or equal to about 10^-8M, or less than or equal to about 10^-9M、10^-10M、10^-11M, or 10^-12M or less KD binds to the target molecule. The binding affinity of a probe for its target analyte can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using Surface Plasmon Resonance (SPR) techniques (e.g., BIAcore 2000 instrument, using general procedures outlined by the manufacturer), by radioimmunoassay, and the like.

The probe set includes an appropriate number of probes for specifically binding a plurality of unique circulating target analytes. According to certain embodiments, a probe set comprises a suitable number of probes for specifically binding 4 to 5 analytes, 6 to 10 analytes, 10 to 15 analytes, 15 to 20 analytes, 20 to 25 analytes, 25 to 30 analytes, 30 to 35 analytes, 35 to 40 analytes, 40 to 45 analytes, 45 to 50 analytes, 50 to 60 analytes, 60 to 70 analytes, 70 to 80 analytes, 80 to 90 analytes, 90 to 100 analytes, 100 to 200 analytes, 200 to 300 analytes, 300 to 400 analytes, 400 to 500 analytes, or 500 to 1000 analytes.

In certain embodiments, a probe set comprises probes for specifically binding 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, or 25 or more analytes. According to some embodiments, a probe set comprises probes for specifically binding 200 or fewer analytes, 150 or fewer analytes, 125 or fewer analytes, 100 or fewer analytes, 75 or fewer analytes, 50 or fewer analytes, 40 or fewer analytes, 30 or fewer analytes, 25 or fewer analytes, 20 or fewer analytes, 15 or fewer analytes, or 10 or fewer analytes.

According to some embodiments, a probe set comprises probes for specifically binding two or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more) of: carcinoembryonic antigen (CEA), C-X-C motif chemokine ligand 4(CXCL 4-also known as platelet factor 4 (or PF4)), C-X-C motif chemokine ligand 7(CXCL 7-also known as neutrophil activating protein 2 (or NAP2)), C-X-C motif chemokine ligand 10(CXCL 10-also known as interferon gamma-inducing protein 10 (or IP10)), Epidermal Growth Factor Receptor (EGFR), pro-surfactant protein B (pro-SFTPB), metalloproteinase 1 tissue inhibitor (TIMP1), anti-angiopoietin-like protein 3 antibody (anti-ANGPTL 3), anti-14-3-protein theta antibody (anti-YAQWH), anti-laminin alpha 1 antibody (anti-LRMR 1), human epididymin 4(HE4), pro-gradin 2(AGR2), chromogranin A (CHGA), glycoprotein leucine rich alpha-632-1 (G1), Anti-annexin 1 antibody (anti-ANXA 1), anti-ubiquitin 1 antibody (anti-UBQLN 1), interleukin 6(IL6), interleukin 8(IL8), C-X-C motif chemokine ligand 2(CXCL2), C-X-C motif chemokine ligand 12(CXCL12), C-X-C motif chemokine ligand 14(CXCL14), defensin, β 1(DEFB1), fibroblast growth factor 2(FGF2), cluster of differentiation 97(CD97), pre-platelet basic protein (PPBP), Procalcitonin (PCT), advanced glycosylation end product Receptor (RAGE), ges 100 calcium binding protein a4(S100a4), S100 calcium binding protein a8(S100a8), and Osteopontin (OPN).

In certain embodiments, a probe set comprises probes for specifically binding one, two, three, or each of CEA, CXCL4, CXCL7, and CXCL10 in any desired combination. According to some embodiments, such probe sets further comprise probes for specifically binding one, two, or each of EGFR, pro-SFTPB, and TIMP1 in any desired combination. In certain embodiments, such a panel of probes further comprises one or more probes that bind to one or any combination of additional analytes in any desired combination selected from the group consisting of anti-ANGPTL 3, anti-ywaq, anti-LAMR 1, HE4, AGR2, CHGA, LRG1, anti-ANXA 1, anti-UBQLN 1, IL6, IL8, CXCL2, CXCL12, CXCL14, DEFB1, FGF2, CD97, PPBP, PCT, RAGE, S100a4, S100a8, and OPN, wherein the method further comprises detecting the presence or absence of binding of one or any combination of additional analytes to the probe panel to generate a circulating analyte profile in the subject.

According to some embodiments, a probe set comprises one or more probes for binding one or more types of circulating cells. Target circulating cells include, but are not limited to, circulating tumor cells and circulating stem cells. "circulating tumor cells" (CTCs) refer to cancer cells that have shed from a solid tumor in a subject and are found in the circulation of the subject, such as the peripheral blood, bone marrow, etc., of the subject. The probes can bind to circulating cells (e.g., CTCs) with the aid of the probes' specificity for known cell surface molecules (e.g., receptors, adhesion molecules, etc.) expressed by the target circulating cells. When the circulating cells are CTCs, the probes (e.g., antibody probes) can specifically bind to a tumor-associated or tumor-specific antigen expressed by the CTCs. By "tumor-associated antigen" is meant a cell surface molecule expressed on malignant cells that has limited expression on cells of normal tissue, or a cell surface molecule expressed on malignant cells at a much higher density than on normal cells. A "tumor-specific antigen" is an antigen that is present on the surface of malignant cells but not on non-malignant cells. The type of CTCs that can be bound by the probes of a probe set can vary, for example, depending on the type of solid tumor that the CTCs shed. In certain aspects, a probe set may include probes that specifically bind CTCs, which specifically bind epithelial cell adhesion molecule (EpCAM) and/or any other useful cell surface CTC molecule. Thus, in some embodiments, the probe set further comprises probes for binding circulating tumor cells, wherein the method further comprises detecting the presence or absence of binding of circulating tumor cells to the probe set probes to generate a circulating analyte profile of the subject. In certain embodiments, detecting the presence or absence of binding of circulating tumor cells comprises quantifying the detected circulating tumor cells.

According to certain embodiments, a probe set comprises one or more probes for binding one or more types of circulating nucleic acids. The target circulating nucleic acid includes circulating double-stranded or single-stranded DNA, circulating double-stranded or single-stranded RNA, circulating DNA-RNA hybrid, and the like. In certain aspects, a set includes one or more probes for specifically binding to one or more circulating tumor dna (ctdna). Dying tumor cells release small fragments of their DNA into the bloodstream, and the amount/concentration of ctDNA in the blood generally increases with increasing cancer stage. According to certain embodiments, a probe set comprises probes for specifically binding ctDNA comprising somatic mutations known to be associated with (or specific for) a target tumor type. Clinically relevant ctDNA includes those described in Bettegowda et al (2014) scientific transformation medicine (sci. trans. med.) 6(224) 224ra 24. Thus, in some embodiments, the probe set further comprises probes for binding to tumor DNA, wherein the method further comprises detecting the presence or absence of binding of tumor DNA to the probes of the probe set to generate a circulating analyte profile of the subject. In certain embodiments, detecting the presence or absence of binding of tumor DNA comprises quantifying the detected tumor DNA.

The methods of the present disclosure include detecting the presence or absence of binding of an analyte to the probes of the probe set to generate a circulating analyte profile of the subject. In certain aspects, detecting comprises quantifying the analyte detected. Any of a variety of suitable assay formats and detection methods may be employed. In certain aspects, the probes of a probe set may be attached directly or indirectly to a solid support, such as a bead (e.g., microparticle, nanoparticle, etc.) or a substantially planar solid support/substrate. According to certain embodiments, the probes may be attached to a solid support as an array. For example, a probe set can be a probe set provided as an addressable probe array.

In certain aspects, a sandwich assay is used to detect the presence or absence of binding of an analyte of the two or more analytes to the probes of the probe set. For example, the probes of a probe set may be attached to a solid surface (e.g., as an array) to capture the analyte, and a detection reagent is added that binds (e.g., specifically binds) to the analyte (if present in the blood sample) at a site where the analyte is not bound by the probes. In certain aspects, the detection reagent is a detection antibody that binds to an epitope of the analyte that is different from the binding site (e.g., epitope) to which the probes of the probe set bind. As a result, the analyte is "sandwiched" between the probe and the detection reagent. The detection reagent may comprise a detectable label such that detecting binding of an analyte of the two or more analytes to the probes of the probe set involves detecting the label of the detection reagent. According to certain embodiments, a second detection reagent is used. Suitable second reagents include labeled second antibodies (e.g., fluorescently labeled antibodies, magnetically labeled antibodies, etc.), second antibodies linked to enzymes that catalyze the conversion of a substrate to a detectable product, and the like. Additional details and design considerations for sandwiches and other assays that can be used to practice the Methods of the present disclosure are described, for example, in Cox et al, (Immunoassay Methods), Eli Lilly & Company and the National Center for Advancing transformational Sciences.

