Biosensor for diagnosing thyroid gland dysfunction
阅读说明：本技术 用于诊断甲状腺功能异常的生物传感器 (Biosensor for diagnosing thyroid gland dysfunction ) 是由 S·斯里瓦斯塔瓦 K·普尼亚尼 M·塔克瓦 于 2019-07-12 设计创作，主要内容包括：本发明涉及一种生物传感器及其用于定量游离甲状腺激素以评价甲状腺功能的应用。本文还公开了用于诊断甲状腺相关疾病的方法和工具。(The invention relates to a biosensor and application thereof in quantifying free thyroid hormone to evaluate thyroid function. Also disclosed herein are methods and tools for diagnosing thyroid-related diseases.)
1. A sensor for quantifying thyroid hormone, the sensor comprising:
a. a substrate, a first electrode and a second electrode,
b. at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4, and
c. optionally, an anti-rT 3 antibody,
wherein the at least 2 iodothyronine deiodinases and the anti-rT 3 antibody are immobilized on the surface of the substrate.
2. The sensor of any one of the preceding claims, wherein the substrate comprises a plurality of surfaces for immobilizing the at least 2 iodothyronine deiodinases, such as a first surface of the substrate and a second surface of the substrate.
3. The sensor of any one of the preceding claims, wherein the at least 2 methyl iodide adeodinases comprise a first methyl iodide adeodinase selected from EC1.21.99.3 and/or EC1.21.99.4 immobilized on the first surface of the substrate and a second methyl iodide adeodinase selected from EC1.21.99.3 and/or EC1.21.99.4 immobilized on the second surface of the substrate.
4. The sensor of any one of the preceding claims, wherein the first iodothyronine deiodinase is selected from EC1.21.99.4 and the second iodothyronine deiodinase is selected from EC 1.21.99.3.
5. The sensor of any one of the preceding claims, wherein the first and second iodothyronine deiodinases are each independently selected from EC 1.21.99.4.
6. The sensor of any one of the preceding claims, wherein the first and second iodothyronine deiodinases are different enzymes selected from EC 1.21.99.4.
7. The sensor of any one of the preceding claims, wherein the anti-rT 3 antibody is immobilized on the third surface of the substrate.
8. The sensor of any one of the preceding claims, wherein the first iodothyronine deiodinase is a type 1 and/or a type 2 iodothyronine deiodinase.
9. The sensor of any one of the preceding claims, wherein the second iodothyronine deiodinase is a type 1 iodothyronine deiodinase, a type 2 iodothyronine deiodinase and/or a type 3 iodothyronine deiodinase.
10. The sensor of any one of the preceding claims, wherein the first iodothyronine deiodinase is type 1 iodothyronine deiodinase and the second iodothyronine deiodinase is type 2 iodothyronine deiodinase.
11. The sensor of any one of the preceding claims, wherein the first iodothyronine deiodinase is type 1 iodothyronine deiodinase and the second iodothyronine deiodinase is type 3 iodothyronine deiodinase.
12. The sensor of any one of the preceding claims, wherein the first iodothyronine deiodinase is type 2 iodothyronine deiodinase and the second iodothyronine deiodinase is type 3 iodothyronine deiodinase.
13. The sensor of any one of the preceding claims, wherein the thyroid hormone is selected from free T4, free T3, trans T3(rT3), and combinations thereof.
14. A sensor according to any preceding claim, wherein the substrate is one or more electrodes and/or chips.
15. The sensor of any preceding claim, wherein the substrate comprises at least one electrode, for example at least 2 electrodes, for example at least 3 electrodes.
16. The sensor of any preceding claim, wherein the substrate comprises at least one chip, for example at least 2 chips, for example at least 3 chips.
17. The sensor according to any one of the preceding claims, wherein the substrate comprises or consists of three electrodes, and wherein the first surface is a surface of a first electrode, the second surface is a surface of a second electrode, and the third surface is a surface of a third electrode.
18. The sensor of any one of the preceding claims, wherein the substrate comprises or consists of three chips, and wherein the first surface is a surface of a first chip, the second surface is a surface of a second chip, and the third surface is a surface of a third chip.
19. The sensor of any preceding claim, wherein the electrodes are made of carbon, gold or platinum.
20. The sensor of any preceding claim, wherein the electrodes are screen printed electrodes.
21. The sensor of any preceding claim, wherein the chip is a glass chip.
22. The sensor of any preceding claim, wherein the first, second and/or third surface of the substrate is a modified surface.
23. The sensor of any one of the preceding claims, wherein the modified surface is a surface comprising a plurality of nano-and/or micro-sized topographical features.
24. The sensor of any one of the preceding claims, wherein the plurality of nano-and/or micro-sized topographical features are selected from the group consisting of: microparticles, nanoparticles, microwires, nanowires, microtubes, nanotubes, nanorods, and combinations thereof.
25. The sensor of any one of the preceding claims, wherein the surface is assembled by sintering, producing the plurality of nano-and/or micro-sized topographical features on the surface of the substrate.
26. The sensor of any one of the preceding claims, wherein the plurality of nano-and/or micro-sized topographical features are created on the surface of the substrate by surface etching.
27. The sensor of any one of the preceding claims, wherein the plurality of nano-and/or micro-sized topographical features are created on the surface of the substrate by particle deposition.
28. The sensor of any preceding claim, wherein the modified surface is a gold-coated surface.
29. The sensor of any one of the preceding claims, wherein the modified surface is a surface modified with nanoparticles selected from gold, silver, copper oxide, graphene, iron oxide, and combinations thereof.
30. The sensor of any one of the preceding claims, wherein the modified surface is a gold-coated surface, and wherein the surface is further modified with nanoparticles selected from gold, silver, copper oxide, graphene, iron oxide, and combinations thereof.
31. The sensor of any one of the preceding claims, wherein the at least 2 iodothyronine deiodinases are immobilized on the surface by a linker comprising a nanoparticle.
32. The sensor of any one of the preceding claims, wherein the at least 2 iodothyronine deiodinases and/or the anti-rT 3 antibody are immobilized on the substrate by a linker comprising a nickel-histidine (Ni-His) covalent coordination bond.
33. The sensor of any one of the preceding claims, wherein the at least 2 iodothyronine deiodinases and/or the anti-rT 3 antibody are immobilized on the substrate by a linker comprising:
a. cysteamine bound to a substrate, and
b. nanoparticles bound to the cysteamine and the iodothyronine deiodinase, optionally via one or more additional cysteamines.
34. The sensor of any one of the preceding claims, wherein the at least 2 iodothyronine deiodinases and/or the anti-rT 3 antibody is mammalian.
35. The sensor of any one of the preceding claims, wherein the at least 2 iodothyronine deiodinases and/or the anti-rT 3 antibody are human.
36. The sensor according to any one of the preceding claims, wherein the at least 2 iodothyronine deiodinases and/or the anti-rT 3 antibody are recombinantly produced, e.g. by means of cell-free expression.
37. The sensor of any one of the preceding claims, wherein the at least 2 iodothyronine deiodinases and/or the anti-rT 3 antibody are each individually conjugated to a further moiety.
38. The sensor of any one of the preceding claims, wherein the further moiety is a peptide, such as a peptide tag, such as a histidine tag.
39. A sensor according to any preceding claim, wherein the further portion is a marker.
40. The sensor of any one of the preceding claims, wherein the sensor comprises the at least 2 iodothyronine deiodinases each between 10 and 100 IU.
41. The sensor of any one of the preceding claims, wherein the sensor comprises between 10 and 100IU of methyladenine iodide deiodinase type 1 and type 2.
42. The sensor of any one of the preceding claims, wherein the sensor comprises between 10 and 100IU of methyladenine iodide deiodinases type 2 and 3.
43. The sensor of any one of the preceding claims, wherein the sensor comprises between 10 and 100IU of methyladenine iodide deiodinases type 1 and 3.
44. A sensor according to any preceding claim, configured such that the substrate can be connected to a bench-top, a handheld electrochemical workstation, a surface plasmon resonance detector or a measurement circuit.
45. A sensor according to any preceding claim, wherein the substrate comprises at least three electrodes, and wherein the electrodes are configured such that they can be connected to an electrochemical workstation.
46. The sensor of any preceding claim, wherein the substrate comprises at least 3 chips, and wherein the chips are configured such that they can be connected to a surface plasmon resonance detector.
47. The sensor of any one of the preceding claims, wherein the sensor is configured for quantifying thyroid hormone.
48. A method of quantifying thyroid hormone in a sample, the method comprising the steps of:
a. providing a sample comprising or suspected of comprising a thyroid hormone,
b. contacting the sensor according to any one of the preceding claims with the sample,
c. measuring a signal from the sensor, an
d. Using the signal to determine the level and/or concentration of one or more thyroid hormones in the sample to detect the thyroid hormone.
49. A method of diagnosing a thyroid-related disorder in a subject, comprising the steps of:
a. providing a sample obtained from a subject,
b. determining the level and/or concentration of the thyroid hormone in the sample using the method of claim 48,
thereby diagnosing one or more thyroid-related conditions.
50. A method of monitoring a thyroid-related disorder in a subject, comprising the steps of:
a. administering to the subject a compound that stimulates the thyroid gland,
b. collecting a sample from the subject after performing step a,
c. determining the level and/or concentration of the thyroid hormone in the sample using the method of claim 48,
thereby monitoring the thyroid-related disorder.
51. The method of claim 50, wherein steps a.through c.are performed more than once.
52. Use of a sensor according to any one of claims 1 to 47 for quantifying thyroid hormone.
53. Use according to claim 52, wherein the quantification of thyroid hormone is carried out according to the method of claim 48.
54. The method of any one of claims 48 to 50, further comprising the step of: using the concentration of thyroid hormone in the sample to calculate the concentration of the thyroid hormone in vivo.
55. The method of any one of claims 48 to 54, wherein the level and/or concentration of the thyroid hormone in the sample is determined from the reaction kinetics between the thyroid hormone and the iodinated methionine deiodinase.
56. A method according to any one of claims 48 to 55, wherein the concentration of the thyroid hormone is determined after the subject has received a medicament comprising a thyroid stimulating compound.
57. The method of any one of claims 50 to 56, wherein the time after the subject has received the drug is from 5 minutes to 48 hours.
58. The method of any one of claims 49-57, further comprising the steps of: comparing the level and/or concentration of the thyroid hormone in the sample to a threshold interval to diagnose a thyroid-related disorder in the subject, wherein the threshold interval is determined from a range of concentrations of thyroid hormone in healthy human individuals (e.g., human individuals not having a thyroid-related disorder),
wherein a level and/or concentration falling outside of the cut-off interval is indicative of the presence of the thyroid-related disorder.
59. The method of claim 58, wherein the cut-off value of free T3 is 2.8-4.4 pg/mL.
60. The method of any one of claims 58 and 59, wherein the cut-off interval for free T4 is between 0.8 and 2.0 ng/mL.
61. The method of any one of claims 58 to 60, wherein the threshold interval of rT3 is 10 to 24 ng/mL.
62. The method of any one of claims 58 to 61, wherein concentrations below the threshold interval are considered low, concentrations within the threshold interval are considered normal, and concentrations above the threshold interval are considered high.
63. The method of any one of claims 49-62, further comprising the step of treating the thyroid-related disorder.
64. The method of claim 63, wherein said treating comprises administering a drug in a therapeutically effective amount.
65. The method of claim 64, wherein the drug is a thyroid stimulating compound.
66. The method of any one of claims 50 to 65, wherein the thyroid stimulating compound is selected from T3, T4, TSH, thyroid autoantibodies (TRAb, TPOAb and TgAb) and thyroglobulin.
67. The method of any one of claims 49-66, wherein the subject is a human subject.
68. The method of claim 67, wherein the human subject is a child or an adult.
69. The method of any one of claims 49-68, wherein said subject is a horse, cow, sheep, pig, goat, cat, or dog.
70. The method of any one of claims 48 to 69, wherein the sample is a blood sample, a serum sample or a plasma sample, optionally wherein the sample has been processed prior to analysis.
71. The method of any one of claims 48 to 70, wherein treatment prior to analysis comprises filtration, removal of rT3, and/or adjustment of pH.
72. The method of any one of claims 48-71, wherein the thyroid hormone is detected using Surface Plasmon Resonance (SPR).
73. The method of any one of claims 48 to 72, wherein the concentration of one or more thyroid hormones is determined using SPR readings.
74. The method of any one of claims 48 to 73, wherein said thyroid hormone is quantified or monitored by electrochemical transduction.
75. The method of any one of claims 49-74, wherein the thyroid-related disorder is selected from the list of: hypothyroidism, hyperthyroidism, clinical depression, goiter, Graves-Basedow disease, Hashimoto's thyroiditis, diseases of normal thyroid function, and polar T3 syndrome.
76. The method of claim 75, wherein the hyperthyroidism is characterized by high free T4, high free T3, and low TSH.
77. The method of claim 75, wherein the disease of normal thyroid function is characterized by low free T3 and high rT 3.
78. The method of claim 75, wherein the hypothyroidism is primary or secondary.
79. The method of claim 78, wherein primary hypothyroidism is characterized by low free T4, normal or low free T3, and high TSH.
80. The method of claim 78, wherein secondary hypothyroidism is characterized by low free T4, normal or low free T3, and normal or low TSH.
81. A method of making a sensor comprising at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4, the method comprising:
a. providing a substrate, wherein the substrate is provided,
b. providing at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4, and optionally an anti-rT 3 antibody,
c. immobilizing the iodothyronine deiodinase and the anti-rT 3 antibody on the surface of the substrate,
thereby producing a sensor comprising at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and EC 1.21.99.4.
82. The method of claim 81, wherein the substrate is as defined in any one of the preceding claims.
83. The process of any one of claims 81 to 82, wherein said at least 2 iodothyronine deiodinases are as defined in any one of the preceding claims.
84. A hand-held device for quantifying and/or monitoring thyroid hormone, the device comprising:
a. a sample inlet;
b. a sensor, comprising:
i. a substrate, a first electrode and a second electrode,
a first iodothyronine deiodinase selected from EC1.21.99.4,
a second iodothyronine deiodinase selected from EC1.21.99.3 and EC1.21.99.4, and
optionally, an anti-rT 3 antibody,
c. a detector configured to receive signals from said sensor and convert them to a user-readable format;
d. optionally, a device for separating cellular components from a sample.
85. A hand-held device according to claim 84, wherein the sensor is as defined in any one of the preceding claims.
86. The handheld device of any one of claims 84 and 85, wherein the first iodothyronine deiodinase, the second iodothyronine deiodinase and the anti-rT 3 antibody are as defined in any one of the preceding claims.
Technical Field
The invention relates to a biosensor and application thereof in quantifying free thyroid hormone to evaluate thyroid function.
Background
The thyroid gland is a bileaflet ductless gland located in the front of the neck behind the laryngeal prominence. It is involved in the synthesis and secretion of the iodine-containing thyroid hormones triiodothyronine (T3) and thyroxine (T4), T3 and T4 affecting the overall metabolic rate and protein synthesis. Secretion of thyroid hormones is governed by the hypothalamic-pituitary-thyroid axis (HPT axis), where the hypothalamus and pituitary glands stimulate the thyroid gland by releasing thyroxine-releasing hormone (TRH) and Thyroid Stimulating Hormone (TSH).
T3, also known as triiodothyronine or [ o- (4-hydroxy-3, 5-iodophenyl) 3, 5-diiodophenyltyrosine ], has effects on increasing basal metabolic rate, protein turnover, lipolysis, cardiac output and fetal and infant development; whereas T4, also known as thyroxine or [ o- (4-hydroxy-3, 5-diiodophenyl) 3,5 diiodophenyl tyrosine ] is a prohormone that migrates to the liver and kidney and serves as a substrate for site-specific synthesis of T3.
Thyroid hormones act on almost every cell in the body. Their effects are to increase basal metabolic rate, influence protein synthesis, help regulate long bone growth (synergistic with growth hormone) and neural maturation, and increase the body's sensitivity to catecholamines (e.g. epinephrine) through radiocommunication. Thyroid hormones are essential for the normal development and differentiation of all cells in the human body. These hormones also regulate the metabolism of proteins, fats and carbohydrates, affecting how high energy compounds are used by human cells. They also stimulate the metabolism of vitamins. A number of physiological and pathological stimuli affect the synthesis of thyroid hormones.
Both hyperthyroidism and hypothyroidism can cause disease.
Hyperthyroidism (Hyperthyroidism), which is usually caused by Graves' Disease, is a clinical syndrome characterized by circulating free thyroxine (fT4), free triiodothyronine (fT3), or both, being excessive and a reduction in TSH. This is a common disease affecting approximately 2% of women and 0.2% of men. Thyrotoxicosis (Thyrotoxicosis) is often used interchangeably with hyperthyroidism, but with subtle differences. While thyrotoxicosis also refers to an increase in circulating thyroid hormones, it may be due to ingestion of thyroxine tablets or due to hyperthyroidism, which refers only to hyperthyroidism.
Hypothyroidism (hypothyroidim), which is usually caused by Hashimoto's thyroiditis, is a condition of thyroxine, triiodothyronine or both deficiency.
Hypothyroidism can sometimes cause clinical depression. T is3Are found at synaptic junctions and regulate the amount and activity of 5-hydroxytryptamine, norepinephrine, and gamma-aminobutyric acid (GABA) in the brain (Dratman M, Gordon J (1996). "thyroloid horrons as neurotransimitters". Thyroid.6(6): 639-47).
Alopecia is sometimes attributable to T3And T4The functional disorder of (1). The normal hair growth cycle may be affected, thereby disrupting hair growth.
Premature delivery may suffer from neurodevelopmental disorders due to a lack of maternal thyroid hormone when the premature infant's own thyroid gland is unable to meet its postnatal needs. Also, in normal pregnancy, adequate maternal thyroid hormone levels are of vital importance in order to ensure availability of thyroid hormone to the fetus and its developing brain. In 1 out of 1600-3400 neonates, congenital hypothyroidism occurs, most of which are asymptomatic at birth and associated symptoms appear weeks after birth.
Therefore, the ability to quantify the amounts of T3 and T4 in humans is important for the diagnosis of thyroid disorders. The routine diagnosis of thyroid dysfunction is performed by immunoassays for thyroid hormone and TSH. Current fT3 and fT4 tests attempt to competitively measure free hormone, either after physically separating bound hormone, or by indirect estimation, often with imprecision. Therefore, there is a drive to find new alternative methods for quantifying free thyroid hormone.
A biosensor is a sensor that utilizes the molecular recognition function of biological materials (e.g., microorganisms, enzymes, antibodies, DNA, and RNA) and uses such biological materials as a molecular recognition element. In other words, the biosensor recognizes a reaction occurring when a target substrate is recognized using an immobilized biomaterial, oxygen consumed by respiration of a microorganism, an enzyme reaction, luminescence, and the like. Among biosensors, practical uses of enzyme sensors are under development. For example, enzyme sensors for glucose, lactate, uric acid, and amino acids can be used in medical instrumentation and in the food processing industry.
For example, in an enzyme sensor, electrons generated by a reaction of a substrate contained in a sample liquid (i.e., an analyte) with an enzyme or the like are reduced to an electron acceptor, and a measuring device electrochemically measures the amount of the reduced electron acceptor. Thus, quantitative analysis of the analyte was performed. An example of such a biosensor is the one proposed in patent application No. PCT/JP 00/08012.
Different techniques can be used to track the reaction between, for example, an enzyme bound to an electrode and a target substrate. One of these techniques relies on Surface Plasmon Resonance (SPR). In SPR, a molecular partner (e.g., a protein) is immobilized on a metal (chip). Photo-exciting surface plasmons in the metal; this causes a change in the detectable surface plasmon signal when the binding partner binds to the immobilized molecule. Another of these techniques relies on electrochemical transduction, in which the content of a biological sample can be analyzed as a result of the direct conversion of a biological event into an electronic signal. The most common techniques in electrochemical biosensing include cyclic voltammetry, chronoamperometry, chronopotentiometry, impedance spectroscopy, and field effect transistor-based methods as well as nanowire or magnetic nanoparticle-based biosensing.
Disclosure of Invention
The present inventors have found that by immobilization on an electrode, enzymes involved in thyroid hormone conversion can be used in a method for direct quantification of free T3 and free T4. These findings allow for more accurate and specific quantification of thyroid hormones, as previous methods quantified free T3 and free T4 by multiple steps, indirect measurements or simple estimation. Accordingly, the present disclosure provides an enhanced system for diagnosing thyroid-related diseases.
The present inventors have found that the presence of at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4 on the sensor (e.g. on a separate surface of the substrate) is necessary for the quantification of fT3 and/or fT 4. In fact, in the case where one hormone cannot be quantified, the other hormone cannot be quantified either, because there is some overlap in the enzymatic reactions used for quantification, as can be seen in fig. 2. Furthermore, the third thyroid hormone, transtriiodothyronine (rT3), plays an important role in quantification, since EC1.21.99.4 iodothyronine deiodinase also catalyzes rT3 deiodination. Thus, rT3 can advantageously be removed or quantified separately before quantifying fT3 and fT4 with the sensor of the present invention. Alternatively, in some embodiments, the sensors of the invention further comprise an anti-rT 3 antibody that, together with at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4, allows for the specific quantification of rT3, fT3 and fT4 in a single step.
One aspect of the present disclosure provides a sensor for quantifying thyroid hormone, the sensor comprising:
a. a substrate, a first electrode and a second electrode,
b. at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4, and
wherein the at least 2 iodothyronine deiodinases are immobilized on the surface of the substrate.
One aspect of the present disclosure is to provide a sensor for quantifying thyroid hormone, the sensor comprising:
a. a substrate, a first electrode and a second electrode,
b. at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4, and
c. (ii) an anti-rT 3 antibody,
wherein the at least 2 iodothyronine deiodinases and the anti-rT 3 antibody are immobilized on the surface of a substrate.
One aspect of the present disclosure provides a sensor for quantifying thyroid hormone, the sensor comprising an iodothyronine deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the iodothyronine deiodinase is immobilized on the sensor.
Another aspect of the present disclosure provides a sensor for detecting thyroid hormone, the sensor comprising a methyl iodide adenylate deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the methyl iodide adenylate deiodinase is immobilized on the sensor.
Yet another aspect of the present disclosure provides a method of quantifying thyroid hormone in a sample, the method comprising the steps of:
a. providing a sample comprising or suspected of comprising a thyroid hormone,
b. in contact with the sensor of the present disclosure,
c. measuring a signal from the sensor, an
d. The signal is used to determine the level and/or concentration of one or more thyroid hormones in the sample, thereby detecting the thyroid hormone.
Yet another aspect of the present disclosure provides a method of diagnosing a thyroid-related disorder in a subject, the method comprising the steps of:
a. providing a sample obtained from a subject,
b. determining the level and/or concentration of the thyroid hormone in the sample using the methods of the present disclosure, thereby diagnosing one or more thyroid-related disorders.
Yet another aspect of the present disclosure provides a method of diagnosing a thyroid-related disorder in a subject, the method comprising the steps of:
a. providing a sample obtained from a subject,
b. contacting a sensor according to the present disclosure with the sample,
c. detecting one or more thyroid hormones in the sample,
d. determining the level and/or concentration of said thyroid hormone in the sample,
thereby diagnosing one or more thyroid-related conditions.
Yet another aspect of the present disclosure provides a method of monitoring a thyroid-related disorder in a subject, the method comprising the steps of:
a. administering to the subject a compound that stimulates the thyroid gland,
b. collecting a sample from the subject after performing step a,
c. determining the level and/or concentration of the thyroid hormone in the sample using the methods disclosed herein, thereby monitoring the thyroid-related disorder.
Yet another aspect of the present disclosure provides a method of monitoring a thyroid-related disorder in a subject, the method comprising the steps of:
a. administering to the subject a compound that stimulates the thyroid gland,
b. collecting a sample from the subject after performing step a),
c. contacting a sensor according to the present disclosure with the sample,
d. the signal is measured and the measured signal is,
e. the signal is used to determine the concentration of thyroid hormone in the sample, thereby monitoring the thyroid-related condition.
Yet another aspect of the present disclosure provides use of a sensor of the present disclosure for quantifying thyroid hormone.
Another aspect of the present disclosure provides a method of detecting thyroid hormone in a sample, the method comprising the steps of:
a. providing a sample comprising or suspected of comprising a thyroid hormone,
b. contacting a sensor according to the present disclosure with the sample,
c. measuring a signal from the sensor, thereby detecting the thyroid hormone.
Yet another aspect of the present disclosure provides a method of making a sensor comprising at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4, the method comprising:
a. providing a substrate, wherein the substrate is provided,
b. providing at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4, and optionally an anti-rT 3 antibody,
c. immobilizing a methyladenine iodide deiodinase and an anti-rT 3 antibody on the surface of the substrate, thereby manufacturing a sensor comprising at least 2 methyladenine iodide deiodinases selected from EC1.21.99.3 and/or EC 1.21.99.3.
Yet another aspect of the present disclosure provides a method of manufacturing a sensor comprising an iodothyronine deiodinase, the method comprising:
a. providing an electrode, and providing a first electrode,
b. providing at least one iodothyronine deiodinase,
c. the iodothyronine deiodinase was immobilized on the electrode, thereby manufacturing a sensor comprising the iodothyronine deiodinase.
Another aspect of the present disclosure provides a handheld device for quantifying and/or monitoring thyroid hormone, the device comprising:
a. a sample inlet;
b. a sensor, comprising:
i. a substrate, a first electrode and a second electrode,
a first iodothyronine deiodinase selected from EC1.21.99.3 and EC1.21.99.4,
a second iodothyronine deiodinase selected from EC1.21.99.4, and
optionally, an anti-rT 3 antibody,
c. a detector configured to receive the signal from the sensor and convert it to a user-readable format;
d. optionally, a device for separating cellular components from a sample.
Another aspect of the present disclosure provides a handheld device for detecting, quantifying, and/or monitoring thyroid hormone, the device comprising:
a. a sample inlet;
b. a sensor comprising an iodothyronine deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the iodothyronine deiodinase is immobilized on the sensor, and wherein the inlet is configured to contact the sample with the sensor;
c. a detector configured to receive the signal from the sensor and convert it to a user-readable format;
d. optionally, a device for separating cellular components from a sample.
Drawings
Figure 1 hypothalamic-pituitary-thyroid axis.
Figure 2. three isoforms of iodothyronine deiodinase catalyze thyroid hormone by iodination.
FIG. 3. Current response of IDII amperometric biosensors with increasing concentration of T4.
FIG. 4. Current response of IDII voltammetric biosensors with increasing concentrations of T4.