In certain aspects, the detection reagent that binds to the analyte bound to the probe is an antibody. Such detection reagents may be modified antibodies. The modified antibody may be configured to specifically bind to the analyte of interest and may also include one or more additional members of a specific binding pair. One or more members of the specific binding pair may be configured to specifically bind to a complementary member of the specific binding pair. In certain instances, the complementary member of the specific binding pair is bound to a magnetic label, for example, when the method is carried out using a magnetic sensor device. The antibody detection reagent may be modified to include biotin, which will specifically bind to streptavidin, for example modified to include a magnetic label of streptavidin. Thus, in certain aspects, the detection reagent specifically binds to the analyte (e.g., via an antibody-antigen interaction) and specifically binds to the label (e.g., a magnetic label) via a selected interaction (e.g., via a streptavidin-biotin interaction). The detection reagent can be configured to bind the analyte and a label (e.g., a magnetic label). In other words, the detection reagent can be configured such that specific binding of the analyte to the detection reagent does not significantly interfere with the ability of the detection reagent to specifically bind to the label. Similarly, the detection reagent can be configured such that specific binding of the label to the detection reagent does not significantly interfere with the ability of the detection reagent to bind to the analyte.

The analyte in the blood sample may be determined qualitatively or quantitatively. Qualitative determinations include providing a user with a simple yes/no result determination of whether an analyte is present in a sample. Quantitative assays include semi-quantitative assays in which a user is provided with coarse-scale results, e.g., low, medium, and high-scale results, regarding the amount of analyte in a sample, and a user is provided with fine-scale results of an accurate measurement of the analyte concentration.

The circulating analyte profile can be generated from a blood sample (e.g., a whole blood sample, a plasma sample, or a serum sample) obtained from any of a variety of subjects. Typically such individuals are "mammals" or "mammals," where these terms are used broadly to describe organisms within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the circulating analyte profile is generated from a blood sample obtained from a human subject.

According to some embodiments, the subject whose circulating analyte profile is generated is from a population with a high risk of lung cancer. Due to a variety of genetic, behavioral, and/or environmental factors, a subject may be at high risk for lung cancer. According to certain embodiments, the subject is from a population having a high risk of lung cancer because the subject is a previous smoker (e.g., a past heavy smoker) or a current smoker. By "prior smoker" is meant that the subject is not a smoker when a blood sample is obtained from the subject for use in the method. My "current smoker" means that the subject is a smoker at the time the blood sample for the method is obtained from the subject. According to certain embodiments, a subject from a population with a high risk of lung cancer means that the subject is 55 to 74 years old, has a minimum smoking history of 30 pack years or more (where "pack year" equals the number of cigarette packs smoked per day x the number of years smoked), is currently smoking or is quitting smoking within the last 15 years, and is apparently free of disease when a circulating analyte profile is generated. For example, a heavy smoker in the past may have a history of smoking for 30 packets of years or more.

In certain aspects, the subject whose circulating analyte profile is generated has a lung nodule (or "lesion"), e.g., an indeterminate lung nodule/lesion. In some cases, the uncertain lung nodules are identified/detected by Low Dose Computed Tomography (LDCT), chest X-ray, chest CT scan, chest MRI, chest Positron Emission Tomography (PET), or other suitable imaging method scan. Indeterminate nodules can be benign (non-cancer) and caused by scarring, inflammation, infection, and the like. In other cases, the nodule may be malignant, such as lung cancer (e.g., early stage lung cancer) or a cancer that spreads from another cancer in the body to the lung. As further described herein and demonstrated in the experimental section below, the circulating analyte profile of a subject can form the basis (e.g., complete or partial basis) for assessing the risk of lung nodule malignancy.

Method based on magnetic sensor

According to certain embodiments, the method of the present disclosure is performed using a magnetic sensor device. For example, the probe sets may be arranged (e.g., provided as an addressable probe array) on a magnetic sensor chip of a magnetic sensor device. The magnetic sensor device can have two or more magnetic sensors with a set of probes (e.g., the same or different arrays of capture probes) attached to its surface. Any of the above-described probe sets may be used. In certain aspects, each of the two or more magnetic sensors having a set of capture probes attached to its surface includes a capture probe for binding the same circulating analyte.

Methods of the present disclosure employing a magnetic sensor device can include contacting a magnetic sensor device having a capture probe set attached to a surface thereof (e.g., an array) with a blood sample and detecting a signal indicative of binding of an analyte (if present in the blood sample) to the capture probe set. In some cases, the magnetic sensor device includes a sensor configured to detect the presence of a nearby magnetic marker without any direct physical contact between the magnetic sensor and the magnetic marker. The magnetic label may be directly or indirectly bound to the analyte, which in turn may be directly or indirectly bound to the magnetic sensor. If the bound magnetic label is located within the detection range of the magnetic sensor, the magnetic sensor may provide a signal indicating the presence of the bound magnetic label and thus the presence of the analyte.

In certain aspects, the methods of the present disclosure are performed using a sandwich assay, wherein a probe set is attached to the surface of the sensing region of the magnetic sensor device. The blood sample is dispensed on the sensing region to contact the blood sample with the probe set under conditions in which the analytes of the two or more analytes (if present in the blood sample) bind to their respective probes. With or without washing, a detection reagent may be added that binds to the analyte of the two or more analytes that is bound to the probes of the probe set. In some cases, the detection reagent is directly bound to the magnetic label. In other aspects, the detection reagent is not directly bound to the magnetic label, but rather a secondary magnetically labeled detection reagent that is bound to the detection reagent is employed. For example, the detection reagent can specifically bind to the analyte (e.g., via an antibody-antigen interaction) and to the magnetic label via a selected interaction (e.g., via a streptavidin-biotin interaction). Binding of the detection reagent to the surface-bound analyte locates the magnetic label within the detection range of the magnetic sensor such that a detectable signal is induced in the magnetic sensor indicative of the presence of the analyte.

In certain embodiments, the electrical signal is generated in response to a magnetic marker near the surface of the magnetic sensor. For example, the magnetic sensor may be configured to detect a change in resistance of the magnetic sensor induced by a change in the local magnetic field. In some cases, the binding of a magnetic label (e.g., a magnetic nanoparticle label) in close proximity to the magnetic sensor induces a detectable change in the resistance of the magnetic sensor. For example, magnetic labels in the vicinity of the magnetic sensor may be magnetized in the presence of an applied external magnetic field. The local magnetic field of the magnetized magnetic labels may induce a detectable change in the resistance of the underlying magnetic sensor. Thus, the presence of the magnetic label can be detected by detecting a change in the resistance of the magnetic sensor. As will be described in further detail below, magnetic sensor devices that may be used to implement the methods of the present disclosure may include magnetoresistive elements. Non-limiting examples of magnetoresistive elements that may be employed include spin valve magnetoresistive elements and Magnetic Tunnel Junction (MTJ) magnetoresistive elements.

In some cases, the method is a wash-free method of assessing the presence of an analyte in a blood sample. By "wash-free" is meant that no washing step is performed after the reagent and/or blood sample is contacted with the magnetic sensor. Thus, the step of removing unbound reagents (e.g., unbound magnetic labels) or unbound sample from the magnetic sensor surface is not performed during the assay of these embodiments. Thus, although the method may include sequential contact of one or more different reagents and/or samples with the magnetic sensor surface, at no time during the assay is the sample surface contacted with the fluid in a manner that removes unbound reagents or sample from the magnetic sensor surface. For example, in certain embodiments, no washing step is performed after the magnetic sensor surface is contacted with the blood sample. In some cases, the method does not include a washing step after contacting the magnetic sensor surface with the magnetic labels. In some cases, no washing step is performed after the magnetic sensor surface is contacted with the detection reagent.

In certain embodiments where a wash step is performed, the wash step does not substantially alter the signal from the magnetic sensor. The washing step may not result in a substantial change in the signal from the magnetic sensor, because in some cases, the unbound magnetic labels do not have a substantially detectable signal as described herein. For example, if a washing step is performed, in some cases, the washing step results in a signal change of 25% or less, e.g., 20% or less, or 15% or less, or 10% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less, as compared to the signal obtained prior to the washing step. In some embodiments, the washing step results in a reduction in the signal from the magnetic sensor of 25% or less, such as 20% or less, or 15% or less, or 10% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less.

Embodiments of the method may also include obtaining a real-time signal from the magnetic sensor device. By "real-time" is meant that the signal is observed as it is generated. For example, the real-time signal is obtained from its start-up instant and is obtained continuously over a given period of time. Thus, certain embodiments include observing the real-time evolution of a signal associated with the occurrence of a target binding interaction (e.g., binding of two or more analytes of interest to a magnetic sensor and/or binding of a magnetic label to a target analyte). The real-time signal may include two or more data points obtained over a given time period, where in certain embodiments the obtained signal is a set of consecutive data points (e.g., in the form of a trajectory) that are consecutively obtained over a target given time period. The target time period may vary, in some cases ranging from 0.5 minutes to 60 minutes, such as from 1 minute to 30 minutes, including from 1 minute to 15 minutes, or from 1 minute to 10 minutes. For example, the time period may begin at the time of initiation of the real-time signal and may continue until the sensor reaches a maximum or saturation level (e.g., all analyte binding sites on the sensor are occupied). For example, in some cases, the time period begins when a blood sample comes into contact with the sensor. In some cases, the time period may begin before the blood sample contacts the sensor, e.g., to record a baseline signal before the sample contacts the sensor. The number of data points in the signal may also vary, where in some cases the number of data points is sufficient to provide a continuous extension of the data over the time course of the real-time signal. "continuous" means that data points are repeatedly obtained at a repetition rate of 1 data point per minute or more, such as 2 data points per minute or more, including 5 data points per minute or more, or 10 data points per minute or more, or 30 data points per minute or more, or 60 data points per minute or more (e.g., 1 data point per second or more), or 2 data points per second or more, or 5 data points per second or more, or 10 data points per second or more, or 20 data points per second or more, or 50 data points per second or more, or 75 data points per second or more, or 100 data points per second or more.

The real-time signal may be a real-time analyte-specific signal. The real-time analyte-specific signal is the above-described real-time signal obtained from only a specific analyte of the two or more analytes of interest. In these embodiments, the unbound analyte and unbound magnetic label do not produce a detectable signal. Thus, the real-time signal obtained is only from the specific magnetically-labeled target analyte bound to the magnetic sensor, and substantially no signal is obtained from unbound magnetic labels or other reagents (e.g., analyte not specifically bound to the sensor).

In some embodiments, the signal is observed when the assay device is in a wet condition. By "wet" or "wet condition" is meant that the assay composition (e.g., an assay composition comprising a blood sample, magnetic labels, and one or more detection reagents) remains in contact with the surface of the magnetic sensor. Thus, no washing step is required to remove unbound fraction of non-targets or excess unbound magnetic label or capture probe. In certain embodiments, as described above, the use of magnetic labels and magnetic sensors facilitates "wet" detection, as the signal induced in the magnetic sensor by the magnetic labels decreases as the distance between the magnetic labels and the surface of the magnetic sensor increases. For example, as described above, the use of magnetic labels and magnetic sensors may facilitate "wet" detection, as the magnetic field generated by the magnetic labels decreases as the distance between the magnetic labels and the surface of the magnetic sensor increases. In some cases, the magnetic field of the magnetic labels bound to the surface-bound analyte significantly exceeds the magnetic field from unbound magnetic labels dispersed in the solution. For example, as described above, a real-time analyte-specific signal may be obtained from only the specific magnetically-labeled target analyte bound to the magnetic sensor, and substantially no signal may be obtained from unbound magnetic labels dispersed in the solution (e.g., not specifically bound to the sensor). Unbound magnetic labels dispersed in the solution may be further away from the surface of the magnetic sensor and may undergo brownian motion, which may reduce the ability of the unbound magnetic labels to induce a detectable change in the resistance of the magnetic sensor. Unbound magnetic labels may also be suspended in solution, e.g. as a colloidal suspension (e.g. due to having nanoscale dimensions), which may reduce the ability of the unbound magnetic labels to induce a detectable change in the resistance of the magnetic sensor.

The magnetic labels useful in the various methods (e.g., as described herein) can vary and include any type of label that induces a detectable signal in a magnetic sensor when the magnetic label is positioned near the surface of the magnetic sensor. A magnetic label is a label moiety that, when sufficiently bound to a magnetic sensor, can be detected by the magnetic sensor and cause the magnetic sensor to output a signal. For example, the presence of magnetic labels near the surface of the magnetic sensor may induce a detectable change in the magnetic sensor, such as, but not limited to, a change in resistance, conductance, inductance, impedance, and the like. In some cases, the presence of a magnetic label near the surface of the magnetic sensor induces a detectable change in the resistance of the magnetic sensor. The target magnetic label may be sufficiently bound to the magnetic sensor if the distance between the centre of the magnetic label and the sensor surface is 1000nm or less, such as 800nm or less, for example 400nm or less, including 100nm or less, or 75nm or less, or 50nm or less, or 25nm or less, or 10nm or less.

In some cases, the magnetic labels comprise one or more materials selected from paramagnetic, superparamagnetic, ferromagnetic, ferrimagnetic, antiferromagnetic materials, combinations thereof, and the like. For example, the magnetic labels may comprise superparamagnetic material. In certain embodiments, the magnetic labels are configured to be non-magnetic in the absence of an external magnetic field. By "non-magnetic" is meant that the magnetization of the magnetic labels is zero or averaged to zero over a certain period of time. In some cases, the magnetic labels may be non-magnetic due to the random flipping of the magnetization of the magnetic labels over time. Magnetic labels configured to be non-magnetic in the absence of an external magnetic field may facilitate dispersion of the magnetic labels in solution, as the non-magnetic labels generally do not agglomerate in the absence of an external magnetic field or even in the presence of a small magnetic field where thermal energy is still dominant. In certain embodiments, the magnetic labels comprise superparamagnetic or synthetic antiferromagnetic materials. For example, the magnetic labels may comprise two or more layers of antiferromagnetically coupled ferromagnets.

In certain embodiments, the magnetic labels are high-moment magnetic labels. The magnetic moment of a magnetic marker is a measure of its tendency to align with an external magnetic field. By "high moment" is meant that the magnetic labels are more prone to align with external magnetic fields. Magnetic labels having a high magnetic moment may facilitate the detection of the presence of magnetic labels near the surface of the magnetic sensor, since the magnetization of the magnetic labels is more easily induced with an external magnetic field.

In certain embodiments, the magnetic labels include, but are not limited to, Co alloys, ferrites, cobalt nitride, cobalt oxide, Co-Pd, Co-Pt, iron oxide, iron alloys, Fe-Au、Fe-Cr、Fe-N、Fe₃O₄Fe-Pd, Fe-Pt, Fe-Zr-Nb-B, Mn-N, Nd-Fe-B, Nd-Fe-B-Nb-Cu, Ni alloys, combinations thereof, and the like. Examples of high magnetic moment magnetic labels include, but are not limited to, Co, Fe or CoFe nanocrystals, which can be superparamagnetic at room temperature, and synthetic antiferromagnetic nanoparticles.

In some embodiments, the surface of the magnetic label is modified. In some cases, the magnetic labels may be coated with a layer configured to facilitate stable binding of the magnetic labels to one member of a binding pair as described above. For example, the magnetic label may be coated with a gold layer, a poly-L-lysine modified glass layer, dextran, or the like. In certain embodiments, the magnetic label comprises one or more iron oxide cores embedded in the dextran polymer. Furthermore, the surface of the magnetic label may be modified with one or more surfactants. In some cases, the surfactant helps to increase the water solubility of the magnetic label. In certain embodiments, the surface of the magnetic labels is modified with a passivation layer. The passivation layer may promote chemical stability of the magnetic label under assay conditions. For example, the magnetic labels may be coated with a passivation layer comprising gold, iron oxide, a polymer (e.g., polymethylmethacrylate film), and the like.

In certain embodiments, the magnetic markers have a spherical shape. Alternatively, the magnetic marker may be a disc, rod, coil or fiber. In some cases, the magnetic labels are of a size such that the magnetic labels do not interfere with the target binding interaction. For example, the magnetic labels may be of a size comparable to the analyte and the capture probes, such that the magnetic labels do not interfere with the binding of the capture probes to the analyte. In some cases, the magnetic label is a magnetic nanoparticle, or contains a plurality of magnetic nanoparticles bound together by a suitable binding agent. In some embodiments, the magnetic labels have an average diameter of 5nm to 250nm, such as 5nm to 150nm, including 10nm to 100nm, such as 25nm to 75 nm. For example, magnetic labels having an average diameter of 5nm, 10nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 70nm, 80nm, 90nm, or 100nm, as well as magnetic labels having an average diameter ranging between any two of these values, can be used with the subject methods. In some cases, the average diameter of the magnetic labels is 50 nm.

Magnetic labels and their conjugation to biomolecules are further described in U.S. patent No. 9,863,939 entitled "Analyte Detection With Magnetic Sensors," the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

Risk assessment

The methods of the present disclosure may further comprise assessing the risk of the subject for having a disease or disorder based on the circulating analyte profile. For example, as described above and demonstrated in the experimental section below, a subject whose circulating analyte profile was generated may have an indeterminate lung nodule/lesion (detected prior to generating the circulating analyte profile of the subject, e.g., by Low Dose Computed Tomography (LDCT)), and the method may further comprise assessing the risk of the lung nodule being malignant (e.g., non-small cell lung cancer (NSCLC) or other malignancy) based on the circulating analyte profile of the subject. For example, the circulating analyte profile may be compared to one or more reference profiles, and based on the comparison, the risk of unsure of whether a lung nodule is malignant (as opposed to benign) may be determined. Risk assessment may be based on a profile of circulating analytes above or below a threshold. Thus, in certain embodiments, the circulating analyte profile of the subject indicates that the pulmonary nodule of the subject is malignant. In some embodiments, a circulating analyte profile is generated and then made available to a third party, such as the subject whose circulating analyte profile is generated, his/her guardian OR representative, a doctor OR healthcare worker, a genetic advisor, OR an insurance agent, for example, through a user interface accessible over the internet, and an interpretation of the circulating analyte profile, for example, in the form of a risk metric such as Absolute Risk (AR), Risk Ratio (RR), OR Odds Ratio (OR) that the nodule is malignant. The results of such risk assessment may be reported in numerical form (e.g., by risk values such as absolute risk, relative risk, and/or odds ratio, or by percentage increase in risk compared to a reference), graphically, and/or by other means suitable for explaining risk to a third party.