FIG. 5 Effect of thyroxine-binding globulin (TBG) concentration on T4 detection. The concentration of T4 was constant.
FIG. 6. cyclic voltammetry measurements in fetal calf serum with increasing concentrations of T4.
Detailed Description
Disclosed herein are biosensors and their use for quantifying free thyroid hormones fT3 and fT4 for assessing thyroid function. Furthermore, the present disclosure relates to a method for diagnosing or monitoring a thyroid-related disorder in a subject, the method comprising determining the concentration of thyroid hormone in a sample obtained from said subject.
Thyroid hormone
The present disclosure relates to devices and methods for detecting and/or quantifying the free thyroid hormones fT3 and fT4 and thus for assessing thyroid function.
Thyroid hormones are hormones produced and released by the thyroid gland. They are tyrosine-based hormones, primarily responsible for regulating metabolism.
The thyroid hormones thyroxine (T4) and triiodothyronine (T3) can be measured as free thyroxine (fT4) and free triiodothyronine (fT3), fT4 and fT3 being indicators of the activity of thyroxine and triiodothyronine in vivo. They can also be measured as total thyroxine and total triiodothyronine, which will depend on the amount of thyroxine and triiodothyronine bound to thyroxine-binding globulin. The relevant parameter is the free thyroxine index, which is the total thyroxine multiplied by the thyroid hormone uptake, which in turn is a measure of unbound thyroxine-bound globulin. In addition, thyroid disorders can be detected prenatally using advanced imaging techniques and testing fetal hormone levels.
Transtriiodothyronine (3, 3',5' -triiodothyronine, trans T3 or rT3) is an isomer of triiodothyronine (3,5, 3' triiodothyronine, T3).
Trans T3 is the third most common iodothyronine released into the blood by the thyroid gland, with rT3 at 0.9%; tetraiodothyronine (levothyroxine, T4) accounted for 90%, and T3 was 9%. However, 95% of rT3 in human blood is produced elsewhere in the human body. Hormone production by the thyroid gland is controlled by the hypothalamus and pituitary. The physiological activity of thyroid hormones is determined by activation, inactivation, or simply discarding prohormone T4And further functionally modifying T3And rT3Regulation of the enzyme system of (a). These enzymes act in a complex direction in the system, including neurotransmitters, hormones, metabolic markers and immune signals. The level of rT3 is increased in conditions such as thyroid-normal pathological syndrome (euthyroid sine), because its clearance is reduced while its production remains unchanged.
In a healthy adult individual (also referred to as a healthy subject), the reference interval for thyroid hormones is:
fT 3: 2.8-4.4pg/mL (>. 1 year, available from Mayo clinical),
fT 4: 0.8-2.0ng/dL (all ages, available from Mayo Clinic),
rT 3: 10-24ng/dL (available from Mayo Clinic),
as described in the following references: demrs LM, Spencer Cl, The thoroid, pathvision and thoroid functioning description in Tietz Textbook of Clinical Chemistry and Molecular diagnostics in fourth edition, edition by CA Burtis, ER Ashwood, DE Bruns.St.Louis, Elsevier Saunders company.2006, pp 2053 + 2087; stockigt JR Free cloved hormone measurment. A critical apresaal. Clin Endocrinol Metab 2001 Jun; 30: 265-; and Moore WT, Eastman RC: Diagnostic Endocrinology, St.Louis, Mosby,1990, pp 182-.
Infants and children may use different reference intervals. Also, different laboratories may adjust the reference interval by ± 0.4 units.
Thus, the concentration and/or level of thyroid hormone within the interval is considered normal, while concentrations and/or levels below the interval are considered low and concentrations and/or levels above the interval are considered high.
Iodothyronine deiodinase
The present disclosure relates to a sensor for quantifying thyroid hormone, comprising a substrate, at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4, and optionally an anti-rT 3 antibody, wherein the at least 2 iodothyronine deiodinases and the anti-rT 3 antibody are immobilized on the surface of the substrate.
The present disclosure relates to a sensor comprising an iodothyronine deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the iodothyronine deiodinase is immobilized on the sensor for the detection and/or quantification of thyroid hormones.
Iodothyronine deiodinases (EC 1.21.99.4 and EC 1.21.99.3) are a subfamily of deiodinases that play an important role in the activation and inactivation of thyroid hormones. The precursor thyroxine (T4) to 3,5,3' -triiodothyronine (T3) is converted to T3 by deiodinase activity. T3 affects the expression of genes in almost every vertebrate cell by binding to the nuclear thyroid hormone receptor. The unusual feature of iodothyronine deiodinases is that these enzymes contain selenium, in the form of the otherwise rare amino acid selenocysteine.
EC1.21.99.4 iodothyronine deiodinase as referred to herein is an enzyme capable of catalyzing the following reaction:
3,5,3 '-triiodo-L-thyronine + iodide + a + H (+) < > L-thyroxine + AH (2) EC1.21.99.4 methionine iodide deiodinase showed enzymatic activity only in the 5' -deiodination direction, which made the thyroid hormone more active. EC 1.21.99.4A-iodothyronine deiodinase includes type I and type II enzymes, both of which contain selenocysteine, but with different kinetics. For a type I enzyme, the first reaction is reductive deiodination, converting the-Se-H group of the enzyme to a-Se-I group; the reducing agent then converts it to-Se-H, releasing iodide. The following enzymes were included in the EC1.21.99.4 panel:
diiodothyronine 5' -deiodinase;
iodothyronine 5' -deiodinase;
an iodothyronine outer ring single deiodinase;
l-thyroxine iodohydrolase (reduction);
thyroxine 5-deiodinase;
type I iodothyronine deiodinase;
type II iodothyronine deiodinase.
EC1.21.99.3 iodothyronine deiodinase as referred to herein is an enzyme capable of catalyzing the following reaction:
3,3',5' -triiodo-L-thyronine + iodide + receptor + H (+) < > L-thyroxine + reduction receptor EC1.21.99.3 methionine iodide deiodinase has shown enzymatic activity in the 5-deiodination direction. This removal of 5-iodine, i.e., from the inner ring, greatly inactivates the hormone thyroxine. The following enzymes were included in group EC1.21.99.4:
diiodothyronine 5' -deiodinase;
iodothyronine 5-deiodinase;
iodinated methionine inner ring monodeiodinase;
type III iodothyronine deiodinase.
Type I deiodinases, also known as deiodinase type I (DI or D1), are commonly found in the liver and kidney and are capable of deiodinating both the inner and outer rings of thyroid hormones. The terms "inner ring" and "outer ring" are visualized in fig. 2 and refer to the different benzene rings present in thyroid hormones.
Type II deiodinases, also known as deiodinase type II (DII or D2), are common in the heart, skeletal muscle, central nervous system, fat, thyroid, and pituitary. It is only known to deiodinate the outer loop of the hormone protothyroxine and it is the main activating enzyme (the already inactivated transtriiodothyronine is also further degraded by DII).
Type III deiodinases, also known as deiodinase type III (DIII or D3), are common in fetal tissues and placenta; it is also present throughout the brain, except the pituitary. It is only known to deiodinate the inner ring of thyroxine or triiodothyronine.
In tissues, deiodinases activate or inactivate thyroid hormones. Activation occurs by the conversion of the hormone thyroxine (T4) to the active hormone triiodothyronine (T3) through the iodine atom on the exclusionary ring. Inactivation of thyroid hormone occurs by removal of the iodine atom from the inner ring, converting thyroxine to inactive transtriiodothyronine (rT3), or converting active triiodothyronine to diiodothyronine (T2).
The major part of thyroxine deiodination occurs intracellularly.
The activity of DII can be modulated by ubiquitination: covalent attachment of ubiquitin inactivates DII by disrupting dimerization and targets it to degradation of the proteosome. Deubiquitination to remove ubiquitin from DII restores its activity and prevents proteosome degradation.
DI both activates T4 to produce T3 and deactivates T4. In addition to increasing the function of producing additional thyroid T3 in hyperthyroid patients, the function is not as well understood as either DII or DIII. DII converts T4 to T3 and is the primary source of the cytoplasmic T3 pool. DIII prevents T4 activation and inactivates T3. https:// en. wikipedia.org/wiki/Iodothyronine _ deiodinase-cite _ note-url _ Bianco _ Lab-9DII and D3 play an important role in the maintenance of T3 levels at the plasma and cellular levels in homeostatic regulation. In hyperthyroidism, D2 was down-regulated and D3 was up-regulated to clear additional T3, while in hypothyroidism, D2 was up-regulated and D3 was down-regulated to increase cytoplasmic T3 levels. Serum T3 levels remained fairly constant in healthy individuals, but D2 and D3 could modulate tissue-specific intracellular T3 levels to maintain homeostasis, as T3 and T4 levels may vary from organ to organ. Deiodinase also provides spatiotemporal developmental control of thyroid hormone levels. The level of D3 was highest early in development and decreased with time, whereas the level of D2 was higher when there was a clear allergic change in the tissue. Thus, D2 was able to generate sufficient T3 at the necessary point in time, while D3 protected the tissue from overexposure to T3.
DII also plays an important role in thermogenesis of Brown Adipose Tissue (BAT). DII increases fatty acid oxidation in response to sympathetic nerve stimulation, temperature reduction, or excessive BAT feeding, and uncouples oxidative phosphorylation by uncoupling proteins, resulting in mitochondrial thermogenesis. DII increases during cold stress of BAT and increases intracellular T3 levels. In the DII-deficient model, shivering is a behavioral adaptation to cold. However, thermogenesis is much less efficient than uncoupling lipid oxidation.
An aspect of the present disclosure is to provide a sensor for detecting thyroid hormone, the sensor comprising an iodothyronine deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the iodothyronine deiodinase is immobilized on the sensor.
In one embodiment, the iodothyronine deiodinase is type 1 iodothyronine deiodinase [ EC1.21.99.4] or a fragment thereof.
In one embodiment, the iodothyronine deiodinase is type 2 iodothyronine deiodinase [ EC1.21.99.4] or a fragment thereof.
In one embodiment, the iodothyronine deiodinase is type 3 iodothyronine deiodinase [ EC1.21.99.3 ] or a fragment thereof.
In one embodiment, the EC1.21.99.4 iodothyronine deiodinase is a type 2 iodothyronine deiodinase or a type 1 iodothyronine deiodinase.
In one embodiment, the EC1.21.99.4 iodothyronine deiodinase is type 2 iodothyronine deiodinase.
In one embodiment, the EC1.21.99.3 iodothyronine deiodinase is type 3 iodothyronine deiodinase.
In some embodiments of the disclosure, the iodothyronine deiodinase is mammalian. In some embodiments of the disclosure, the iodothyronine deiodinase is human.
In some embodiments of the disclosure, at least two iodinated methionine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4 and/or anti-rT 3 antibodies are mammalian.
In some embodiments of the disclosure, at least two iodinated methionine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4 and/or anti-rT 3 antibodies are human.
In some embodiments of the present disclosure, at least two iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4 and/or anti rT3 antibodies are conjugated to additional moieties.
In some embodiments of the present disclosure, at least two iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4 and/or anti rT3 antibodies are each conjugated to an additional moiety, respectively. For example, the additional moiety may facilitate immobilization of iodothyronine deiodinase on a sensor or detection of thyroid hormone.
In some embodiments of the present disclosure, the substrate comprises a plurality of surfaces for immobilizing the at least 2 iodothyronine deiodinases, for example a first surface of the substrate and a second surface of the substrate.
In some embodiments of the present disclosure, the first surface, the second surface, and optionally the third surface are on the same substrate.
In some embodiments of the present disclosure, the first surface, the second surface, and optionally the third surface are on different substrates.
In some embodiments of the disclosure, the at least 2 methyl iodide adeodinases include a first methyl iodide adeodinase selected from EC1.21.99.3 and/or EC1.21.99.4 immobilized on a first surface of a substrate, and a second methyl iodide adeodinase selected from EC1.21.99.3 and/or EC1.21.99.4 immobilized on a second surface of the substrate.
In some embodiments of the present disclosure, the first iodothyronine deiodinase is selected from EC1.21.99.4 and the second iodothyronine deiodinase is selected from EC 1.21.99.3.
In some embodiments of the present disclosure, the first and second iodothyronine deiodinases are each independently selected from EC 1.21.99.4.
In some embodiments of the disclosure, the first and second iodothyronine deiodinases are different enzymes selected from EC 1.21.99.4.
In some embodiments of the disclosure, the first iodothyronine deiodinase is a type 1 iodothyronine deiodinase and/or a type 2 iodothyronine deiodinase.
In some embodiments of the disclosure, the second iodothyronine deiodinase is a type 1 iodothyronine deiodinase, a type 2 iodothyronine deiodinase, and/or a type 3 iodothyronine deiodinase.
In some embodiments of the disclosure, the first iodothyronine deiodinase is a type 1 iodothyronine deiodinase and the second iodothyronine deiodinase is a type 2 iodothyronine deiodinase.
In some embodiments of the disclosure, the first iodothyronine deiodinase is a type 1 iodothyronine deiodinase and the second iodothyronine deiodinase is a type 3 iodothyronine deiodinase.
In some embodiments of the disclosure, the first iodothyronine deiodinase is a type 2 iodothyronine deiodinase and the second iodothyronine deiodinase is a type 3 iodothyronine deiodinase.
In some embodiments of the disclosure, t is
In some embodiments of the disclosure, t is
In some embodiments of the disclosure, t is
In some embodiments of the disclosure, t is
In some embodiments, the additional moiety is a peptide, such as a polyhistidine tag (His-tag).
In some embodiments, the additional moiety is a label, also referred to as a fluorescent tag or probe.
Polyhistidine tags can be successfully used to immobilize proteins on a surface, such as a metal surface (e.g., a nickel or cobalt coated microtiter plate), or on an array of proteins.
In some embodiments, a sensor according to the present disclosure includes both a type 2 and a type 3 iodothyronine deiodinase, or a fragment thereof. The presence of both DII and DIII can lead to more accurate diagnosis of thyroid disease.
In some embodiments, a sensor according to the present disclosure includes both a type 1 and a type 2 iodothyronine deiodinase, or a fragment thereof. The presence of both DI and DII can lead to more accurate diagnosis of thyroid disorders.
In some embodiments, a sensor according to the present disclosure includes both a type 1 and a type 3 iodothyronine deiodinase, or a fragment thereof. The presence of both DI and DIII can lead to more accurate diagnosis of thyroid disorders.
In some embodiments, a sensor according to the present disclosure comprises a first and a second iodothyronine deiodinase, both independently selected from EC 1.21.99.4.
In some embodiments, a sensor according to the present disclosure comprises a first and a second iodothyronine deiodinase, both independently selected from EC 1.21.99.3.
In some embodiments, a sensor according to the present disclosure comprises a first and a second iodothyronine deiodinase, both independently selected from EC1.21.99.3 and EC 1.21.99.4.
In some embodiments, a sensor according to the present disclosure includes a first iodothyronine deiodinase independently selected from EC1.21.99.4 and a second iodothyronine deiodinase independently selected from EC1.21.99.3 and EC 1.21.99.4.
In some embodiments, a sensor according to the present disclosure includes a first iodothyronine deiodinase immobilized on a first surface of a substrate and a second iodothyronine deiodinase immobilized on a second surface of the substrate.
In some embodiments, the type 1 iodothyronine deiodinase comprises or consists of a polypeptide or fragment thereof having an amino acid sequence identical to SEQ ID NO: 1, at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, such as about 100% sequence identity.
In some embodiments, the type 2 iodothyronine deiodinase comprises or consists of a polypeptide or fragment thereof having an amino acid sequence identical to SEQ ID NO: 2 at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, such as about 100% sequence identity.
In some embodiments, the type 3 iodothyronine deiodinase comprises or consists of a polypeptide or fragment thereof having an amino acid sequence identical to SEQ ID NO: 3 at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, such as about 100% sequence identity. .
In some embodiments, the iodothyronine deiodinase is recombinantly produced, e.g., via cell-free expression.
In some embodiments, EC1.21.99.3, EC1.21.99.4, and/or the anti-rT 3 antibody are recombinantly produced, e.g., via cell-free expression.
Cell-free expression, also known as cell-free protein synthesis or CFPS, is the biological production of proteins in a cell-free system (i.e., without the use of living cells). The in vitro protein synthesis environment is not constrained by the cell wall or homeostatic conditions necessary to maintain cell viability. Thus, CFPS is able to directly access and control the translation environment, which is advantageous for many applications, including co-translational solubilization of membrane proteins, optimization of protein production, incorporation of unnatural amino acids, selectivity, and site-specific labeling.
Regarding sequence identity: a high level of sequence identity indicates the likelihood that the first sequence is derived from the second sequence. Amino acid sequence identity requires that two aligned sequences have the same amino acid sequence. Thus, a candidate sequence having at least 95% amino acid identity to a reference sequence requires that at least 95% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence after alignment. Identity can be determined by means of computer analysis, such as, but not limited to, ClustalW computer alignment programs (Higgins D., Thompson J., Gibson T., Thompson J.D., Higgins D.G., Gibson T.J.,1994.CLUSTAL W: optimizing the sensitivity of progressive multiple sequence alignment third through sequence weighing, position-specific gap weights and weight matrix acids.22: 4673 Res 4680), and default parameters suggested therein.
Biosensor concept
The present disclosure relates to a biosensor which is a sensor comprising a substrate, at least 2 iodothyronine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4 and an anti-rT 3 antibody, wherein the at least 2 iodothyronine deiodinases and anti-rT 3 antibody are immobilized on the surface of the substrate.
The present disclosure relates to a biosensor which is a sensor comprising a methyl iodide adenylate deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] immobilized on one of its surfaces.
Various devices for detecting ligand/receptor interactions are known. The most basic of these are pure chemical/enzymatic assays, in which the presence or amount of an analyte is detected by measuring or quantifying a detectable reaction product. Ligand/receptor interactions can also be detected and quantified by radiolabeling assays.
This type of quantitative binding assay involves two separate components: a reaction substrate, such as a solid phase test strip, chip or electrode, and a separate reader or detector device, such as a scintillation counter or spectrophotometer. The substrate is generally not suitable for use in a variety of assays or miniaturized for processing a variety of analyte assays in small body fluid samples.
In contrast, in biosensors, the assay substrate and the detector surface are integrated into a single device. One common type of biosensor employs an electrode surface coupled to a current or impedance measuring element to detect a change in current or impedance in response to the presence of a ligand-receptor binding event. Another type of biosensor may employ a chip, such as a glass chip, in combination with an optical detector (e.g., in combination with surface plasmon resonance).
"biosensor", sometimes referred to herein as a "sensor", refers to a system that includes a sensor and a biological component (element). Biosensors are actually a cumbersome, expensive, complex and unsuitable alternative to conventional analytical techniques for in situ surveillance. A biosensor is a chemical analysis device, implying that a biological component is integrated with a transducer system. It integrates biological components within or in close contact with the transducer, producing an electronic signal proportional to the individual analytes, which is further transmitted to a detector.
A biosensor comprises three basic components, namely a biological receptor (biological component), a transducer and an electronic circuit. Biological receptors or biological components are biomolecules, such as enzymes, DNA, proteins, whole cells, antibodies, etc., that are embedded in the transducer. In the present application, the biological receptor is an iodothyronine deiodinase. Transducers are devices that reproduce one form of energy into another, for example, chemical energy into electrical energy. For example, the transducer is a detector. The detectors encompassed by the methods of the present disclosure are optical detectors, such as surface plasmon resonance detectors, electrochemical detectors, and measurement circuits. The electronic circuit includes a signal processing system that converts the electrical signal into a processable signal.
Biosensors based on Surface Plasmon Resonance (SPR) effects exploit shifts in the SPR surface reflection angle that occur due to perturbations (e.g., binding events) at the SPR interface. Finally, biosensors may also exploit changes in the optical properties of the biosensor surface.
Electrochemical biosensors are generally based on the enzymatic action (oxidoreductases) of reactions that produce or consume electrons. The sensor substrate typically contains three electrodes: a reference electrode, a working electrode, and a counter electrode. The target analyte participates in a reaction that occurs at the surface of the active electrode and this reaction may result in electron transfer across the bilayer (creating a current) or can contribute to the bilayer potential (creating a voltage). The current can be measured where the flow rate of electrons is proportional to the analyte concentration at a fixed potential, or the potential can be measured at zero current, giving a logarithmic response. Further, the use of biologically functionalized ion sensitive field effect transistors allows label-free direct electrical detection of small peptides and proteins by their intrinsic charge.
Potentiometric biosensors, which produce a potential at zero current, give a high dynamic range logarithmic response. Such biosensors are generally manufactured by screen printing an electrode pattern on a plastic substrate coated with a conductive polymer and then attached with some protein (enzyme or antibody). They have only two electrodes and are very sensitive and reliable. They are capable of detecting analytes at levels previously achievable only by HPLC and LC/MS, without the need for stringent sample preparation. Since the biosensor components are highly selective for the analyte of interest, all biosensors typically require very little sample preparation. The signal is generated as a result of electrochemical and physical changes in the conductive polymer layer due to changes in the sensor surface. These changes can be attributed to ionic strength, pH, hydration, and redox reactions. Because binding of an analyte to the gate region of a Field Effect Transistor (FET) results in a change in drain-source current, FETs in which the gate region has been modified by an enzyme or antibody can also detect very low concentrations of various analytes.
Biosensors have many potential advantages over integrated assay systems having separate reaction substrates and reader devices. An important advantage is the ability to manufacture small-scale, but highly reproducible biosensor units using microchip manufacturing methods.
There are many potential applications for various types of biosensors. The main requirements of the biosensor approach that are valuable in research and commercial applications are the identification of target molecules, the availability of suitable biorecognition elements and in some cases the preferred potential of disposable portable detection systems by laboratory-based sensitive technologies.
In some embodiments, the present disclosure relates to a biosensor for detecting and/or quantifying thyroid hormone, wherein the thyroid hormone is selected from the group consisting of free T4, free T3, and trans T3(rT 3).
In some embodiments, the present disclosure relates to a biosensor for quantifying thyroid hormone, wherein the thyroid hormone is selected from the group consisting of free T4, free T3, and trans T3(rT 3).
In some embodiments, the present disclosure relates to a biosensor, wherein the biosensor comprises a sensor comprising a substrate; at least 2 iodinated methionine deiodinases selected from EC1.21.99.3 and/or EC1.21.99.4, or fragments thereof; and an anti-rT 3 antibody or fragment thereof, wherein at least 2 of the iodothyronine deiodinases and the anti-rT 3 antibody are immobilized on the surface of the substrate.
In some embodiments, the present disclosure relates to a biosensor, wherein the biosensor comprises a sensor comprising an electrode and an iodothyronine deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or fragment thereof immobilized on the electrode.
In some embodiments, a sensor according to the present disclosure comprises between 10 and 100IU of iodothyronine deiodinase, such as between 10 and 15IU, such as between 15 and 20IU, such as between 20 and 25IU, such as between 25 and 30IU, such as between 30 and 35IU, such as between 35 and 40IU, such as between 40 and 45IU, such as between 45 and 50IU, such as between 50 and 55IU, such as between 55 and 60IU, such as between 60 and 65IU, such as between 65 and 70IU, such as between 70 and 75, such as between 75 and 80IU, such as between 80 and 85IU, such as between 85 and 90IU, such as between 90 and 95, such as between 95 and 100 IU.
In some embodiments, a sensor according to the present disclosure comprises between 10 and 100IU of EC1.21.99.3 and EC1.21.99.4 of the apo-lyme-dium iodide, e.g., between 10 and 15IU, e.g., between 15 and 20IU, e.g., between 20 and 25IU, e.g., between 25 and 30IU, e.g., between 30 and 35IU, e.g., between 35 and 40IU, e.g., between 40 and 45IU, e.g., between 45 and 50IU, e.g., between 50 and 55IU, e.g., between 55 and 60IU, e.g., between 60 and 65IU, e.g., between 65 and 70IU, e.g., between 70 and 75IU, e.g., between 75 and 80IU, e.g., between 80 and 85, e.g., between 85 and 90IU, e.g., between 90 and 95IU, e.g., between 95 and 100 IU.
In some embodiments, a sensor according to the present disclosure comprises between 10 and 100IU of the type 2 and type 3 iodothyronine deiodinase, e.g., between 10 and 15IU, e.g., between 15 and 20IU, e.g., between 20 and 25IU, e.g., between 25 and 30IU, e.g., between 30 and 35IU, e.g., between 35 and 40IU, e.g., between 40 and 45IU, e.g., between 45 and 50IU, e.g., between 50 and 55IU, e.g., between 55 and 60IU, e.g., between 60 and 65IU, e.g., between 65 and 70, e.g., between 70 and 75IU, e.g., between 75 and 80IU, e.g., between 80 and 85IU, e.g., between 85 and 90, e.g., between 90 and 95IU, e.g., between 95 and 100 IU.
IU denotes international units, and it is a unit of measure of the amount of a substance; the mass or volume constituting an international unit varies depending on the substance to be measured, and the difference is based on the biological activity or effect, for the purpose of simpler comparison between substances.
In some embodiments of the present disclosure, a sensor for quantifying thyroid hormone is disclosed, comprising:
-a first electrode comprising a first surface;
-a second electrode comprising a second surface;
-a first iodothyronine deiodinase selected from EC1.21.99.4 immobilized on the first surface of the first electrode;
-a second iodothyronine deiodinase selected from EC1.21.99.3 and EC1.21.99.3 immobilized on the second surface of the second electrode.
In some embodiments of the present disclosure, a sensor for quantifying thyroid hormone is disclosed, comprising:
-a first electrode comprising a first surface;
-a second electrode comprising a second surface;
-a third electrode comprising a third surface;
-a first iodothyronine deiodinase selected from EC1.21.99.4 immobilized on the first surface of the first electrode;
-a second iodothyronine deiodinase selected from EC1.21.99.3 and EC1.21.99.3 immobilized on the second surface of the second electrode, and
-an anti-rT 3 antibody immobilized on the third surface of the third electrode.
One aspect of the present disclosure provides a method of manufacturing a sensor comprising an iodothyronine deiodinase, the method comprising:
a) providing an electrode, and providing a plurality of electrodes,
b) providing at least one iodothyronine deiodinase,
c) the iodothyronine deiodinase is fixed on an electrode,
thereby manufacturing a sensor comprising the iodothyronine deiodinase.
In a particular embodiment, the electrodes are as defined in accordance with embodiments of the present disclosure. In a particular embodiment, the iodothyronine deiodinase is as defined according to embodiments of the present disclosure.