The risk assessment may be based solely on the circulating analyte profile, or may be based in part on the circulating analyte profile. Where the risk assessment is based in part on the circulating analyte profile, the risk assessment may be further based on clinical parameters of the subject selected from the group consisting of age of the subject, size of the nodule, nodule edge (whether there is a burr), nodule location, subject gender, subject cancer history, subject cancer family history, smoking status (e.g., previous and current smokers), smoking history (including smoking intensity), and any combination thereof.

Treatment of

The methods of the present disclosure may further comprise treating a subject who developed a circulating analyte profile thereof. In certain aspects, the subject has an indeterminate lung nodule, and the method comprises assessing the risk of the indeterminate lung nodule being malignant or benign. If the assessed risk of malignancy of a lung nodule meets a threshold criterion, the nodule may be biopsied to diagnose whether the lung nodule is malignant or benign. In some embodiments, the method comprises making such a diagnosis. If the lung nodule is diagnosed as malignant, in some embodiments, the method includes treating the subject after diagnosis, e.g., based on the diagnosis. Treatment can include, for example, administering to the subject a therapeutically effective amount of an agent (e.g., a chemotherapeutic agent (e.g., crizotinib, ceritinib, erlotinib, bugatitinib, loratinib, erlotinib, gefitinib, afatinib, dactinotinib, crizotinib, dabrafenib, trimetatinib, etc.), a small molecule, a biologic (e.g., an antibody), an engineered cell, etc.), radiation therapy, and the like. Alternatively or additionally, treatment may include removing all or part of a tissue (e.g., tumor tissue) or organ that contributes to (e.g., causes) a disease or condition from a subject. Treatment may include surgical removal of all or a portion of the cancer (e.g., by a lung resection, a lobectomy, a segmental or wedge resection, a sleeve resection, etc.); radiofrequency ablation (RFA) of all or part of a tumor; and so on.

Device for measuring the position of a moving object

As summarized above, aspects of the present disclosure include sensor devices (e.g., magnetic sensor devices). The sensor device includes a set of probes for specific binding to an analyte. The sensor device of the present invention can comprise any of the probe sets described above in the methods section of the present disclosure and the experimental section below. According to some embodiments, the sensor device comprises a set of capture probes provided as an addressable array of probes, e.g. in a sensing area of the sensor device.

According to some embodiments, the devices of the present disclosure comprise a set of probes (e.g., a set of capture probes provided as an addressable probe array) comprising probes for specifically binding two or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more) of: carcinoembryonic antigen (CEA), C-X-C motif chemokine ligand 4(CXCL 4-also known as platelet factor 4 (or PF4)), C-X-C motif chemokine ligand 7(CXCL 7-also known as neutrophil activating protein 2 (or NAP2)), C-X-C motif chemokine ligand 10(CXCL 10-also known as interferon gamma-inducing protein 10 (or IP10)), Epidermal Growth Factor Receptor (EGFR), pro-surfactant protein B (pro-SFTPB), metalloproteinase 1 tissue inhibitor (TIMP1), anti-angiopoietin-like protein 3 antibody (anti-ANGPTL 3), anti-14-3-protein theta antibody (anti-YAQWH), anti-laminin alpha 1 antibody (anti-LRMR 1), human epididymin 4(HE4), pro-gradin 2(AGR2), chromogranin A (CHGA), glycoprotein leucine rich alpha-632-1 (G1), Anti-annexin 1 antibody (anti-ANXA 1), anti-ubiquitin 1 antibody (anti-UBQLN 1), interleukin 6(IL6), interleukin 8(IL8), C-X-C motif chemokine ligand 2(CXCL2), C-X-C motif chemokine ligand 12(CXCL12), C-X-C motif chemokine ligand 14(CXCL14), defensin, β 1(DEFB1), fibroblast growth factor 2(FGF2), cluster of differentiation 97(CD97), pre-platelet basic protein (PPBP), Procalcitonin (PCT), advanced glycosylation end product Receptor (RAGE), ges 100 calcium binding protein a4(S100a4), S100 calcium binding protein a8(S100a8), and Osteopontin (OPN).

In certain embodiments, the devices of the present disclosure comprise a set of probes (e.g., a set of capture probes provided as an addressable probe array) comprising probes for specific binding to one, two, three, or each of CEA, CXCL4, CXCL7, and CXCL10 in any desired combination. According to some embodiments, such probe sets further comprise probes for specifically binding one, two, or each of EGFR, pro-SFTPB, and TIMP1 in any desired combination. In certain embodiments, such a probe set further comprises one or more probes for specifically binding to one or any combination of additional analytes selected from the group consisting of anti-ANGPTL 3, anti-ywaq, anti-LAMR 1, HE4, AGR2, CHGA, LRG1, anti-ANXA 1, anti-UBQLN 1, IL6, IL8, CXCL2, CXCL12, CXCL14, DEFB1, FGF2, CD97, PPBP, PCT, RAGE, tras 100a4, S100a8, and OPN in any desired combination.

According to certain embodiments, the device comprises a set of probes for specifically binding 4 to 5 analytes, 6 to 10 analytes, 10 to 15 analytes, 15 to 20 analytes, 20 to 25 analytes, 25 to 30 analytes, 30 to 35 analytes, 35 to 40 analytes, 40 to 45 analytes, 45 to 50 analytes, 50 to 60 analytes, 60 to 70 analytes, 70 to 80 analytes, 80 to 90 analytes, 90 to 100 analytes, 100 to 200 analytes, 200 to 300 analytes, 300 to 400 analytes, 400 to 500 analytes, or 500 to 1000 analytes.

In certain embodiments, the device comprises a set of probes for specifically binding 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, or 25 or more analytes. According to some embodiments, a probe set comprises probes for specifically binding 200 or fewer analytes, 150 or fewer analytes, 125 or fewer analytes, 100 or fewer analytes, 75 or fewer analytes, 50 or fewer analytes, 40 or fewer analytes, 30 or fewer analytes, 25 or fewer analytes, 20 or fewer analytes, 15 or fewer analytes, or 10 or fewer analytes.

The set of probes included in the sensor devices of the present disclosure can further include probes for binding circulating cells, such as Circulating Tumor Cells (CTCs), circulating stem cells, and the like, and/or circulating nucleic acids, such as circulating DNA (e.g., circulating tumor DNA) and/or circulating RNA, as described above.

Magnetic sensor device

According to certain embodiments, the sensor device of the present disclosure is a magnetic sensor device. The magnetic sensor device of the present disclosure may comprise a magnetic sensor chip comprising a set of probes (e.g., attached to a surface of the magnetic sensor chip), including any set of probes described elsewhere herein. In certain aspects, the magnetic sensor chip comprises two or more magnetic sensors having capture probes attached to their surfaces (e.g., as an addressable array of capture probes). Each of the two or more magnetic sensors having capture probes attached to its surface may comprise capture probes for binding the same circulating analyte. Aspects of the magnetic sensor device and system will now be described.

Magnetic sensor

In certain aspects, the magnetic sensor devices of the present disclosure include one or more magnetic sensors. In some cases, the one or more magnetic sensors are configured to detect the presence of nearby magnetic labels without any direct physical contact between the magnetic sensors and the magnetic labels. In certain embodiments, the magnetic sensor is configured to detect the presence of an analyte of two or more circulating analytes that may be present in the blood sample. For example, the magnetic label may be directly or indirectly bound to the analyte, which in turn may be directly or indirectly bound to the magnetic sensor. If the bound magnetic label is located within the detection range of the magnetic sensor, the magnetic sensor may provide a signal indicating the presence of the bound magnetic label and thus the presence of the analyte.

In some cases, the magnetic sensor has a detection range from 1nm to 1000nm from the magnetic sensor surface, such as from 1nm to 800nm, including from 1nm to 500nm, e.g., from 1nm to 300nm, including from 1nm to 100nm, or from 1nm to 75nm, or from 1nm to 50nm, or from 1nm to 25nm, or from 1nm to 10nm from the magnetic sensor surface. In some cases, minimization of the detection range of the sensor may facilitate detection of specifically bound analytes while minimizing detectable signals from non-target analytes. "detection range" refers to the distance from the surface of the magnetic sensor where the presence of a magnetic label will induce a detectable signal in the magnetic sensor. In some cases, a magnetic label placed close enough to the magnetic sensor surface to be within the detection range of the magnetic sensor will induce a detectable signal in the magnetic sensor. In some cases, a magnetic marker placed at a distance from the surface of the magnetic sensor that is greater than the detection range of the magnetic sensor will not induce a detectable or non-negligible signal in the magnetic sensor. For example, a magnetic marker may have a magnetic flux proportional to 1/r3, where r is the distance between the magnetic sensor and the magnetic marker. Thus, only those magnetic labels located in close proximity (e.g., within the detection range of the magnetic sensor) will induce a detectable signal in the magnetic sensor.