Step c) above comprises immobilizing the iodothyronine deiodinase on an electrode, for example a sensor. This step may be considered as including a step of functionalizing the electrode, and then immobilizing the iodothyronine deiodinase on the functionalized electrode. Examples of methods that can be used to immobilize the iodothyronine deiodinase on the electrode are described in detail in this disclosure.
Enzyme immobilization
Immobilization of biological components (e.g. target enzymes) on the sensor surface (which may be metal, polymer or glass) is an essential critical step in biosensor design. There are different fixing techniques depending on the substrate used, which are known to the person skilled in the art.
One aspect of the present disclosure provides a sensor for detecting thyroid hormone, the sensor comprising a substrate,
a. a first iodothyronine deiodinase selected from EC1.21.99.4,
b. a second iodothyronine deiodinase selected from EC1.21.99.3 or EC1.21.99.4, and
c. optionally, an anti-rT 3 antibody,
wherein the first iodothyronine deiodinase, the second iodothyronine deiodinase and the anti-rT 3 antibody are immobilized on the surface of the substrate.
In a first aspect, there is provided a sensor for quantifying thyroid hormone, the sensor comprising an iodothyronine deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the iodothyronine deiodinase is immobilized on the sensor.
One aspect of the present disclosure provides a sensor for detecting thyroid hormone, the sensor comprising a methyl iodide adenylate deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the methyl iodide adenylate deiodinase is immobilized on the sensor.
In some embodiments, a substrate according to the present disclosure includes one or more electrodes and/or chips.
In some embodiments, a substrate according to the present disclosure comprises at least 1 electrode, for example at least 2 electrodes, for example at least 3 electrodes.
In some embodiments, a substrate according to the present disclosure comprises at least 1 chip, such as at least 2 chips, such as at least 3 chips.
In some embodiments, a substrate according to the present disclosure comprises or consists of 3 electrodes, and wherein the first surface is a surface of a first electrode, the second surface is a surface of a second electrode, and the third surface is a surface of a third electrode.
In some embodiments, the substrate according to the present disclosure comprises or consists of three chips, and wherein the first surface is a surface of a first chip, the second surface is a surface of a second chip, and the third surface is a surface of a third chip.
In some embodiments, a sensor according to the present disclosure includes a substrate having a modified surface. The substrate is modified so that the iodothyronine deiodinase can be immobilized on its surface.
In some embodiments, the substrate according to the present disclosure is an electrode or a chip. In a further embodiment, the chip is a glass chip. As used herein, the term "glass" is equivalent to quartz or silicon dioxide, and encompasses materials having the bulk chemical formula SiO2Silicon and oxygen atoms in a continuous framework.
In some embodiments, a sensor according to the present disclosure comprises one electrode, e.g., two electrodes, e.g., three electrodes, wherein a first surface of the first electrode is a modified surface, wherein a second surface of the second electrode is a modified surface, and/or wherein a third surface of the third electrode is a modified surface.
When reference is made to "electrodes" in this disclosure, it refers to the first electrode, the second electrode, the third electrode and/or more electrodes on which the biological components of the sensor (i.e., the first iodothyronine deiodinase selected from EC1.21.99.4, the second iodothyronine deiodinase selected from EC1.21.99.3 and EC1.21.99.4, and optionally the anti-rT 3 antibody) are immobilized.
When reference is made to a "surface" in the present disclosure, it refers to the first surface, the second surface, the third surface and/or more surfaces of the substrate (i.e. of the electrodes and/or the chip) on which the biological components of the sensor are immobilized, i.e. the first iodothyronine deiodinase selected from EC1.21.99.4, the second iodothyronine deiodinase selected from EC1.21.99.3 and EC1.21.99.4 and optionally the anti-rT 3 antibody.
In some embodiments, the electrodes are made of carbon, gold, or platinum.
In another embodiment, the electrode is a screen printed electrode.
In some embodiments, a sensor according to the present disclosure includes at least one surface of a chip or electrode coated with a gold layer or a gold monolayer. In other embodiments, the surface of the chip or electrode is coated with a material selected from the group consisting of silver, copper oxide, graphene, iron oxide, and combinations thereof.
In some embodiments according to the present disclosure, the first, second and/or third surface of the substrate of the sensor is a modified surface.
The sensor surface, e.g., the surface of the electrodes and/or the surface of the chip) may be subjected to any type of treatment prior to use. The treatment may include deposition of nanoparticles, which may be performed by, for example, coating, dip coating, spin coating, Langmuir-Blodgett, self-assembly, solvent evaporation, doctor blade coating, chemical vapor deposition, transfer printing, direct deposition, deposition-precipitation methods, use of an adhesion layer between the electrode surface and the nanoparticle-containing polyelectrolyte, covalent immobilization, for example, by amide bond immobilization, electrostatic immobilization, polymer brush immobilization, sol-gel/polymer network immobilization, van der waals force immobilization, hydrophobic/hydrophilic immobilization, deposition by evaporation and/or dewetting, electrodeposition (e.g., photo-induced conductive deposition), Turkevich-fresns method, Brust-Schiffrin method, layer-by-layer sequential ionic layer deposition, chemical method, photochemical method, sonochemical method, or a combination thereof.
The surface of the sensor may be further patterned, for example by micro-machining techniques, prior to depositing the nanoparticles, so that at least a portion of the nanoparticles are fixed in a specific pattern on the surface of the sensor. The surface of the sensor may be further treated to induce chemical changes, such as functionalization or activation by, for example, amine functionalization, thiol functionalization, hydroxylation, silylation, oxidation, and/or plasma activation, or combinations thereof. The surface modification may be performed at any point during the surface treatment, for example before nanoparticle deposition.
The sensor may be further assembled or processed according to any method useful for assembly of a sensor, for example by sintering, printing (e.g., 3d printing, screen printing, and/or inkjet printing), casting, electrodeposition, thin film techniques, network formation, dealloying, photolithography (e.g., optical lithography and/or imprint lithography), sputtering, stamping, thermal annealing, electrolysis, anodization, etching (e.g., electrochemical etching, wet etching, and dry etching), or a combination thereof.
In some embodiments according to the present disclosure, the modified surface is a surface comprising a plurality of nano-and/or micro-sized topographical features. Such modified surfaces comprising a plurality of nano-and/or micro-sized topographical features are also referred to as roughened or roughened surfaces. Roughening the surface is beneficial for enzyme immobilization because it minimizes van der waals forces that could otherwise cause the immobilized enzyme to break down.
In some embodiments according to the present disclosure, the plurality of nano-and/or micro-sized topographical features are selected from the group consisting of: microparticles, nanoparticles, microwires, nanowires, microtubes, nanotubes, nanorods, and combinations thereof.
In some embodiments according to the invention, the surface is assembled by sintering, producing a plurality of nano-and/or micro-sized topographical features on the surface of the substrate.
In some embodiments according to the present disclosure, wherein the plurality of nano-and/or micro-sized topographical features are created on the surface of the substrate by surface etching. For example, the surface etching may be wet etching or dry etching.
In some embodiments according to the present disclosure, wherein the plurality of nano-and/or micro-sized topographical features are created on the surface of the substrate by particle deposition. For example, a plurality of nano-and/or micro-sized topographical features may be created on the surface of the substrate by electrophoretic deposition.
In some embodiments according to the present disclosure, the surface modified therein is a surface coated with a gold layer.
In some embodiments according to the present disclosure, wherein the modified surface is a surface coated with nanoparticles selected from the group consisting of gold, silver, copper oxide, graphene, iron oxide, and combinations thereof.
In some embodiments, a sensor according to the present disclosure includes at least one surface of a substrate (chip or electrode) coated with a gold layer or gold monolayer, and the surface is further modified with nanoparticles selected from the group consisting of gold, silver, copper oxide, graphene, iron oxide, and combinations thereof.
In other words, the substrate (electrode or chip) may be coated with a gold layer or gold monolayer on which nanoparticles, for example, nanoparticles selected from gold, silver, copper oxide, graphene, iron oxide, and combinations thereof, may be present, and the methyladenine iodide deiodinase molecule may be immobilized on the nanoparticles.
In one embodiment, at least one surface of the chip or electrode is modified with nanoparticles selected from the group consisting of gold, silver, copper oxide, graphene, iron oxide, and combinations thereof.
In some embodiments according to the present disclosure, wherein the modified surface is a surface coated with a gold layer, and wherein the surface is further modified with nanoparticles selected from the group consisting of gold, silver, copper oxide, graphene, iron oxide, and combinations thereof.
The nanoparticles disclosed herein may be capped with a capping agent. The capping agent may be an organic molecule, such as citrate, and may be used to prevent nanoparticle growth to control its size. In some embodiments, the nanoparticles are citrate-capped, amino-capped, or citrate-capped and amino-capped.
In one embodiment, the sensor comprises a chip with a modified surface, wherein the chip is a chemically modified glass substrate, and wherein the chip is used in combination with surface plasmon resonance to detect and/or quantify thyroid hormone.
In one embodiment, the sensor comprises a chip having a modified surface, wherein the chip is a glass substrate modified with nanoparticles, wherein the nanoparticles may be selected from the group consisting of gold, silver, copper oxide, graphene, iron oxide, and combinations thereof, and wherein the chip is used in conjunction with surface plasmon resonance to detect and/or quantify thyroid hormone.
In one embodiment, the sensor comprises a chip having a modified surface, wherein the chip is a glass substrate modified with a layer or monolayer, wherein the layer or monolayer is made of a material selected from the group consisting of gold, silver, copper oxide, graphene, iron oxide, and combinations thereof, and wherein the chip is used in conjunction with surface plasmon resonance to detect and/or quantify thyroid hormone.
In one embodiment, the sensor comprises a chip with a modified surface, wherein said chip is a glass substrate modified with gold nanoparticles, and wherein said chip is used in combination with surface plasmon resonance to detect and/or quantify thyroid hormone.
In one embodiment, the sensor comprises a chip having a modified surface, wherein the chip comprises a glass substrate modified with a gold layer or a gold monolayer, and wherein the chip is used in conjunction with surface plasmon resonance to detect and/or quantify thyroid hormone.
In one embodiment, the sensor comprises an electrode having a modified surface, wherein the electrode comprises a layer or monolayer surface made of gold, and wherein the electrode is used in conjunction with electrochemical transduction to detect and/or quantify thyroid hormone.
In another embodiment, the electrode comprises a layer or monolayer surface made of silver, wherein the electrode is used in conjunction with electrochemical transduction to detect and/or quantify thyroid hormone according to the present disclosure.
In another embodiment, the electrode comprises a layer or monolayer surface made of copper oxide, wherein the electrode is used in conjunction with electrochemical transduction to detect and/or quantify thyroid hormone according to the present disclosure.
In another embodiment, the electrode comprises a layer or monolayer surface made of graphene, wherein said electrode is used in conjunction with electrochemical transduction to detect and/or quantify thyroid hormone according to the present disclosure.
In another embodiment, the electrode comprises a layer or monolayer surface made of iron oxide, wherein the electrode is used in conjunction with electrochemical transduction to detect and/or quantify thyroid hormone according to the present disclosure.
In another embodiment, the electrode comprises a layer or monolayer surface made of a combination of metals, such as gold and silver, wherein the electrode is used in conjunction with electrochemical transduction to detect and/or quantify thyroid hormone according to the present disclosure.
In some embodiments, an iodothyronine deiodinase according to the present disclosure is immobilized on a substrate.
In some embodiments, an iodothyronine deiodinase according to the present disclosure is immobilized on the surface of a sensor by a linker comprising nanoparticles.
In some embodiments, an iodothyronine deiodinase according to the present disclosure is immobilized on the surface of a sensor by a linker comprising a nickel-histidine (Ni-His) covalent coordination bond. This may be particularly suitable when the iodothyronine deiodinase comprises a histidine tag.
In some embodiments according to the present disclosure, wherein the nanoparticles have a size of 1nm to 50nm, preferably a size of 5nm to 45nm, preferably a size of 10nm to 40nm, preferably a size of 10nm to 35nm, preferably a size of 10nm to 30 nm. The size of the nanoparticles can be determined by TEM microscopy. Using nanoparticles of this size range as linkers between the substrate surface and the iodothyronine deiodinase may give the substrate surface a preferred curvature to immobilize the iodothyronine deiodinase.
In some embodiments, an iodothyronine deiodinase according to the present disclosure is immobilized on a substrate by ionic interaction.
In some embodiments, an iodinated methionine deiodinase according to the present disclosure is immobilized on a substrate by non-covalent interactions.
In some embodiments, an iodothyronine deiodinase according to the present disclosure is covalently immobilized on a substrate.
In some embodiments, the iodothyronine deiodinase is immobilized on a substrate via a linker comprising one or more nanoparticles. The presence of at least one nanoparticle between the substrate and the iodothyronine deiodinase prevents unfolding of the protein.
In some embodiments, the iodothyronine deiodinase is immobilized on the substrate by a linker comprising:
cysteamine combined with an electrode, and
nanoparticles bound to the cysteamine and iodothyronine, optionally via one or more additional cysteamines.
In some embodiments, the at least 2 iodothyronine deiodinases and/or anti rT3 antibodies are immobilized on the substrate by a linker comprising:
cysteamine combined with an electrode, and
nanoparticles bound to the cysteamine and iodothyronine, optionally via one or more additional cysteamines.
In some embodiments, the sensors of the present disclosure further comprise an anti-rT 3 antibody immobilized on a surface of the sensor, in particular on a surface of an electrode (e.g., on a third surface of a third electrode) or on a surface of a chip (e.g., on a third surface of a third chip).
Various techniques are known to those skilled in the art that can be used to immobilize antibodies on an electrode surface or chip surface.
In some embodiments of the disclosure, the anti-rT 3 antibody is immobilized on the surface of the substrate by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) -N-hydroxysuccinimide (NHS) chemistry.
In some embodiments of the present disclosure, the anti-rT 3 antibody can be an azide-modified antibody, and it can be immobilized on the surface of a substrate by click chemistry on alkyne or heavy chain-associated glycans.
In some embodiments of the disclosure, the anti-rT 3 antibody is immobilized directly on the surface of the substrate, wherein the surface of the substrate is a positively charged amine-modified surface.
In some embodiments of the disclosure, the anti-rT 3 antibody is immobilized on the surface of the substrate by biotin-avidin binding, in which case the anti-rT 3 antibody is biotinylated.
In some embodiments, the iodinated methionine deiodinase is bound to the linker via the C-terminus or the N-terminus. In some embodiments, the iodothyronine deiodinase is bound to the linker through the N-terminus by an amide bond with cysteamine.
For example, an iodothyronine deiodinase may be immobilized on the surface of a substrate by applying any of the following procedures:
·carbon electrode: can use H2SO4/HNO3(aq) oxidizing the electrode to introduce hydroxyl groups; washing; in the dark byReacting with cysteamine hydrochloride (aq) to introduce free thiol groups; washing; covalently immobilizing the citrate-terminated nanoparticles using a thiol/gold coupling; washing; introducing free amine groups using cysteamine hydrochloride (aq); washing; immobilizing the iodothyronine deiodinase or fragment thereof by formation of its carboxylate residue via an amide bond; washing; blocking with bovine serum albumin. See also Sharma S, Zapatero-Rodr I guez J, Saxena R, O' Kennedy R, Srivastava S2018. ultrasensive direct electrochemical sensors for detection of server HER2&Bioelectronics 106:78-85。
·Gold electrode: introducing an amino group by Au/SH coupling by reaction with cysteamine hydrochloride (aq); washing; covalently linking the citrate-terminated nanoparticles by amide bond formation; washing; the iodothyronine deiodinase or fragment thereof is immobilized by covalent coupling between a primary amine of the iodothyronine deiodinase or fragment thereof and a free carboxylate residue on the nanoparticle. See also Raghav R, Srivastava S,2016. Immobilisation Stratagene for Enhancing Sensitivity of biosensors L-Asparagine-AuNPs as a promoting activity of EDC-NHS activated citrate-AuNPs for Antibody mobilization biosensors&Bioelectronics 15；78:396-403。
·Gold electrode: introducing an amino group by Au/SH coupling by reaction with cysteamine hydrochloride (aq); washing; covalently linking the citrate-terminated nanoparticles by amide bond formation; washing; adding an aqueous solution of EDC crosslinker (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) and sulfo-NHS (N-hydroxysulfosuccinimide); the iodothyronine deiodinase or fragment thereof is immobilized by covalent coupling between the primary amine of the iodothyronine deiodinase or fragment thereof and the NHS ester intermediate. See also Raghav R, Srivastava S,2016. Immobilisation Stratagene for Enhancing Sensitivity of biosensors L-Asparagine-AuNPs as a promoting activity of EDC-NHS activated citrate-AuNPs for Antibody mobilization biosensors&Bioelectronics 15；78:396-403。
·Gold electrode: introducing an amino group by Au/SH coupling by reaction with cysteamine hydrochloride (aq); washing machine(ii) a Covalently linking the amino group and the citrate-terminated nanoparticle by forming an amide bond; washing; the iodothyronine deiodinase or fragment thereof is immobilized by formation of an amide bond by covalent coupling. See also Raghav R, Srivastava S,2016. Immobilisation Stratagene for Enhancing Sensitivity of biosensors L-Asparagine-AuNPs as a promoting activity of EDC-NHS activated citrate-AuNPs for Antibody mobilization biosensors&Bioelectronics 15；78:396-403。
·Gold electrode: the amino group was introduced by au/sh coupling by reaction with cysteamine hydrochloride (aq); washing; covalently linking the citrate-terminated nanoparticles by amide bond formation; washing; proteins or antibodies are immobilized by covalent coupling through formation of amide bonds. See also Raghav R, Srivastava S,2015, Core-shell gold-silver nanoparticles for pigment antigen CA125.Sensors and activators B, Chemical 220: 557-.
Although the above procedure involves carbon and gold electrodes, other types of electrodes and similar procedures may be used.
In some embodiments, a sensor according to the present disclosure is configured such that the electrodes can be connected to a bench top, a handheld electrochemical workstation, a surface plasmon resonance detector, or a measurement circuit.
In some embodiments, a sensor according to the present disclosure comprises a substrate comprising at least 3 electrodes, wherein the electrodes are configured such that they can be connected to an electrochemical workstation.
In some embodiments, a sensor according to the present disclosure includes a substrate comprising at least 3 chips, and wherein the chips are configured such that they can be connected to a surface plasmon resonance detector.
In one embodiment, the sensor is configured to detect and/or quantify thyroid hormone.
Method of producing a composite material
The present disclosure relates to a sensor for detecting and/or quantifying thyroid hormone, comprising an iodothyronine deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the iodothyronine deiodinase is immobilized on the sensor, and the use of said sensor in the diagnosis and/or monitoring of thyroid related diseases.
The present invention relates to a sensor for detecting and/or quantifying thyroid hormone, comprising a substrate, a first iodothyronine deiodinase selected from EC1.21.99.4, a second iodothyronine deiodinase selected from EC1.21.99.3 and EC1.21.99.4, and optionally an anti-rT 3 antibody, wherein the iodothyronine deiodinase and the anti-rT 3 antibody are immobilized on the surface of the substrate, and the use of said sensor in the diagnosis and/or monitoring of thyroid related diseases.
One aspect of the present disclosure provides a method of diagnosing a thyroid-related disorder in a subject, the method comprising the steps of:
a) providing a sample obtained from a subject,
b) contacting with the sample as disclosed herein,
c) detecting one or more thyroid hormones in the sample,
d) determining the level and/or concentration of said thyroid hormone in the sample,
thereby diagnosing one or more thyroid-related conditions.
Yet another aspect of the present disclosure provides a method of monitoring a thyroid-related disorder in a subject, the method comprising the steps of:
a) administering to the subject a compound that stimulates the thyroid gland,
b) collecting a sample from the subject after performing step a),
c) contacting a sensor according to the present disclosure with the sample,
d) the signal is measured and the measured signal is,
e) using the signal to determine the concentration of thyroid hormone in the sample,
thereby monitoring the thyroid-related condition. In a particular embodiment of the method according to the present disclosure, steps b) -e) are performed more than once.
Another aspect of the present disclosure provides a method of detecting thyroid hormone in a sample, the method comprising the steps of:
a) providing a sample comprising or suspected of comprising a thyroid hormone,
b) contacting a sensor as disclosed herein with the sample,
c) the signal from the sensor is measured and,
thereby detecting thyroid hormone.
Another aspect of the present disclosure provides a method of quantifying thyroid hormone in a sample, the method comprising the steps of:
a. providing a sample comprising or suspected of comprising a thyroid hormone,
b. contacting a sensor as disclosed herein with the sample,
c. measuring a signal from the sensor, an
d. Using the signal to determine the level and/or concentration of one or more thyroid hormones in the sample,
thereby detecting thyroid hormone.
In a particular embodiment, the method for detecting thyroid hormone in a sample according to the present disclosure further comprises step d): the signal is used to determine the concentration of one or more thyroid hormones in the sample.
In a particular embodiment, the method according to the present disclosure further comprises the steps of: the concentration of thyroid hormone in the sample is used to calculate the concentration of thyroid hormone in vivo.
In some embodiments of the methods according to the present disclosure, the concentration of thyroid hormone in the sample is determined according to the kinetics of the reaction between the thyroid hormone and the iodothyronine deiodinase.
In certain embodiments, the concentration of thyroid hormone is determined after the subject has received a drug comprising a thyroid stimulating compound.
In a particular embodiment, the time after the subject has received the drug is between 5 minutes and 48 hours, such as between 5 minutes and 45 hours, such as between 5 minutes and 40 hours, such as between 5 minutes and 36 hours, such as between 5 minutes and 32 hours, such as between 5 minutes and 30 hours, such as between 5 minutes and 28 hours, such as between 5 minutes and 24 hours, such as between 5 minutes and 20 hours, such as between 5 minutes and 18 hours, such as between 5 minutes and 16 hours, such as between 5 minutes and 14 hours, such as between 5 minutes and 12 hours, such as between 5 minutes and 11 hours, such as between 5 minutes and 10 hours, such as between 5 minutes and 9 hours, such as between 5 minutes and 8 hours, such as between 5 minutes and 7 hours, such as between 5 minutes and 6 hours, such as between 5 minutes and 5 hours, such as between 5 minutes and 4 hours, such as between 5 minutes and 3 hours, such as between 5 minutes and 2 hours, such as between 5 minutes and 1 hour, for example 5 minutes to 45 minutes, for example 5 minutes to 30 minutes.
In particular embodiments, the time after the subject has received the drug is from 5 minutes to 48 hours, e.g., 15 minutes to 48 hours, e.g., 30 minutes to 48 hours, e.g., 45 minutes to 48 hours, for example 60 minutes to 48 hours, for example 1 to 48 hours, for example 2 to 48 hours, for example 3 to 48 hours, e.g., 4 to 48 hours, e.g., 5 to 48 hours, e.g., 6 to 48 hours, e.g., 7 to 48 hours, e.g., 8 to 48 hours, e.g., 9 to 48 hours, e.g., 10 to 48 hours, for example 11 to 48 hours, for example 12 to 48 hours, for example 14 to 48 hours, for example 16 to 48 hours, e.g., 18 to 48 hours, e.g., 20 to 48 hours, e.g., 24 to 48 hours, e.g., 28 to 48 hours, for example 32 to 48 hours, for example 36 to 48 hours, for example 40 to 48 hours, for example 44 to 48 hours.
In one embodiment of the method according to the present disclosure, the subject does not receive a drug comprising a thyroid stimulating hormone prior to determining the concentration of the thyroid hormone.
In certain embodiments, the method according to the present disclosure further comprises the steps of: comparing the level and/or concentration of the thyroid hormone in the sample to a cut-off interval (cut-off interval) to diagnose a thyroid-related disorder in the subject, wherein the cut-off interval is determined according to the range of concentrations of thyroid hormone in healthy human individuals, e.g. human individuals not suffering from a thyroid-related disorder,
wherein a level and/or concentration falling outside of a threshold interval is indicative of the presence of the thyroid-related disease.
In some embodiments of the methods according to the present disclosure, the threshold interval of free T3 is 2.8 to 4.4pg/mL, the threshold interval of free T4 is 0.8 to 2.0ng/mL, and the threshold interval of rT3 is 10 to 24 ng/mL. In some embodiments, concentrations below the threshold interval are considered low, concentrations within the threshold interval are considered normal, and concentrations above the threshold interval are considered high.
In some embodiments of the methods according to the present disclosure, the threshold interval of free T3 is 2.4 to 4.2pg/mL, the threshold interval of free T4 is 0.8 to 1.8ng/mL, and the threshold interval of rT3 is 10 to 24 ng/mL. In some embodiments, concentrations below the threshold interval are considered low, concentrations within the threshold interval are considered normal, and concentrations above the threshold interval are considered high.
In some embodiments of the methods according to the present disclosure, the threshold interval of free T3 is 2.8 to 4.0pg/mL, the threshold interval of free T4 is 0.8 to 2.2ng/mL, and the threshold interval of rT3 is 10 to 24 ng/mL. In some embodiments, concentrations below the threshold interval are considered low, concentrations within the threshold interval are considered normal, and concentrations above the threshold interval are considered high.
In one embodiment, the method according to the present disclosure further comprises the step of treating the thyroid-related disorder. In a particular embodiment, the treatment comprises administering the drug in a therapeutically effective amount. In a further embodiment, the drug is a thyroid stimulating compound.
In some embodiments, the thyroid stimulating compound is selected from T3, T4, TSH, thyroid autoantibodies (TRAb, TPOAb, and TgAb), and thyroglobulin.
Test subject
One aspect of the present disclosure provides a method according to the present disclosure, wherein the subject is a human subject. In particular embodiments, the human subject is a child or an adult.
In other embodiments of the methods according to the present disclosure, the subject is a horse, cow, sheep, pig, goat, cat, or dog.
Sample (I)
In a particular embodiment of the method according to the present disclosure, the sample is a blood sample, a serum sample or a plasma sample, optionally wherein the sample has been processed prior to the analysis.
In particular embodiments, the treatment prior to analysis comprises filtration, removal of rT3, and/or adjustment of pH. It will be appreciated by those skilled in the art that filtration of a sample, such as a blood sample, may provide a means for removing blood cells. The pH can be adjusted by adding a suitable acid or base to the sample until the desired pH is obtained. Suitable acids and bases for adjusting the pH are known to those skilled in the art.
Detection techniques
In some embodiments of the methods according to the present disclosure, the thyroid hormone is detected using Surface Plasmon Resonance (SPR). In particular embodiments, surface plasmon resonance readings are used to determine the concentration of one or more thyroid hormones.