As described, the probes of the probe set may be bound to a magnetic sensor surface. For example, cationic polymers such as Polyethyleneimine (PEI) can be used to non-specifically bind charged probes (e.g., antibodies, antigens, ligands, nucleic acids, etc.) to the sensor surface by physisorption (physical adsorption). Alternatively, covalent chemistry can be used to covalently bind the analyte-specific probe to the magnetic sensor surface using free amine or free thiol groups on the analyte-specific probe. For example, a N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) coupling system can be used to covalently bind analyte-specific probes to the magnetic sensor surface.

In certain embodiments, the magnetic sensor is configured to generate an electrical signal in response to a magnetic label in close proximity to the magnetic sensor surface. For example, the magnetic sensor may be configured to detect changes in the magnetic sensor resistance induced by changes in the local magnetic field. In some cases, as described above, binding of a magnetic label (e.g., a magnetic nanoparticle label) in close proximity to the magnetic sensor induces a detectable change in the resistance of the magnetic sensor. For example, magnetic marks in the vicinity of the magnetic sensor may be magnetized in the presence of an applied external magnetic field. The local magnetic field of the magnetized magnetic labels may induce a detectable change in the resistance of the underlying magnetic sensor. Thus, the presence of the magnetic label can be detected by detecting a change in the resistance of the magnetic sensor. In certain embodiments, the magnetic sensor is configured to detect a change in resistance of 1Ohm or less, such as 500mOhm or less, including 100mOhm or less, or 50mOhm or less, or 25mOhm or less, or 10mOhm or less, or 5mOhm or less, or 1mOhm or less. In certain embodiments, the change in resistance can be expressed in Parts Per Million (PPM) relative to the original sensor resistance, such as a change in resistance of 2PPM or greater, or 20PPM or greater, or 200PPM or greater, or 400PPM or greater, or 600PPM or greater, or 1000PPM or greater, or 2000PPM or greater, or 4000PPM or greater, or 6000PPM or greater, or 10,000PPM or greater, or 20,000PPM or greater, or 40,000PPM or greater, or 60,000PPM or greater, or 100,000PPM or greater, or 200,000PPM or greater.

The magnetic sensor may comprise a magneto-resistive element. Suitable magnetoresistive elements include, but are not limited to, spin valve magnetoresistive elements and Magnetic Tunnel Junction (MTJ) magnetoresistive elements.

In certain embodiments, the magnetic sensor elements are spin valve magnetoresistive elements. In some cases, the spin valve element is a multilayer structure including a first ferromagnetic layer, a nonmagnetic layer disposed on the first ferromagnetic layer, and a second ferromagnetic layer disposed on the nonmagnetic layer. The first ferromagnetic layer may be configured to have a magnetization vector fixed in a certain direction. In some cases, the first ferromagnetic layer is referred to as the "pinned layer". In certain embodiments, the spin valve element includes a pinned layer having a magnetization substantially parallel to the width of the magnetic sensor element. The second ferromagnetic layer may be configured such that its magnetization vector may rotate freely under an applied magnetic field. In some cases, the second ferromagnetic layer is referred to as the "free layer". In some cases, the first ferromagnetic layer (which may be referred to as the "pinned layer") is replaced by a synthetic or artificial antiferromagnet consisting of two antiparallel ferromagnetic layers separated by a nonmagnetic spacer: one ferromagnetic layer (which may be referred to as the "reference layer") is located below the nonmagnetic layer below the "free layer"; the other ferromagnetic layer (the other "pinned layer") is typically "pinned" by a natural antiferromagnet, such as IrMn, PtMn, FeMn, or NiO.

In some cases, the resistance of a spin valve element depends on the relative orientation of the magnetization vectors of the free and pinned layers. When the two magnetization vectors are parallel, the resistance is lowest; when the two magnetization vectors are anti-parallel, the resistance is highest. The relative change in resistance is known as the Magnetoresistive (MR) ratio. In certain embodiments, the spin valve element has an MR ratio of 1% to 20%, such as 3% to 15%, including 5% to 12%. In some cases, the MR ratio of the spin valve element is 10% or more in a small magnetic field such as 100 Oe. As described above, a change in the resistance of the spin valve element due to the presence of magnetic marks near the surface of the spin valve element can be detected.

In certain embodiments, the signal from the spin valve element due to the magnetic marks is dependent on the distance between the magnetic marks and the free layer of the spin valve element. In some cases, the voltage signal from the magnetic labels decreases as the distance from the center of the magnetic label to the midplane of the free layer increases. Therefore, in some cases, the free layer in the spin valve element is located at the surface of the spin valve element. Placing the free layer on the surface of the spin valve element may minimize the distance between the free layer and any bound magnetic labels, which may facilitate the detection of the magnetic labels.

In some embodiments, the spin valve element may include a passivation layer disposed on one or more spin valve element surfaces. In some cases, the passivation layer has a thickness of 60nm or less, e.g., 50nm or less, including 40nm or less, 30nm or less, 20nm or less, 10nm or less. For example, the passivation layer may have a thickness of 1nm to 10nm, such as 1nm to 5nm, including 1nm to 3 nm. In certain embodiments, the passivation layer comprises gold, tantalum, SiO₂、Si₃N₄Combinations thereof, and the like.

In certain embodiments, the magnetic sensor element is a Magnetic Tunnel Junction (MTJ) magnetoresistive element (also referred to herein as an MTJ element). In some cases, the MTJ element includes a multilayer structure including a first ferromagnetic layer, an insulating layer disposed on the first ferromagnetic layer, and a second ferromagnetic layer disposed on the insulating layer. The insulating layer may be a thin insulating tunnel barrier and may comprise alumina, MgO, or the like. In some cases, electron tunneling between the first and second ferromagnetic layers depends on the relative magnetization of the two ferromagnetic layers. For example, in some embodiments, the tunneling current is high when the magnetization vectors of the first and second ferromagnetic layers are parallel and the tunneling current is low when the magnetization vectors of the first and second ferromagnetic layers are anti-parallel. In some cases, the first ferromagnetic layer may be replaced by a synthetic or artificial antiferromagnet comprising two antiparallel ferromagnetic layers separated by a non-magnetic spacer: one ferromagnetic layer may be below the tunnel barrier; the other ferromagnetic layer may be "pinned" by a natural antiferromagnet, such as IrMn, PtMn, or FeMn.

In some cases, the MTJ element has a Magnetoresistance Ratio (MR) of 1% to 300%, e.g., 10% to 250%, including 25% to 200%. As described above, a change in resistance of the MTJ element due to the presence of the magnetic mark near the surface of the MTJ element can be detected. In some cases, the MTJ element has an MR of 50% or greater, or 75% or greater, or 100% or greater, or 125% or greater, or 150% or greater, or 175% or greater, or 200% or greater, or 225% or greater, or 250% or greater, or 275% or greater, or 200% or greater. For example, the MTJ element may have an MR of 225% or greater.

In some embodiments, the second ferromagnetic layer (e.g., the MTJ element layer at the MTJ element surface) includes two or more layers. For example, the second ferromagnetic layer may include a first layer, a second layer disposed on the first layer, and a third layer disposed on the second layer. In some cases, the first layer is a thin ferromagnetic layer (e.g., NiFe, CoFe, CoFeB, etc.). The thin metal layer may have a thickness of 6nm or less, such as 5nm or less, including 4nm or less, 3nm or less, 2nm or less, or 1nm or less, or 0.5nm or less. The second layer can include a conductive metal such as copper, aluminum, palladium alloys, palladium oxide, platinum alloys, platinum oxide, ruthenium alloys, ruthenium oxide, silver alloys, silver oxide, tin alloys, tin oxide, titanium alloys, titanium oxide, tantalum alloys, tantalum oxide, combinations thereof, and the like. The second layer can have a thickness of 2nm or less, e.g., 0.5nm or less, including 0.4nm or less, 0.3nm or less, 0.2nm or less, or 0.1nm or less. The third layer may comprise a ferromagnetic material such as, but not limited to, NiFe, CoFe, CoFeB, and the like. The third layer may have a thickness of 6nm or less, for example 5nm or less, including 4nm or less, 3nm or less, 2nm or less or 1nm or less or 0.5nm or less.

In some cases, the MTJ element is configured such that the distance between the bonded magnetic label and the top surface of the free layer ranges from 5nm to 1000nm or from 10nm to 800nm, such as from 20nm to 600nm, including from 40nm to 400nm, such as from 60nm to 300nm, including from 80nm to 250 nm.

The MTJ element may include a passivation layer disposed on one or more MTJ element surfaces. In some cases, the passivation layer has a thickness of 60nm or less, e.g., 50nm or less, including 40nm or less, 30nm or less, 20nm or less, 10nm or less. For example, the passivation layer may have a thickness of 1nm to 50nm, such as 1nm to 40nm, including 1nm to 30nm, or 1nm to 20 nm. In some cases, the passivation layer has a thickness of 30 nm. In some cases, the passivation layer comprises gold, tantalum alloy, tantalum oxide, aluminum alloy, aluminum oxide, SiO₂、Si₃N₄、ZrO₂Combinations thereof, and the like. In certain embodiments, a passivation layer having a thickness as described above facilitates maximizing the signal detected by magnetic labels specifically bound to the sensor surface while minimizing the signal from magnetic labels not specifically bound.