Surface plasmon resonance is a resonant oscillation under excitation of incident light that conducts electrons at the interface between a negative dielectric constant material and a positive dielectric constant material. SPR is the basis of many standard tools for measuring the adsorption of materials on a planar metal (e.g. gold or silver) surface or on the surface of metal nanoparticles. It is the rationale behind many color-based biosensor applications, different sensors and diatom photosynthesis. SPR can be used to detect biomolecule binding interactions. In SPR, a molecular chaperone such as a protein is immobilized on a metal membrane. Photo-exciting surface plasmons in the metal; this causes a change in the detectable surface plasmon signal when the binding partner binds to the immobilized molecule.
In some embodiments of the methods according to the present disclosure, the iodothyronine deiodinase is immobilized on a metal surface or on a nanoparticle layer on a substrate. In some embodiments, the substrate is a glass chip.
In some embodiments of the methods according to the present disclosure, the thyroid hormone is detected or monitored by electrochemical transduction.
Electrochemical biosensors, also known as biosensors using electrochemical transduction, provide an attractive means of analyzing the content of a biological sample by directly converting a biological event into an electronic signal. The most common techniques in electrochemical biosensing include cyclic voltammetry, chronoamperometry, chronopotentiometry, impedance spectroscopy, and field effect transistor-based methods as well as nanowire or magnetic nanoparticle-based biosensing. Other measurement techniques that may be used in conjunction with electrochemical detection may further include electrochemical forms of surface plasmon resonance, optical waveguide optical mode spectroscopy, ellipsometry, quartz crystal microbalance, and scanning probe microscopy.
The general performance of electrochemical transduction and electrochemical sensors is generally determined by the surface structure that connects the electrodes to the nanoscale biological sample. The modification of the electrode surface, various electrochemical transduction mechanisms, and the choice of biological components bound to the electrode all affect the ultimate sensitivity of the sensor.
Thyroid-related disorders
In some embodiments, the thyroid-related disease is selected from the following list: hypothyroidism, hyperthyroidism, clinical depression, Goitre (Goitre), Graves-base disease, hashimoto's thyroiditis, diseases of euthyroidic function (eutheroid sick), and Polar T3syndrome (Polar T3 syndrome).
In particular embodiments, hyperthyroidism is characterized by high free T4, high free T3, and low TSH. In one embodiment, the disease of normal thyroid function is characterized by low free T3 and high rT 3. In another embodiment, hypothyroidism is primary or secondary. In one embodiment, primary hypothyroidism is characterized by low free T4, normal or low free T3, and high TSH. In one embodiment, secondary hypothyroidism is characterized by low free T4, normal or low free T3, and normal or low TSH.
Household appliance
Yet another aspect of the invention provides a handheld device for detecting, quantifying, and/or monitoring a thyroid hormone selected from the group consisting of fT3, fT4, and rT3, the device comprising:
a. a sample inlet;
b. a sensor, comprising:
i. a substrate, a first electrode and a second electrode,
a first iodothyronine deiodinase selected from EC1.21.99.3 and EC1.21.99.4, and
a second iodothyronine deiodinase selected from EC 1.21.99.4;
c. a detector configured to receive the signal from the sensor and convert it to a user-readable format;
d. optionally, a device for separating cellular components from a sample.
Yet another aspect of the invention provides a handheld device for detecting, quantifying, and/or monitoring a thyroid hormone selected from the group consisting of fT3, fT4, and rT3, the device comprising:
e. a sample inlet;
f. a sensor, comprising:
i. a substrate, a first electrode and a second electrode,
a first iodothyronine deiodinase selected from EC1.21.99.3 and EC1.21.99.4,
a second iodothyronine deiodinase selected from EC1.21.99.4, and
an anti-rT 3 antibody;
g. a detector configured to receive the signal from the sensor and convert it to a user-readable format;
h. optionally, a device for separating cellular components from a sample.
Yet another aspect of the present disclosure provides a handheld device for detecting, quantifying, and/or monitoring a thyroid hormone, wherein the thyroid hormone is selected from the group consisting of fT3, fT4, and rT3, the device comprising:
a) a sample inlet;
b) a sensor comprising an iodothyronine deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the iodothyronine deiodinase is immobilized on the sensor, and wherein the inlet is configured to place the sample in contact with the sensor;
c) a detector configured to receive the signal from the sensor and convert it to a user-readable format;
d) optionally, a device for separating cellular components from a sample.
In a particular embodiment, a handheld device according to the present disclosure includes a sensor as defined in any one of the embodiments of the present disclosure.
Item
1. A sensor for quantifying thyroid hormone, the sensor comprising an iodothyronine deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the iodothyronine deiodinase is immobilized on the sensor.
2. A sensor for detecting thyroid hormone, the sensor comprising an iodothyronine deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the iodothyronine deiodinase is immobilized on the sensor.
3. The sensor of any one of the preceding items, wherein the iodothyronine deiodinase is mammalian.
4. The sensor of any one of the preceding items, wherein the iodothyronine deiodinase is human.
5. The sensor of any one of the preceding items, wherein the iodothyronine deiodinase is conjugated to a further moiety.
6. The sensor of any one of the preceding items, wherein the additional moiety is a peptide.
7. The sensor of any one of the preceding items, wherein the further portion is a marker.
8. The sensor of any one of the preceding items, wherein the sensor comprises 10 to 100IU of iodothyronine deiodinase.
9. The sensor of any one of the preceding items, wherein the iodothyronine deiodinase is type 2 iodothyronine deiodinase [ EC1.21.99.3 ].
10. The sensor of any one of the preceding items, wherein the iodothyronine deiodinase is type 3 iodothyronine deiodinase [ EC1.21.99.4 ].
11. The sensor of any one of the preceding items, wherein the sensor comprises both a type 2 and a type 3 iodothyronine deiodinase.
12. The sensor of any one of the preceding items, wherein the type 1 iodothyronine deiodinase comprises or consists of a polypeptide or fragment thereof having an amino acid sequence identical to SEQ ID NO: 1, at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity entity, such as about 100% sequence identity.
13. The sensor of any one of the preceding items, wherein the type 2 iodothyronine deiodinase comprises or consists of a polypeptide or fragment thereof having an amino acid sequence identical to SEQ ID NO: 2 at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity entity, such as about 100% sequence identity.
14. The sensor of any one of the preceding items, wherein the type 3 iodothyronine deiodinase comprises or consists of a polypeptide or fragment thereof having an amino acid sequence identical to SEQ ID NO: 3 at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity entity, such as about 100% sequence identity.
15. The sensor according to any one of the preceding items, wherein the iodothyronine deiodinase is recombinantly produced, e.g. by means of cell-free expression.
16. The sensor of any one of the preceding items, wherein the thyroid hormone is selected from free T4, free T3, and trans T3(rT 3).
17. The sensor of any one of the preceding items, wherein the sensor comprises a substrate, and wherein the substrate is an electrode or a chip.
18. The sensor of any one of the preceding items, wherein the chip is a glass chip.
19. The sensor of any one of the preceding items, wherein the substrate has a modified surface.
20. The sensor of any one of the preceding items, wherein the electrodes are made of carbon, gold or platinum.
21. The sensor of any one of the preceding items, wherein the electrodes are screen printed electrodes.
22. A sensor according to any preceding claim, wherein at least one surface of the substrate is coated with a layer of gold.
23. The sensor of any one of the preceding items, wherein at least one surface of the substrate is modified with nanoparticles selected from gold, silver, copper oxide, graphene, iron oxide, and combinations thereof.
24. The sensor of any one of the preceding items, wherein at least one surface of the substrate is coated with a layer of gold, and wherein the surface is further modified with nanoparticles selected from gold, silver, copper oxide, graphene, iron oxide, and combinations thereof.
25. The sensor of any one of the preceding items, wherein the iodothyronine deiodinase is immobilized on a substrate.
26. The sensor of any one of the preceding items, wherein the iodothyronine deiodinase is immobilized on the substrate by a linker comprising nanoparticles.
27. The sensor of any one of the preceding items, wherein the iodothyronine deiodinase is immobilized on the substrate by a linker comprising:
a. cysteamine bound to a substrate, and
b. nanoparticles bound to the cysteamine and iodothyronine, optionally via one or more additional cysteamines.
28. The sensor of any one of the preceding items, configured such that the substrate can be connected to a bench top, a handheld electrochemical workstation, a surface plasmon resonance detector, or a measurement circuit.
29. The sensor according to any one of the preceding items, wherein the substrate is an electrode, and wherein the electrode is configured such that it can be connected to an electrochemical workstation.
30. The sensor according to any one of the preceding items, wherein the substrate is a chip, and wherein the chip is configured such that it can be connected to a surface plasmon resonance detector.
31. The sensor according to any one of the preceding items, wherein the sensor is configured for detecting and/or quantifying thyroid hormone.
32. A method of diagnosing a thyroid-related disorder in a subject, the method comprising the steps of:
a. providing a sample obtained from a subject,
b. contacting the sensor according to any one of the preceding items with the sample,
c. detecting one or more thyroid hormones in the sample,
d. determining the level and/or concentration of said thyroid hormone in the sample,
thereby diagnosing one or more thyroid-related conditions.
33. A method of monitoring a thyroid-related disorder in a subject, the method comprising the steps of:
a. administering to the subject a compound that stimulates the thyroid gland,
b. collecting a sample from the subject after performing step a,
c. contacting the sensor according to any one of the preceding items with the sample,
d. the signal is measured and the measured signal is,
e. using the signal to determine the concentration of thyroid hormone in the sample,
thereby monitoring the thyroid-related condition.
34. The method of clause 32, wherein steps b.
35. A method of detecting thyroid hormone in a sample, the method comprising the steps of:
a. providing a sample comprising or suspected of comprising a thyroid hormone,
b. contacting the sensor according to any one of the preceding items with the sample,
c. the signal from the sensor is measured and,
thereby detecting thyroid hormone.
36. The method of item 34, further comprising the step of d.: the signal is used to determine the concentration of one or more thyroid hormones in the sample.
37. The method of item 35, further comprising the steps of: the concentration of thyroid hormone in the body is calculated using the concentration of thyroid hormone in the sample.
38. The method of any of clauses 31-36, wherein the concentration of thyroid hormone in the sample is determined according to the kinetics of the reaction between the thyroid hormone and iodothyronine deiodinase.
39. The method of any of clauses 31-37, wherein the concentration of thyroid hormone is determined after the subject has received a drug comprising a thyroid stimulating compound.
40. The method of any of clauses 31-38, wherein the time after the subject has received the drug is 5 minutes to 48 hours.
41. The method of any of items 31-39, further comprising the steps of: comparing the level and/or concentration of the thyroid hormone in the sample to a threshold interval to diagnose a thyroid-related disorder in the subject, wherein the threshold interval is determined based on the range of concentrations of thyroid hormone in healthy human individuals (e.g., human individuals not having a thyroid-related disorder),
wherein a level and/or concentration falling outside of a threshold interval is indicative of the presence of the thyroid-related disorder.
42. The method of any one of clauses 31-40, wherein the cutoff interval for free T3 is 2.8 to 4.4 pg/mL.
43. The method of any one of clauses 31 to 40, wherein the cut-off interval for free T4 is 0.8 to 2.0 ng/mL.
44. The method of any one of clauses 31 to 40, wherein rT3 has a cut-off interval of 10 to 24 ng/mL.
45. The method of any of items 31 to 43, wherein concentrations below a threshold interval are considered low, concentrations within the threshold interval are considered normal, and concentrations above the threshold interval are considered high.
46. The method of any of clauses 31-44, further comprising the step of treating the thyroid-related disorder.
47. The method of clause 45, wherein the treatment comprises administering a drug in a therapeutically effective amount.
48. The method of clause 46, wherein the drug is a thyroid stimulating compound.
49. The method of any one of clauses 31 to 47, wherein the thyroid stimulating compound is selected from T3, T4, TSH, thyroid autoantibodies (TRAb, TPOAb and TgAb) and thyroglobulin.
50. The method of any of items 31-48, wherein the subject is a human subject.
51. The method of any of clauses 31-49, wherein the human subject is a child or an adult.
52. The method of any one of items 31 to 50, wherein the subject is a horse, cow, sheep, pig, goat, cat, or dog.
53. The method of any of clauses 31 to 51, wherein the sample is a blood sample, a serum sample, or a plasma sample, optionally wherein the sample has been processed prior to analysis.
54. The method of any one of clauses 31 to 52, wherein the treatment prior to analysis comprises filtration, removal of rT3, and/or adjustment of pH.
55. The method of any one of items 31 to 53, wherein thyroid hormone is detected using Surface Plasmon Resonance (SPR).
56. The method of any of clauses 31 to 54, wherein the concentration of one or more thyroid hormones is determined using SPR readings.
57. The method of any one of items 31 to 55, wherein the iodothyronine deiodinase is immobilized on a chip or electrode.
58. The method of any one of items 31 to 56, wherein thyroid hormone is detected or monitored by electrochemical transduction.
59. The method of any of clauses 31-57, wherein the thyroid-related disorder is selected from the list; hypothyroidism, hyperthyroidism, clinical depression, goiter, Graves-Basedow disease, Hashimoto's thyroiditis, diseases of normal thyroid function, and polar T3 syndrome.
60. The method of clause 58, wherein hyperthyroidism is characterized by high free T4, high free T3 and low TSH.
61. The method of clause 58, wherein the disease of normal thyroid function is characterized by low free T3 and high rT 3.
62. The method of clause 58, wherein the hypothyroidism is primary or secondary.
63. The method of clause 61, wherein primary hypothyroidism is characterized by low free T4, normal or low free T3, and high TSH.
64. The method of clause 61, wherein secondary hypothyroidism is characterized by low free T4, normal or low free T3, and normal or low TSH.
65. A method of making a sensor comprising an iodothyronine deiodinase, the method comprising:
a. providing a substrate, wherein the substrate is provided,
b. providing at least one iodothyronine deiodinase,
c. the iodothyronine deiodinase was immobilized on a substrate to produce a sensor comprising the iodothyronine deiodinase.
66. The method of item 64, wherein the substrate is as defined in any of the preceding items.
67. The method of any one of items 64 to 65, wherein the iodothyronine deiodinase is as defined in any one of the preceding items.
68. A hand-held device for detecting, quantifying, and/or monitoring thyroid hormone, the device comprising:
a. a sample inlet;
b. a sensor comprising an iodothyronine deiodinase [ EC1.21.99.3 and/or EC1.21.99.4] or a fragment thereof, wherein the iodothyronine deiodinase is immobilized on the sensor, and wherein the inlet is configured to place the sample in contact with the sensor;
c. a detector configured to receive the signal from the sensor and convert it to a user-readable format;
d. optionally, a device for separating cellular components from a sample.
69. The handheld device of item 67 wherein the sensor is as defined in any one of the preceding items.
70. The handheld device of any one of items 67 and 68, wherein the iodothyronine deiodinase is as defined in any one of the preceding items.
Examples
Example 1 estimation of T4 Using an IDII amperometric biosensor
IDII was extracted from rat brain as a coarse microsomal fraction and used to fabricate amperometric biosensors.
An electrochemical biosensor was fabricated using a CH604E electrochemical analyzer/workstation (CH instruments). The carbon electrode was amino-functionalized with 10uL (3-aminopropyl) triethoxysilane (5mM, APTES) and incubated for 2 hours in the dark. The electrode was rinsed with double distilled water to remove unbound 3-APTES, and then 0.5 μ g citrate-capped AuNP was added. The NP surface was amino-terminated with 20. mu.g of 2mg/mL cysteamine hydrochloride, incubated for 2 hours and washed, and then 10. mu.L of a cross-linking agent (10% (v/v) glutaraldehyde in water) was added, followed by air drying. Finally, the rat brain crude extract (microsomal fraction) was added to the electrode and allowed to dry at ambient temperature for 2 hours. Fig. 3 shows the effect of the concentration of T4 on the current response.
Results: a linear change in current was observed with increasing concentration of T4.
Example 2 estimation of T4 Using IDII voltammetric biosensors
Fig. 4 shows the cyclic voltammetry measurements used to quantify T4.
Results: as the concentration of T4 increased, the oxidation peak current decreased.
Example 3 free vs. bound T4
Further, we investigated the interference of thyroxine-binding globulin (TBG) on the detection of T4. Figure 5 shows that the current response decreases with increasing TBG concentration.
Results: the data demonstrate that the enzyme catalyzes the deiodination of fT4 only, and not the bound form (tT 4). Thus, this strategy allows direct estimation of fT 4.
Example 4 interference with serum proteins
Cyclic voltammetry measurements were performed in the presence of fetal bovine serum to study the interference of serum proteins on quantification. Figure 6 shows these measurements in fetal calf serum as the concentration of T4 increased.
Results: the data demonstrate that the oxidation peak current still follows a consistent trend, overcoming the effect of serum proteins on the measurements.
Example 5 estimation of fT3 and fT4 Using IDII-IDIII-anti-rT 3 voltammetric biosensors
IDII and IDIII were extracted from rat brain as coarse microsomal fractions and used to fabricate amperometric biosensors.
The IDII is directly connected to the surface of an electrode 1, wherein the surface of said electrode 1 is decorated with nanoparticles, or wherein the surface of said electrode is decorated with a gold layer, or wherein the surface of said electrode has been roughened by other means known to the person skilled in the art.
The IDIII is directly connected to the surface of the electrode 2, wherein the surface of said electrode 2 is decorated with nanoparticles, or wherein the surface of said electrode is decorated with a gold layer, or wherein the surface of said electrode has been roughened by other means known to the person skilled in the art.
The anti-rT 3 antibody is directly attached to the surface of the electrode 3, wherein the surface of the electrode 3 is modified with nanoparticles, or wherein the surface of the electrode is modified with a gold layer, or wherein the surface of the electrode has been roughened by other means known to the person skilled in the art. Commercially available anti-rT 3 antibodies (e.g., monoclonal anti-rT 3 antibody, Life span Bioscience, Inc. (US); rT 3/anti-triiodothyronine polyclonal antibody, Life span Bioscience, Inc. (US); anti-triiodothyronine antibody, MyBioSource. com (US); anti-triiodothyronine (rT3) monoclonal antibody, Biomatik (US); anti-triiodothyronine (rT3) polyclonal antibody Biomatik (US)) were used.
An electrochemical biosensor was fabricated using a CH604E electrochemical analyzer/workstation (CH instruments).
DIO2 deiodinase T4 and rT3 in the sample on electrode 1, thus measuring the sum of [ T4+ rT3 ].
DIO3 deiodinases T4 and T3 in the sample on electrode 2, thus measuring the sum of [ T4+ T3 ].
anti-rT 3 binds to rT3 and was therefore measured [ rT3 ].
Based on the obtained [ T4+ rT3], [ T4+ T3] and [ rT3], [ T4] and [ T3] can be determined mathematically.
Sequence listing
<110> Sirolatistated Co., Ltd (Thyrolytics AB)
<120> biosensor for diagnosing thyroid gland dysfunction
<130> P4912PC00
<150> EP 18183439.1
<151> 2018-07-13
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 249
<212> PRT
<213> Intelligent (Homo sapiens)
<220>
<221> PEPTIDE
<222> (1)..(1)
<223> DIO1
<220>
<221> PEPTIDE
<222> (126)..(126)
<223> X = selenocysteine
<400> 1
Met Gly Leu Pro Gln Pro Gly Leu Trp Leu Lys Arg Leu Trp Val Leu
1 5 10 15
Leu Glu Val Ala Val His Val Val Val Gly Lys Val Leu Leu Ile Leu
20 25 30
Phe Pro Asp Arg Val Lys Arg Asn Ile Leu Ala Met Gly Glu Lys Thr
35 40 45
Gly Met Thr Arg Asn Pro His Phe Ser His Asp Asn Trp Ile Pro Thr
50 55 60
Phe Phe Ser Thr Gln Tyr Phe Trp Phe Val Leu Lys Val Arg Trp Gln
65 70 75 80
Arg Leu Glu Asp Thr Thr Glu Leu Gly Gly Leu Ala Pro Asn Cys Pro
85 90 95
Val Val Arg Leu Ser Gly Gln Arg Cys Asn Ile Trp Glu Phe Met Gln
100 105 110
Gly Asn Arg Pro Leu Val Leu Asn Phe Gly Ser Cys Thr Xaa Pro Ser
115 120 125
Phe Met Phe Lys Phe Asp Gln Phe Lys Arg Leu Ile Glu Asp Phe Ser
130 135 140
Ser Ile Ala Asp Phe Leu Val Ile Tyr Ile Glu Glu Ala His Ala Ser
145 150 155 160
Asp Gly Trp Ala Phe Lys Asn Asn Met Asp Ile Arg Asn His Gln Asn
165 170 175
Leu Gln Asp Arg Leu Gln Ala Ala His Leu Leu Leu Ala Arg Ser Pro
180 185 190
Gln Cys Pro Val Val Val Asp Thr Met Gln Asn Gln Ser Ser Gln Leu
195 200 205
Tyr Ala Ala Leu Pro Glu Arg Leu Tyr Ile Ile Gln Glu Gly Arg Ile
210 215 220
Leu Tyr Lys Gly Lys Ser Gly Pro Trp Asn Tyr Asn Pro Glu Glu Val
225 230 235 240
Arg Ala Val Leu Glu Lys Leu His Ser
245
<210> 2
<211> 273
<212> PRT
<213> Intelligent (Homo sapiens)
<220>
<221> PEPTIDE
<222> (1)..(1)
<223> DIO2
<220>
<221> PEPTIDE
<222> (133)..(133)
<223> X = selenocysteine
<220>
<221> PEPTIDE
<222> (266)..(266)
<223> X = selenocysteine
<400> 2
Met Gly Ile Leu Ser Val Asp Leu Leu Ile Thr Leu Gln Ile Leu Pro
1 5 10 15
Val Phe Phe Ser Asn Cys Leu Phe Leu Ala Leu Tyr Asp Ser Val Ile
20 25 30
Leu Leu Lys His Val Val Leu Leu Leu Ser Arg Ser Lys Ser Thr Arg
35 40 45
Gly Glu Trp Arg Arg Met Leu Thr Ser Glu Gly Leu Arg Cys Val Trp
50 55 60
Lys Ser Phe Leu Leu Asp Ala Tyr Lys Gln Val Lys Leu Gly Glu Asp
65 70 75 80
Ala Pro Asn Ser Ser Val Val His Val Ser Ser Thr Glu Gly Gly Asp
85 90 95
Asn Ser Gly Asn Gly Thr Gln Glu Lys Ile Ala Glu Gly Ala Thr Cys
100 105 110
His Leu Leu Asp Phe Ala Ser Pro Glu Arg Pro Leu Val Val Asn Phe
115 120 125
Gly Ser Ala Thr Xaa Pro Pro Phe Thr Ser Gln Leu Pro Ala Phe Arg
130 135 140
Lys Leu Val Glu Glu Phe Ser Ser Val Ala Asp Phe Leu Leu Val Tyr
145 150 155 160
Ile Asp Glu Ala His Pro Ser Asp Gly Trp Ala Ile Pro Gly Asp Ser
165 170 175
Ser Leu Ser Phe Glu Val Lys Lys His Gln Asn Gln Glu Asp Arg Cys
180 185 190
Ala Ala Ala Gln Gln Leu Leu Glu Arg Phe Ser Leu Pro Pro Gln Cys
195 200 205
Arg Val Val Ala Asp Arg Met Asp Asn Asn Ala Asn Ile Ala Tyr Gly
210 215 220
Val Ala Phe Glu Arg Val Cys Ile Val Gln Arg Gln Lys Ile Ala Tyr
225 230 235 240
Leu Gly Gly Lys Gly Pro Phe Ser Tyr Asn Leu Gln Glu Val Arg His
245 250 255
Trp Leu Glu Lys Asn Phe Ser Lys Arg Xaa Lys Lys Thr Arg Leu Ala
260 265 270
Gly
<210> 3
<211> 304
<212> PRT
<213> Intelligent (Homo sapiens)
<220>
<221> PEPTIDE
<222> (1)..(1)
<223> DIO3
<220>
<221> PEPTIDE
<222> (170)..(170)
<223> X = selenocysteine
<400> 3
Met Pro Arg Gln Ala Thr Ser Arg Leu Val Val Gly Glu Gly Glu Gly
1 5 10 15
Ser Gln Gly Ala Ser Gly Pro Ala Ala Thr Met Leu Arg Ser Leu Leu
20 25 30
Leu His Ser Leu Arg Leu Cys Ala Gln Thr Ala Ser Cys Leu Val Leu
35 40 45
Phe Pro Arg Phe Leu Gly Thr Ala Phe Met Leu Trp Leu Leu Asp Phe
50 55 60
Leu Cys Ile Arg Lys His Phe Leu Gly Arg Arg Arg Arg Gly Gln Pro
65 70 75 80
Glu Pro Glu Val Glu Leu Asn Ser Glu Gly Glu Glu Val Pro Pro Asp
85 90 95
Asp Pro Pro Ile Cys Val Ser Asp Asp Asn Arg Leu Cys Thr Leu Ala
100 105 110
Ser Leu Lys Ala Val Trp His Gly Gln Lys Leu Asp Phe Phe Lys Gln
115 120 125
Ala His Glu Gly Gly Pro Ala Pro Asn Ser Glu Val Val Leu Pro Asp
130 135 140
Gly Phe Gln Ser Gln His Ile Leu Asp Tyr Ala Gln Gly Asn Arg Pro
145 150 155 160
Leu Val Leu Asn Phe Gly Ser Cys Thr Xaa Pro Pro Phe Met Ala Arg
165 170 175
Met Ser Ala Phe Gln Arg Leu Val Thr Lys Tyr Gln Arg Asp Val Asp
180 185 190
Phe Leu Ile Ile Tyr Ile Glu Glu Ala His Pro Ser Asp Gly Trp Val
195 200 205
Thr Thr Asp Ser Pro Tyr Ile Ile Pro Gln His Arg Ser Leu Glu Asp
210 215 220
Arg Val Ser Ala Ala Arg Val Leu Gln Gln Gly Ala Pro Gly Cys Ala
225 230 235 240
Leu Val Leu Asp Thr Met Ala Asn Ser Ser Ser Ser Ala Tyr Gly Ala
245 250 255
Tyr Phe Glu Arg Leu Tyr Val Ile Gln Ser Gly Thr Ile Met Tyr Gln
260 265 270
Gly Gly Arg Gly Pro Asp Gly Tyr Gln Val Ser Glu Leu Arg Thr Trp
275 280 285
Leu Glu Arg Tyr Asp Glu Gln Leu His Gly Ala Arg Pro Arg Arg Val
290 295 300
- 上一篇：一种医用注射器针头装配设备
- 下一篇：酶法工业化生产NAD的方法