In some embodiments, the MTJ elements have a size ranging from 1 μm x 1 μm to 200 μm x 200 μm, including 1 μm x 200 μm or less, for example 200 μm x 1 μm or less, such as 150 μm x 10 μm or less, or 120 μm x 5 μm or less, or 120 μm x 0.8 μm or less, or 0.8 μm x 120 μm or less, or 100 μm x 0.7 μm or less, or 100 μm x 0.6 μm or less, or 100 μm x 0.5 μm or less, or 10 μm x 0.6 μm or less, or 10 μm x 0.5 μm or less. In some cases, the MTJ element has a size of 120 μm x 0.8 μm or less, e.g., 2.0 μm x 0.8.8 μm.

U.S. patent No. 9,863,939, the disclosure of which is incorporated herein by reference in its entirety for all purposes, further describes a Magnetic Tunnel Junction (MTJ) detector. The detector is further described in U.S. patent No. 7,906,345, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

In some cases, the magnetic sensor is a multilayer thin film structure. The sensor may include alternating layers of ferromagnetic and non-magnetic materials. Ferromagnetic materials may include, but are not limited to, permalloy (NiFe), iron cobalt (FeCo), nickel iron cobalt (NiFeCo), CoFeB, combinations thereof, and the like. In some cases, the non-magnetic material is a noble metal, such as, but not limited to, Cu, Au, Ag, and the like. In certain embodiments, the ferromagnetic layer has a thickness of 1nm to 10nm, such as 2nm to 8nm, including 3nm to 4 nm. In some cases, the non-magnetic layer has a thickness of 0.2nm to 5nm, such as 1nm to 3nm, including 1.5nm to 2.5nm, or 1.8nm to 2.2 nm.

In certain embodiments, the magnetic sensor device may be configured to include one or more magnetic sensing regions. The magnetic sensing region may correspond to a device region where a magnetic sensor array (e.g., a biosensor array) is located. For example, the magnetic sensing region may be a region of the device surface that is exposed to the blood sample during use and that has an array of magnetic sensors as described above.

The magnetic sensing region may be configured to include a fluid reservoir. The fluid reservoir may be any of a variety of configurations, wherein the fluid reservoir is configured to hold a blood sample in contact with the magnetic sensor array. Accordingly, configurations of the fluid reservoir may include, but are not limited to: cylindrical hole configurations, square hole configurations, rectangular hole configurations, round bottom hole configurations, and the like. For example, the fluid reservoirs may include walls that separate one fluid reservoir from an adjacent fluid reservoir. The walls may be substantially perpendicular relative to the surface of the reservoir plate. In some cases, the walls of each fluid reservoir define a spatial volume that can receive a sample volume that is equal to or less than the spatial volume defined by the fluid reservoir.

In certain embodiments, the fluid reservoir has a volume of 10mL or less, or 5mL or less, or 3mL or less, or 1mL or less, such as 500 μ L or less, including 100 μ L or less, such as 50 μ L or less, or 25 μ L or less, or 10 μ L or less, which is sufficient to contain the same or less volume of sample.

Magnetic sensor system

Aspects of the present disclosure include a magnetic sensor system. In some embodiments, a magnetic sensor system includes a magnetic sensor device and a magnetic field source. The magnetic sensor device includes a support having one or more magnetic sensor arrays (e.g., biosensor arrays) positioned thereon. The system may be configured to obtain signals from one or more magnetic sensor arrays that indicate whether an analyte of the circulating analyte is present in one or more respective blood samples.

In certain embodiments, the system includes a magnetic field source. The magnetic field source may be configured to apply a magnetic field to a magnetic sensor device (e.g., a magnetic sensor array) sufficient to generate a DC and/or AC field in an assay sensing region (e.g., in a region where the magnetic sensor array is located during signal acquisition). In some cases, the magnetic field source is configured to generate a magnetic field having a magnetic field strength of 1Oe or greater, or 5Oe or greater, or 10Oe or greater, or 20Oe or greater, or 30Oe or greater, or 40Oe or greater, or 50Oe or greater, or 60Oe or greater, or 70Oe or greater, or 80Oe or greater, or 90Oe or greater, or 100Oe or greater.

The magnetic field source may be positioned such that, when the magnetic sensor device is used, a magnetic field is generated in the region where the magnetic sensor array is positioned. In some cases, the magnetic field source is configured to generate a uniform, controllable magnetic field around the fluid reservoir set on the reservoir plate where the assay is being performed. The magnetic field source may comprise one or more, such as two or more, three or more, four or more magnetic field generating members. In some cases, the magnetic field source may include one or more electromagnets, such as coil electromagnets. The coil electromagnet may comprise a wound coil. For example, the magnetic field source may comprise two electromagnets arranged in a helmholtz coil geometry.

Embodiments of the system further include a computer-based system. The system may be configured to qualitatively and/or quantitatively assess binding interactions as described above. "computer-based system" refers to hardware, software, and data storage components for analyzing signals from magnetic sensors. The hardware of the computer-based system may include a Central Processing Unit (CPU), input, output, and data storage components. Any of a variety of computer-based systems are suitable for use with the subject system. The data storage component may include any computer readable medium for recording signals from the magnetic sensor array or an accessible storage component that may store signals from the magnetic sensor array.

"recording" data, programs, or other information on a computer-readable medium refers to the process of storing the information using any such method known in the art. Any convenient data storage structure may be selected, depending on the method used to access the stored information. A variety of data processor programs and formats are available for storage, such as word processing text files, database formats, and the like.

In certain embodiments, the system includes an activation and signal processing unit. The activation and signal processing unit may be configured to be operatively coupled to the magnetic sensor device. In some cases, the activation and signal processing unit is electrically coupled to the magnetic sensor device. The activation and signal processing units may be electrically coupled such as to provide bi-directional communication to and from the magnetic sensor device. For example, the activation and signal processing unit may be configured to provide power, activation signals, etc. to components of the magnetic sensor device (such as, but not limited to, a magnetic sensor array). Thus, the activation and signal processing unit may comprise an activation signal generator. The activation signal generator may be configured to provide power, activation signals, etc. to components of the analyte detection device, such as, but not limited to, a magnetic sensor array. In some cases, the activation and signal processing unit is configured to apply a voltage on the magnetic sensor array in a range of 1mV to 10V, such as 100mV to 5V, including 200mV to 1V, for example 300mV to 500 mV. In some cases, the activation and signal processing unit is configured to apply a voltage of 500mV across the magnetic sensor array.

Furthermore, the activation and signal processing unit may be configured to receive signals from the magnetic sensor device, such as signals from a magnetic sensor array of the magnetic sensor device. The signal from the magnetic sensor of the magnetic sensor device may be used to detect the presence of two or more analytes of the circulating analyte in the blood sample. In some cases, the activation and signal processing unit may include a processor configured to output an analyte detection result in response to receiving a signal from the magnetic sensor array. Thus, the processor of the activation and signal processing unit may be configured to receive the signal from the magnetic sensor device, process the signal according to a predetermined algorithm, obtain a result related to the presence of the one or more analytes in the sample, and output the result to a user in a human readable or audible format. Models that may be used, for example, to assess the risk of unsure lung nodules being malignant include those described herein in the experiments.

"processor" refers to any combination of hardware and/or software that will perform one or more programmed functions. For example, any of the processors herein may be a programmable digital microprocessor, such as available in the form of an electronic controller, a mainframe, a server, or a personal computer (e.g., desktop or portable). Where the processor is programmable, suitable programming may be transmitted to the processor from a remote location or pre-stored in a computer program product, such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state based device. For example, a magnetic medium, an optical disk, or a solid state storage device may carry the programming and may be read by a suitable reader in communication with the processor.

In some cases, the subject systems are configured to modulate a current (e.g., a sense current) applied to the magnetic sensor array. The subject systems may also be configured to modulate a magnetic field generated by a magnetic field source. Modulating the sensing current and the magnetic field may facilitate minimization of signal noise and, thus, maximization of signal-to-noise ratio. Other aspects of modulating the sensing current and magnetic field are described in more detail in U.S. application No. 12/759,584 entitled "method and Devices for Detecting the Presence of an Analyte in a Sample," filed on 13.4.2010, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

Embodiments of the subject system can also include the following components: (a) a wired or wireless communication module configured to transmit information between the system and one or more users, such as through a user computer as described below; and (b) a processor for performing one or more tasks relating to qualitative and/or quantitative analysis of the signal from the magnetic sensor. In certain embodiments, a computer program product is provided that includes a computer usable medium having control logic (e.g., a computer software program, including program code) stored therein. When executed by a processor of a computer, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. The implementation of a hardware state machine to perform the functions described herein may be accomplished using any convenient method and technique.

In addition to the magnetic sensor device and the activation and signal processing unit, the system may comprise many additional components, such as, but not limited to: data output devices such as monitors, speakers, etc.; data input devices such as interface ports, buttons, switches, keyboards, etc.; fluid handling components, such as microfluidic components; a power source; a power amplifier; wired or wireless communication means, etc. For example, the system may include fluid handling components, such as microfluidic fluid handling components. In certain embodiments, the fluid handling components of the microfluidic are configured to deliver fluid to the fluid reservoirs of the reservoir plate. In some cases, the fluid includes one or more of: an assay composition, a blood sample, one or more detection reagents (e.g., detection antibodies, magnetic labels, etc.). In some cases, the fluid handling components of the microfluidic are configured to deliver a small volume of fluid, e.g., 1mL or less, e.g., 500 μ L or less, including 100 μ L or less, e.g., 50 μ L or less, or 25 μ L or less, or 10 μ L or less.

In certain embodiments, the system is a high sensitivity analyte detector. By "high sensitivity" is meant that the system is configured to detect an analyte in a sample, wherein the concentration of the analyte in the sample is low. In some cases, the system is configured to produce a detectable signal indicative of the presence of an analyte of interest in a sample at a concentration of 1 μ M or less, such as 100nM or less, or 10nM or less, or 1nM or less, including 100pM or less, or 10pM or less, or 1pM or less, e.g., 500fM or less, or 250fM or less, or 100fM or less, or 50fM or less, or 25fM or less, such as 10fM or less, or 5fM or less, or 1fM or less. In other words, the system may be configured to have a detection limit, e.g., a lower limit of quantitation (LLOQ), of 1 μ Μ or less, such as 100nM or less, or 10nM or less, or 1nM or less, including 100pM or less, or 10pM or less, or 1pM or less, e.g., 500fM or less, or 250fM or less, or 100fM or less, or 50fM or less, or 25fM or less, such as 10fM or less, or 5fM or less, or 1fM or less.

In certain embodiments, the system includes a display. The display may be configured to provide a visual indication of the analyte detection results obtained from the activation and signal processing unit described above. The display may be configured to display the qualitative analyte detection result. For example, the qualitative display may be configured to display to a user a qualitative indicator that the sample includes or does not include a particular target analyte. In some embodiments, the display may be configured to display an analyte detection result, wherein the analyte detection result is a quantitative result, such as a quantitative measurement of the concentration of the analyte in the sample. For example, in embodiments where the system is configured to output quantitative analyte detection results, the system may include a display configured to display the quantitative analyte detection results.

The magnetic sensor device optionally includes a programmable memory that can be programmed with relevant information before and during use of the magnetic sensor device, such as: calibration data for each individual sensor; a record of how biochips were prepared with surface-functionalised molecules prior to assay; recording all completed assay steps; a record of which sample was measured; recording of measurement results, etc.

Additional aspects of the magnetic sensor system are described in more detail in U.S. patent nos. 9,151,763 and 9,164,100 and 9,528,995, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Reagent kit

Kits useful, for example, in practicing one or more embodiments of the methods of the disclosure are also provided.

In some embodiments, a kit of the present disclosure includes a set of probes comprising probes for specifically binding two or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more) of the following in any desired combination: carcinoembryonic antigen (CEA), C-X-C motif chemokine ligand 4(CXCL 4-also known as platelet factor 4 (or PF4)), C-X-C motif chemokine ligand 7(CXCL 7-also known as neutrophil activating protein 2 (or NAP2)), C-X-C motif chemokine ligand 10(CXCL 10-also known as interferon gamma-inducing protein 10 (or IP10)), Epidermal Growth Factor Receptor (EGFR), pro-surfactant protein B (pro-SFTPB), metalloproteinase 1 tissue inhibitor (TIMP1), anti-angiopoietin-like protein 3 antibody (anti-ANGPTL 3), anti-14-3-protein theta antibody (anti-YAQWH), anti-laminin alpha 1 antibody (anti-LRMR 1), human epididymin 4(HE4), pro-gradin 2(AGR2), chromogranin A (CHGA), glycoprotein leucine rich alpha-632-1 (G1), Anti-annexin 1 antibody (anti-ANXA 1), anti-ubiquitin 1 antibody (anti-UBQLN 1), interleukin 6(IL6), interleukin 8(IL8), C-X-C motif chemokine ligand 2(CXCL2), C-X-C motif chemokine ligand 12(CXCL12), C-X-C motif chemokine ligand 14(CXCL14), defensin, β 1(DEFB1), fibroblast growth factor 2(FGF2), cluster of differentiation 97(CD97), pre-platelet basic protein (PPBP), Procalcitonin (PCT), advanced glycosylation end product Receptor (RAGE), ges 100 calcium binding protein a4(S100a4), S100 calcium binding protein a8(S100a8), and Osteopontin (OPN).

In certain embodiments, a kit of the present disclosure includes a set of probes comprising probes for specifically binding one, two, three, or each of CEA, CXCL4, CXCL7, and CXCL10 in any desired combination. According to some embodiments, such probe sets further comprise probes for specifically binding one, two, or each of EGFR, pro-SFTPB, and TIMP1 in any desired combination. In certain embodiments, such a probe set further comprises one or more probes for specifically binding to one or any combination of additional analytes selected from the group consisting of anti-ANGPTL 3, anti-ywaq, anti-LAMR 1, HE4, AGR2, CHGA, LRG1, anti-ANXA 1, anti-UBQLN 1, IL6, IL8, CXCL2, CXCL12, CXCL14, DEFB1, FGF2, CD97, PPBP, PCT, RAGE, S100a4, S100a8, and OPN.

In some embodiments, when a kit of the present disclosure includes a set of probes as described above, the set of probes can be a set of capture probes provided as an addressable probe array. For example, a kit may include a set of probes provided as any of the devices and systems of the present disclosure.

The subject kits can vary, and can include various devices (e.g., any of the sensor devices (e.g., magnetic sensor devices) of the present disclosure) and reagents. Reagents and devices include those mentioned herein with respect to magnetic sensor devices or components thereof (such as magnetic sensor arrays), magnetic labels, one or more probe sets, detection reagents, buffers, and the like. The reagents may be provided in separate containers so that the reagents, magnetic labels, probes, etc. may be used separately as desired. Alternatively, one or more reagents, magnetic labels, probes, etc. may be provided in the same container, such that the one or more reagents, magnetic labels, capture probes, etc. are provided to the user in a pre-combination.

In certain embodiments, the kit comprises a magnetic sensor device and a magnetic label as described above. For example, the magnetic labels may be magnetic nanoparticles, as described above. In some cases, a kit includes at least reagents useful in a method (e.g., as described above); and a computer readable medium having a computer program stored thereon, wherein the computer program when loaded into a computer operates the computer to qualitatively and/or quantitatively determine a target binding interaction from a signal (e.g. a real-time signal) obtained from a sensor (e.g. a magnetic sensor); and a physical substrate having an address from which the computer program is obtained.

The kits of the present disclosure may further comprise instructions. In some embodiments, the instructions comprise instructions for contacting a blood sample from the subject with the probe set to generate a circulating analyte profile of the subject. The subject kits can include instructions for performing any of the methods of the present disclosure. In some embodiments, the instructions comprise instructions for contacting a blood sample from a subject from a population at high risk for lung cancer with a probe set to generate a circulating analyte profile of the subject. According to certain embodiments, the instructions include instructions for contacting a blood sample from a subject that is a previous or current smoker with the probe set to generate a circulating analyte profile of the subject. In some embodiments, the instructions include instructions for contacting a blood sample from a subject having a lung nodule (e.g., an indeterminate lung nodule (e.g., detected by Low Dose Computed Tomography (LDCT)) with a probe set to generate a circulating analyte profile of the subject.

The instructions can be present in the subject kits in a variety of forms, one or more of which can be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., one or more sheets of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Another way is a computer readable medium, such as a CD, DVD, blu, a computer readable storage device (e.g., flash drive), etc., on which information is recorded. Another way that may exist is a website address that may be used over the internet to access information on a deleted site. Any convenient means may be present in the kit.

As can be appreciated from the disclosure provided above, the present disclosure has a wide variety of applications. Thus, the following examples are provided for illustrative purposes and are not intended to be construed as limiting the invention in any way. One skilled in the art will readily recognize that various non-critical parameters may be altered or modified to produce substantially similar results. Thus, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.

The following examples are provided by way of illustration and not limitation.

Experiment of

Introduction to

Examples herein relate to protein biomarkers that can be measured in human blood as a feature associated with malignant lung nodules of previous smokers. This information can be used alone or in combination with clinical parameters to calculate the risk that a previous smoker will see in their LDCT scan that the nodule is a malignant lung nodule. Protein biomarkers can be measured using magnetic nanosensor technology developed by MagArray, which overcomes the expense and low throughput of mass spectrometry blood protein measurement technology, and overcomes the detection limit of ELISA-based tests. Since the magnetic nanosensor can multiplex up to 80 individual detectors at a time to achieve high throughput, a single patient blood can be used to simultaneously measure pulmonary nodule-associated protein biomarkers. The measured protein biomarker levels are then combined in a model that provides a risk of malignancy for the lung nodules. The resulting model is robust and has clinical utility for large patient populations for lung cancer assessment. The model may also be useful for screening of high-risk populations of lung cancer and therapy prediction and monitoring of lung cancer patients after diagnosis, either alone or in combination with standard clinical assessments and/or other cancer biomarkers.

The principles of magnetic nanosensor chips and Giant Magnetoresistive (GMR) sensors and their use in measuring biomarkers have been described. Using this technique configured in an MR-813 instrumentation system, a multiplexed reagent set was developed to measure 3 previously reported and 5 recently identified human proteins thought to be associated with lung cancer and likely to be found in the circulation. Previously reported proteins are Epidermal Growth Factor Receptor (EGFR), pro-surface active protein B (pro-SFTPB) and tissue inhibitor of metalloproteinase 1 (TIMP 1). The 5 proteins recently developed and tested were carcinoembryonic antigen (CEA), human epididymis protein 4(HE4), CXCL7 (also known as neutrophil activating protein 2(NAP2)), receptor for advanced glycosylation end products (RAGE), and S100 calcium binding protein a8(S100a 8).

To evaluate the clinical utility of those proteins, a group of over 1100 human plasma samples obtained from a cohort of 8 geographically diverse centers including Stanford University Clinic (Stanford University clinical), Pacific Medical Center, Calif. and Palo Alto vehicles afficians department of Affairs Hospital, San Francisco vehicles afficians Medical Center, University of Pennsyly and Lung Cancer biological sample Resource Network (Lung Cancer research Network) (southern Carolina University of South Carolina), Japan University of Tenn and Vignan University of City University and University of City (University of City). A subset of 405 samples was selected from 1100 samples with the clinical data required to calculate the pre-test probability of malignant lung nodules in a subject using a Mayo clinical risk assessment model ("Mayo model"). 405 samples were from 3 cohorts, balanced for benign and malignant lung nodules, and included current and former smokers, as shown in tables 1 and 2. Previous smoking was defined as no smoking at enrollment, while current smoking was defined as smoking until study enrollment. The entire cohort of previous and current smokers is called a "once smoker".

The overall disease prevalence for the 405 sample group was 48%, close to the target prevalence 50%, with the aim of ensuring a balanced weight of biomarker levels from benign and malignant disease states. Benign diagnosis is defined as two years of nodule stability or nodule regression, and malignant diagnosis is based on pathological reports after resection or biopsy.

Table 1 sample cohort containing 405 subjects from which training and testing groups were selected

TABLE 2405 smoking history for cohorts in subject groups

Overall disease prevalence: 48 percent; total current smokers: 41 percent

Levels of 8 proteins were obtained in 405 samples using an MR813 multiplex assay panel. The samples were run in two studies, 150 samples in the first study and 255 samples in the second study. Samples from the first study were selected to cover the range of pre-test probabilities for malignancy as determined using the Mayo clinical model. The second study contained the remaining 255 samples. In both studies, samples were run in random order by a technician blinded to the clinical information of the samples. Data collection and analysis was performed according to rational biomarker study design principles.

Statistical analysis of assay results began by evaluating the assay Coefficient of Variation (CV) between replicate measurements as an indicator of test reproducibility. The intra-run variability of the assay data is 10% or less, and the overall variability of the in-orbit running controls run with each set of assays is typically less than 15%. Assay replicates showing over 30% CV were repeated. 12 samples were repeated in the first study and 15 samples in the second study, with a total repetition of less than 7% due to the unexpectedly large% CV.

The assay data was analyzed as raw GMR Parts Per Million (PPM) signal and also normalized to the ratio of the sample signal to the run control signal obtained every 3 samples.

Several pre-specified analyses were performed to determine the diagnostic accuracy of the panel to distinguish malignant from benign disease, using logistic regression modeling techniques and cross-validation to generate accuracy, sensitivity, specificity, NPV and PPV indices. The cross-validation technique allows estimation of how much the result will change if the model is applied to other possible queues. Such techniques reduce false discovery rates when defining model components and help ensure that coefficients of the logistic regression algorithm are not overly specific only to the training set.

Example 1 prediction of Lung cancer risk in previous smokers

K2EDTA plasma from a cohort of 405 patients with CT scans showing lung nodules was tested as a retrospective design of case controls collected from three medical centers. Cases were diagnosed as lung cancer from their pathology reports, and controls were negative/normal pathology or subjects with 2 years of stable nodules. Magnetic nanosensors and sandwich immunoassays developed by Stanford and MagArray were used to measure the protein biomarkers Epidermal Growth Factor Receptor (EGFR), pro-surface active protein B (pro-SFTPB), tissue inhibitor of metalloproteinase 1 (TIMP1), carcinoembryonic antigen (CEA), human epididymin 4(HE4), neutrophil activator protein 2(NAP2), receptor for advanced glycosylation end products (RAGE), and S100 calcium binding protein a8(S100a8) in 20 μ L of subject plasma. The levels of protein biomarkers are then analyzed in a sub-cohort of those patients stratified by diagnosis and smoking status to understand the relationship of the biomarkers to diagnosis and smoking status using ANOVA and logistic regression.

The average levels of the eight biomarkers in subjects stratified by smoking status are shown in figure 1. A significant difference in level between benign and malignant diagnosis is represented by a p-value of less than or equal to 0.05. P values greater than 0.05 are not significant and are indicated by "ns".

209 benign diagnostic samples and 196 malignant diagnostic samples comprising 405 sample sets were each randomly divided into 2/3 and 1/3 subgroups for models where the training samples were different from the samples used to test the model. This is done to reduce the likelihood of overly optimistic testing performance of the model that may occur when testing the model on the same data set used to train the model.

Using biomarker levels of previous smokers plus subject clinical data, different variable combinations in the training set using 1/3 sample subgroups were evaluated using a generalized linear modeling process. Models capable of distinguishing between malignant and benign diseases within the test sub-group (2/3 samples) were identified with an accuracy of 79%. The model named #217-3092 consisted of the biomarkers CEA, EGFR, NAP2, ProSB, and TIMP1, as well as clinical factors, which were subject age, nodule size, subject gender, and nodule margin (with or without burrs). The Receiver Operating Characteristic (ROC) curve for model 217 and 3092 is shown in fig. 2, where the area under the curve (AUC) is 0.86, compared to 0.79 for the Mayo model for the same sample.

Exploring model 217_3092 in 93 previous smoking subjects with a pre-malignant probability of 0.05 to 0.65 (intermediate risk range defined in published guidelines) for the Mayo model indicates that the biomarker and clinical factor combination provides an improved AUC compared to the Mayo model (figure 3). The model 217_3092 performance is summarized in table 3, where the model performance index estimates a Negative Predictive Value (NPV) of 91%, disease prevalence of 0.25, and an appreciable Positive Predictive Value (PPV) of 51%. This disease level is based on a study by community pneumologists, where 1/4 the person seeking their care is diagnosed with malignant lung cancer. Other important measures of clinical performance indicate that the model 217_3092 shows excellent sensitivity (76%) and specificity (82%) at a cut-off of 0.485. Finally, the ability of the model to accurately classify an intermediate-risk subject in the Mayo model is taken into account by using a net weight classification metric that determines the net number of correctly classified subjects after subtraction of model misdiagnosed subjects. The reclassification percentage of malignant subjects in the Mayo model intermediate risk category (IDm _ RI) was 6%, while the number of reclassified intermediate risk benign subjects (IDb _ RI) was 48%, resulting in an overall net reclassification index (ID _ NetRI) of 55%.

Table 1 summary of model 217_3092 test group performance in previous smokers

Example 2-prediction of Lung cancer risk in Current smokers

The combination of biomarkers of the evaluation model 217_3092 plus clinical factors (biomarkers: CEA, EGFR, NAP2, ProSB, and TIMP1, and clinical factors: subject age, nodule size, subject gender, and nodule margin (with or without burrs)) in this example predicted the discriminatory ability of malignancy in the current subset of smokers using a generalized logistic regression prediction approach. The training group consisted of the 2/3 cohort, while the test group was the remaining 1/3 cohort of current smokers in the 405 sample group.

The overall performance of the model is summarized in table 4, where an accuracy of 69% and a sensitivity of 61% and a specificity of 77% use a cut-off value of 0.508.

More significant is the re-classification performance, where 41% of net current smokers labeled as intermediate risk (ID _ NetRI) are correctly referred to as benign or malignant. This is the sum of 6% of net malignant intermediate risk current smokers (IDM _ RI) and 35% of net benign intermediate risk current smokers (IDb _ RI) in the Mayo model.

Table 2 summary of model 217_3092 test performance in current smokers

Considering the entire test cohort of current smokers, the model performance measured by the ROC curve AUC was 0.75 compared to 0.72 for the Mayo model (fig. 4).

Evaluation of a medium risk current smoker subject (n-37) on the Mayo risk score using model 217_3092 showed improved performance compared to the Mayo model itself (fig. 5). Compared with the AUC of the Mayo model of 0.69, the AUC of the model is 0.76.

Accordingly, the foregoing merely illustrates the principles of the disclosure. It will thus be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Moreover, all examples and conditional language recited herein are principally intended expressly to aid the reader in understanding the principles of the invention and the concepts contributed by the inventor to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.